The Scanning Transmission Electron Microscope EDITOR-IN-CHIEF PETER W

Advances in IMAGING AND ELECTRON PHYSICS

VOLUME 159

Cold Field Emission and the Scanning Transmission Electron Microscope EDITOR-IN-CHIEF PETER W. HAWKES CEMES-CNRS Toulouse, France Advances in IMAGING AND ELECTRON PHYSICS

VOLUME 159 Cold Field Emission and the Scanning Transmission Electron Microscope

Edited by PETER W. HAWKES CEMES-CNRS, Toulouse, France

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Printed in the United States of America 0910111210987654321 Contents

Preface xi Contributors xv Future Contributions xvii

1. The Work of Albert Victor Crewe on the Scanning Transmission Electron Microscope and Related Topics 1 A. V. Crewe 1. Introduction 2 2. Some Chicago Aberrations: A Personal Collection 6 Acknowledgments 13 3. Electron Microscope Studies: Achievements of the Crewe Lab 13 Introduction 14 Construction (Reference Group A) 16 Source Development (Reference Group B) 17 STEM Development and Atomic Images (Reference Group C) 18 The Field Emission SEM (Reference Group D) 19 Energy Loss and Radiation Damage (Reference Group E) 20 Secondary Electron Production (Reference Group F) 21 DNA Labeling (Reference Group G) 21 Nucleosomes (Reference Group H) 22 Attempts at Aberration Correction (Reference Group I) 23 Theoretical Electron Optics (Reference Group J) 27 Optimization and the Super-High-Resolution SEM (Reference Group K) 28 Image Processing (Reference Group L) 30 Three-Dimensional Reconstruction (Reference Group M) 31 Hemoglobin Work (Reference Group N) 32 References (of Chicago Aberrations) 37 References (of DOE Report) 38 2. A Review of the Cold-Field Electron Cathode 63 L. W. Swanson and G. A. Schwind 1. Introduction 63 2. Work Function 65

v vi Contents

3. Energy Distribution 67 3.1. Theoretical Background 67 3.2. Analytical versus Numerical Results 68 3.3. Measured Values of the FWHM for the Tungsten Cold-Field Electron 72 4. Source Optics 79 5. Column Optics Using the Cold-Field Electron Source 82 6. Current Stability 86 6.1. High-Frequency Current Fluctuations 87 6.2. Long-Term Current Drift 88 6.3. Cold-Field Electron End-of-Life Mechanisms 94 7. Summary 97 Acknowledgments 98 References 98

3. History of the STEM at Brookhaven National Laboratory 101 Joseph S. Wall, Martha N. Simon, and James F. Hainfeld 1. Introduction 101 2. Instrument Design Parameters 102 3. Heavy Metal Cluster Labeling 106 4. Early User/Collaborator Projects 108 5. Recent Work 115 6. Conclusion 115 Acknowledgments 115 References 116

4. Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 123 Hiromi Inada, Hiroshi Kakibayashi, Shigeto Isakozawa, Takahito Hashimoto, Toshie Yaguchi, and Kuniyasu Nakamura 1. Introduction 124 2. The Dawn of Hitachi Electron Microscopes (by Hiromi Inada) 125 2.1. Crewe STEM Shock and Field Emission Development 125 3. Cold FE-SEM Studies and Expansion to Different Fields 128 3.1. CFE-STEM Development at HCRL 128 3.2. CFE-SEM Development at Naka Works 131 3.3. Studies of Field Emission Stability 132 4. Expansion of High-Voltage TEMs and STEMs 137 4.1. Holography Studies by Tonomura with FE-TEMs 137 4.2. Multistage Acceleration CFEG for TEM Applications 138 4.3. Commercialized Analytical CFE-TEM/STEM 139 5. Development of 50-kV STEM in 1970s (by Shigeto Isakozawa) 141 5.1. Development of Hitachi’s 50kV Prototype CFE-STEM 141 Contents vii

5.2. Bright-Field Images Obtained with 50-kV CFE-STEM 142 5.3. Single-Atom Observation 144 5.4. Development of the Electron Energy-Loss Spectrometer 147 5.5. Further Development of 50-kV CFE-STEM 149 6. Hitachi’s First Commercialized Dedicated STEM (by Takahito Hashimoto) 150 6.1. 200-kV Analytical CFE-TEM, HF-2000 150 6.2. ‘‘Gate Viewer,’’ Trigger for Dedicated STEM 154 6.3. Novel Functions for HD Series 161 7. Cutting-Edge Applications with Customized HD Models (by Toshie Yaguchi) 165 7.1. 120-kV FE UHV for Nanotubes 165 7.2. 200-kV FE Ultrahigh-Resolution STEM 165 7.3. 3D Structural and Elemental Analysis 169 8. Aberration-Corrected CFE-STEM (by Kuniyasu Nakamura) 170 8.1. Atomic-Level Characterization Instrument 170 8.2. Theoretical Consideration of Advantages of Aberration-Corrected CFE-STEM 172 8.3. Evaluation of Aberration-Corrected CFE-STEM 173 8.4. Advanced Application Results with HD-2700C 176 9. Conclusion and Future Prospective (by Hiroshi Kakibayashi) 181 Acknowledgments 182 References 182

5. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 187 P. W. Hawkes 1. Introduction 188 2. The AEI STEM-1 188 3. The Siemens ST100F 191 3.1. The New ELMISKOP ST100F Scanning Transmission Electron Microscope 195 Introduction 195 Construction of the ELMISKOP ST100F 195 Advantages of the Scanning Transmission Electron Microscope 198 Imaging with Low Radiation Damage 198 Conclusion 201 3.2. Image Forming Systems 203 General Remarks 203 The Imaging Process 204 Image Forming Properties of Magnetic Lenses 205 Strong Lenses 208 Lens Systems for CTEM and STEM: Similarities and Differences 210 Requirements for Analytical Microscopy 215 Acknowledgments 217 References 217 viii Contents

6. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 221 Ian R. M. Wardell and Peter E. Bovey 1. The Early Days 222 2. Design Considerations for a Commercial STEM 224 2.1. The Objective Lens 225 2.2. The Specimen Stage 225 2.3. The Electron Gun 226 2.4. Location of Analytical Facilities 226 2.5. The Resultant Assembly 227 3. The First STEMs and HB5 Development 227 3.1. The Optical Column 232 3.2. Stage Development 238 3.3. Early Airlock and Manipulator 240 3.4. The Dark-Field Detector 241 3.5. Secondary Electron Detector 245 3.6. Diffraction Facilities 246 3.7. The Energy Analyzer Mk1 249 3.8. Electronics 251 4. Improved Resolution 254 4.1. The MIT HB5 254 4.2. The University of Illinois HB5 255 5. Other Developments 256 5.1. Beam-Blanking Plates 256 5.2. Detectors and the Virtual Objective Aperture 257 5.3. Stage Motor Drives 261 5.4. New Airlock 262 5.5. Energy Analyzer Mk2 264 5.6. Gun Lens 266 5.7. High-Excitation Objective Lens 266 6. Stages and Cartridges 268 6.1. Basic Cartridges 268 6.2. Beryllium Cartridges 269 6.3. Cold Stage 269 6.4. Cryo-Transfer System 270 7. The HB501 271 7.1. General Development 271 7.2. HB501UX and High-Resolution Imaging 273 8. Special and Variant Instruments 273 8.1. University of Glasgow’s HB5 275 8.2. The HB501A 276 9. The HB601 278 Contents ix

10. Postscript 280 Acknowledgments 282 References 283

7. Development of the 300-kV Vacuum Generator STEM (1985–1996) 287 H. S. von Harrach 1. Prelude in Oxford (1973–1975) 288 2. The MIDAS Project (1985–1988) 288 2.1. System 289 2.2. Gun Lens 289 2.3. Objective Lens 290 2.4. Side-Entry Stage 291 2.5. MIDAS Performance 291 3. The HB603 300-kV STEM instruments 292 3.1. The Prototype Design Phase 293 4. MIT and ANL Designs 305 5. Oak Ridge Design 307 6. Lehigh Design 310 7. Testing, Testing 311 8. Record-Breaking Results 316 8.1. Source Brightness 316 8.2. Energy Spread 316 8.3. ADF Resolution 316 8.4. X-Ray Microanalysis 317 9. Conclusions 320 Acknowledgments 321 References 322

8. On the High-Voltage STEM Project in Toulouse (MEBATH) 325 Bernard Jouffrey 1. Introduction 325 2. The Building 328 3. Generator 333 4. The Column 336 4.1. The Source 336 4.2. Accelerating Tube 342 4.3. Lenses 346 4.4. Spectrometers 348 5. Suspension of the Platform and the Microscope 349 6. Recording of The Signal 351 7. Conclusions 352 Acknowledgments 352 References 353 x Contents

9. Scanning Transmission Electron Microscopy: Biological Applications 357 Andreas Engel 1. Introduction 358 2. Image Formation 359 2.1. Electron-Sample Interactions 359 2.2. The Optical System 360 2.3. Detectors 360 2.4. Single-Electron Counting 361 2.5. Imaging Modes 363 3. Imaging Thin Sections 363 4. Imaging Negatively Stained Samples 365 5. Mass Measurements Using the Basel STEM 366 5.1. Principle 366 5.2. Estimate of the Statistical Error 369 5.3. Mass Determination of Biological Samples 371 6. Specific Examples of STEM Imaging and Mass Measurements 373 7. High-Throughput Visual Proteomics 378 8. Conclusions and Perspectives 380 Acknowledgments 382 References 382

10. STEM at Cambridge University: Reminiscences and Reflections from the 1950s and 1960s 387 K. C. A. Smith 1. STEM as a Diagnostic Tool for SEM 387 2. The Observation of Specimens in Water Vapor by Means of STEM 393 3. High-Voltage STEM Using a Single-Field Condenser—Objective Lens 400 References 405

Contents of Volumes 151–158 407

Index 411 Preface

Tom Mulvey (right) with Elmar Zeitler, Berlin 1990* I have the sad duty of recording the death of our other Honorary Associ- ate Editor, Tom Mulvey, who died on 16 July 2009, a few days before his 88th birthday. He was born on 26 July 1921 in Manchester; during the wartime years he served in the Navy and was sent to the Far East where he was responsible for anti-submarine operations. After being demobbed, he was awarded the MSc degree by Manchester University for a substantial study of electrostatic lenses and then spent several years in the Metropolitan–Vickers research establishment at Aldermaston Court. It was during this period that he met Rita, his future wife, for whom he cared devotedly during the last years of her life. In 1965, he joined the Birmingham College of Advanced Technology, later the University of Aston in Birmingham (today Aston University) and was made Emeritus Professor on his retirement in 1986. The last years of his life were spent near his son Nicholas, close to Oxford. In addition to his own research, he took over from V. E. Cosslett as editor of Advances in Optical & Electron Microscopy and later became an Associate Editor of Advances in Imaging & Electron Physics, to which he contributed several biographical articles on the pioneers of electron microscopy: Ernst Ruska, Dennis Gabor and Jan le Poole; he also wrote the Royal Society memoir on V. E. Cosslett and

* Courtesy of Prof. Dr B. Lencova´

xi xii Preface translated Ruska’s book on the early history of the electron microscope. The history of electron microscopy is richer by the presence of his many articles, of which the first (Brit. J. Appl. Phys. 13, 1962, 197–207) is truly remarkable, written before most of the fundamental documents for such a study had been published; a more extended account appeared in Proc. RMS 2 (1967) 201–227. The volume of these Advances that he guest-edited on ‘‘The Growth of Electron Microscopy’’ was a splendid achievement. His research interests were wide-ranging and he introduced many unconventional lens designs. Among the most memorable were boiling-water lenses, pancake lenses, single-polepiece lenses and snorkel lenses, the dimensions of which were often indicated in units of ‘‘England’s Glory’’ matchboxes. For his 80th birthday (but rather belatedly), the Royal Microscopical Society (of which he was an honorary Fellow) brought together many of his friends for a celebration in Birmingham, which is commemorated in a long section of the Proceedings of the society (39, 2004, 206–233). A much fuller biography is to be found there, as well as tributes from many of those present; a bibliography of his publications was published for an earlier birthday and occupies four pages of the Journal of Microscopy (179, 1995, 101–104). One aspect that emerges from the tributes is just how entertaining he could be – he had an unlimited stock of anecdotes about present and past colleagues and jokes ranging from the politically highly incorrect to the purely frivolous. He was also a Distinguished Scientist and Fellow of the Microscopy Society of America and was awarded the gold Krizı´k medal of the Czech Academy of Sciences. He will be greatly missed and we extend our sympathy to all his family. The scanning transmission electron microscope (STEM) has a long history, going back to the 1960s. In the present volume, its development is traced from its inception in the Argonne National Laboratory and the University of Chicago, where A. V. Crewe built the first simple instruments, the success of which owed much to the quality of the field-emission gun. Albert Crewe was unfortunately not able to prepare a new account for this volume and, with his approval, two older documents are reproduced here: ‘‘Some Chicago aberrations’’ and a long report submitted to the US Department of Energy in 1992, which includes a very full list of publications. The field-emission source is the vital element of the STEM, for the brightness of thermionic guns is insufficient for a high-resolution instrument. Chapter 2 offers a review of cold field-emission source properties by L. W. Swanson and G. A. Schwind, both major contributors to our understanding of these guns. They cover both early developments and current research preoccupations. The following group of chapters describes specific instruments and commercial developments. J. S. Wall was a member of Crewe’s team Preface xiii when the first STEMs were being constructed and, as he explains, he was later recruited by Brookhaven National Laboratory to advance the construction of their STEM. Together with M. N. Simon and J. F. Hainfeld, he describes the design of this early home-made instrument and subsequent developments. The spectacular images obtained by Crewe awakened interest worldwide, not least in Japan. Hitachi in particular was stimulated to enter the STEM field and in Chapter 4, H. Inada, H. Kakibayashi, S. Isakozawa, T. Hashimoto, T. Yaguchi and K. Nakamura describe the early work that made STEM development possible and such topics as holography that also benefited from highly coherent sources. The various models are presented, together with some of the most impressive results. The closing paragraphs bring the story up to the aberration-corrected era. This does not exhaust the STEM projects in Japan. For example, a high-voltage instrument constructed at Nagoya University is described by M. Hibino, H. Shimoyaya and S. Maruse ( J. Electron Microsc. Tech. 12, 1989, 296–304) and an aberration-corrected STEM is also available from JEOL. In Europe, Siemens, AEI and Vacuum Generators all embarked on STEM projects. The VG instruments are described at length by I. R. M. Wardell, P. E. Bovey and S. von Harrach in Chapters 6 and 7. But for Siemens and AEI, I was unable to find authors and have therefore written an account of their STEMs myself, based on published information and personal communication from several of those involved (Chapter 5). It was natural that the Laboratoire d’Optique Electronique in Toulouse, already home to two high-voltage transmission electron microscopes, should embark on the design and construction of a high-voltage STEM. In Chapter 8, B. Jouffrey, who launched and piloted this project, describes the principal stages in the preparations for this instrument, which alas was never completed. The foregoing chapters contain much description of instrumentation while Chapter 9 is a reminder that the STEM has proved an invaluable tool for microscopists in many fields of application. Here, A. Engel shows how fruitful it has been in biology. We conclude with a chapter by K. C. A. Smith, who was not only a pioneer of the scanning electron microscope in the Cambridge University Engineering Department but was also responsible for the design and construction of the first Cambridge high-voltage electron microscope, built in the Cavendish Laboratory in the 1960s. It was at that time that the idea of operating the microscope in the STEM mode came to him and it is this little-known early effort that is recorded here. It is worth emphasizing that it was first mentioned in print in 1968, the same year as Crewe’s key paper in the Journal of Applied Physics. This volume should also have contained two more chapters, by M. S. Isaacson and O. L. Krivanek. Circumstances have prevented them xiv Preface from completing their chapters in time and their contributions will therefore appear in later volumes of the Advances. I greatly regret that they do not appear here, for M. S Isaacson was one of the pioneers of the STEM while O. L. Krivanek not only produced the first successful corrector of spherical aberration for the STEM but also suggested to me that a thematic volume on STEM would be timely. As always, I am most grateful to all the contributors for writing such readable and informative accounts.

Peter W. Hawkes Contributors

A. V. Crewe 1 Enrico Fermi Institute and Department of Physics, University of Chicago, Chicago, USA L. W. Swanson and G. A. Schwind 63 FEI Co. Hillsboro, Oregon, USA Joseph S. Wall, Martha N. Simon, and James F. Hainfeld 101 Biology Department, Brookhaven National Laboratory, Upton, New York 11073, USA Hiromi Inada, Hiroshi Kakibayashi, Shigeto Isakozawa, Takahito Hashimoto, Toshie Yaguchi, and Kuniyasu Nakamura 123 Hitachi High-Technologies Corp., Tokyo, Japan P. W. Hawkes 187 CEMES-CNRS, Toulouse, France Ian R. M. Wardell 221 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom Peter E. Bovey 221 Lindfield, West Sussex, United Kingdom H. S. von Harrach 287 FEI Electron Optics, Eindhoven, The Netherlands Bernard Jouffrey 325 Laboratoire de Structures, Sols et Mate´riaux (LMSS-Mat), E´ cole Centrale Paris, Unite´ Mixte de Recherche (UMR), National Center for Scientific Research (CNRS) 8579, Chaˆtenay-Malabry, France Andreas Engel 357 M. E. Mu¨ller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, CH-4056 Switzerland, and Department of Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, Wood Bldg 321D, Cleveland, Ohio 44106, USA K. C. A. Smith 387 Fitzwilliam College, University of Cambridge, Cambridge CB3 0DG, United Kingdom

xv Future Contributions

S. Ando Gradient operators and edge and corner detection K. Asakura Energy-filtering X-ray PEEM W. Bacsa Optical interference near surfaces, sub-wavelength microscopy and spectroscopic sensors Baranczuk, J. Giesen, Z. K. Simon and P. Zolliker (vol. 160) Gamut mapping C. Beeli Structure and microscopy of quasicrystals C. Bobisch and R. Mo¨ller Ballistic electron microscopy G. Borgefors Distance transforms Z. Bouchal Non-diffracting optical beams A. Buchau Boundary element or integral equation methods for static and time-dependent problems B. Buchberger Gro¨bner bases E. Cosgriff, P. D. Nellist, L. J. Allen, A. J. d’Alfonso, S. D. Findlay, and A. I. Kirkland Three-dimensional imaging using aberration-corrected scanning confocal electron microscopy T. Cremer Neutron microscopy C. J. Edgcombe New dimensions for field emission: effects of structure in the emitting surface

xvii xviii Future Contributions

A. N. Evans (vol. 160) Area morphology scale-spaces for colour images A. X. Falca˜o The image foresting transform R. H. A. Farias and E. Recami Introduction of a quantum of time (‘‘chronon’’) and its consequences for the electron in quantum and classical physics R. G. Forbes Liquid metal ion sources C. Fredembach Eigenregions for image classification A. Go¨lzha¨user Recent advances in electron holography with point sources M. Haschke Micro-XRF excitation in the scanning electron microscope L. Hermi, M. A. Khabou and M. B. H. Rhouma (vol. 162) Shape recognition based on eigenvalues of the Laplacian M. I. Herrera The development of electron microscopy in Spain M. S. Isaacson Early STEM development J. Isenberg Imaging IR-techniques for the characterization of solar cells K. Ishizuka Contrast transfer and crystal images A. Jacobo Intracavity type II second-harmonic generation for image processing L. Kipp Photon sieves G. Ko¨gel Positron microscopy T. Kohashi Spin-polarized scanning electron microscopy O. L. Krivanek Aberration-corrected STEM R. Leitgeb Fourier domain and time domain optical coherence tomography B. Lencova´ Modern developments in electron optical calculations Future Contributions xix

H. Lichte New developments in electron holography M. Mankos, V. Spasov and E. Munro (vol. 161) Principles of dual-beam low energy electron microscopy M. Marrocco Discrete diffraction M. Matsuya Calculation of aberration coefficients using Lie algebra S. McVitie Microscopy of magnetic specimens J. D. Mendiola-Santiban˜ez, I. R. Terol-Villalobos and I. M. Santilla´n-Me´ndez (vol. 161) Determination of adequate parameters for connected morphological contrast mappings through morphological contrast measures I. Moreno Soriano and C. Ferreira (vol. 161) Fractional Fourier transforms and geometrical optics M. A. O’Keefe Electron image simulation D. Oulton and H. Owens Colorimetric imaging D. Paganin and T. Gureyev Intensity-linear methods in inverse imaging N. Papamarkos and A. Kesidis The inverse Hough transform K. S. Pedersen, A. Lee and M. Nielsen The scale-space properties of natural images Y. Pu (vol. 160) Harmonic holography G. X. Ritter and G. Urcid (vol. 160) Lattice algebra approach to endmember determination in hyperspectral imagery R. Ru¨denberg (vol. 160) Origin and background of the invention of the electron microscope, Memoir H. G. and P. Rudenberg (vol. 160) Origin and background of the invention of the electron microscope, Commentary R. Shimizu, T. Ikuta and Y. Takai Defocus image modulation processing in real time S. Shirai CRT gun design methods xx Future Contributions

A. S. Skapin The use of optical and scanning electron microscopy in the study of ancient pigments T. Soma Focus-deflection systems and their applications P. Sussner and M. E. Valle Fuzzy morphological associative memories I. Talmon Study of complex fluids by transmission electron microscopy M. E. Testorf and M. Fiddy Imaging from scattered electromagnetic fields, investigations into an unsolved problem N. M. Towghi

Ip norm optimal filters E. Twerdowski Defocused acoustic transmission microscopy Y. Uchikawa Electron gun optics K. Vaeth and G. Rajeswaran Organic light-emitting arrays V. Velisavljevic and M. Vetterli (vol. 161) Space-frequence quantization using directionlets M. H. F. Wilkinson and G. Ouzounis (vol. 161) Second generation connectivity and attribute filters E. Wolf (vol. 162) History and a recent development in the theory of reconstruction of crystalline solids from X-ray diffraction experiments Chapter 4

Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes

Hiromi Inada, Hiroshi Kakibayashi, Shigeto Isakozawa, Takahito Hashimoto, Toshie Yaguchi, and Kuniyasu Nakamura

Contents 1. Introduction 124 2. The Dawn of Hitachi Electron Microscopes (by Hiromi Inada) 125 2.1. Crewe STEM Shock and Field Emission Development 125 3. Cold FE-SEM Studies and Expansion to Different Fields 128 3.1. CFE-STEM Development at HCRL 128 3.2. CFE-SEM Development at Naka Works 131 3.3. Studies of Field Emission Stability 132 4. Expansion of High-Voltage TEMs and STEMs 137 4.1. Holography Studies by Tonomura with FE-TEMs 137 4.2. Multistage Acceleration CFEG for TEM Applications 138 4.3. Commercialized Analytical CFE-TEM/STEM 139 5. Development of 50-kV STEM in 1970s (by Shigeto Isakozawa) 141 5.1. Development of Hitachi’s 50kV Prototype CFE-STEM 141

Hitachi High-Technologies Corp., Tokyo, Japan

123 124 Hiromi Inada et al.

1. INTRODUCTION

Hitachi started researching and developing electron microscopes in 1940 and has since developed and manufactured many electron microscopes. Cold-field emission (CFE) technology developed in a number of different directions. Crewe invented the field emission scanning transmission electron microscope (FE-STEM) and used an early version to observe individual atoms. Stimulated by his reports, Hitachi began developing a CFE-STEM in the mid-1960s and invited Crewe to serve as a consultant to Hitachi. He introduced his CFE technology and helped Hitachi develop and commercialize STEMs and scanning electron microscopes (SEMs). Hitachi’s prototype CFE-STEM opened a new world of analytical electron microscopes equipped with an X-ray analyzer and energy spectrometer. The HFS-2 FE-SEM, which was built at Hitachi’s Naka Works in 1972, was the first step in Hitachi’s development of FE-SEMs. A 50-kV CFE-TEM Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 125 was developed at Hitachi’s Central Research Laboratory (HCRL) by Tonomura and his group in 1973 for application to electron holography, which enabled higher image resolution. To incorporate a sophisticated FE electron gun into microscopes, it was necessary to create an ultrahigh vacuum (UHV) (of the order of 10 8 Pa) to fundamentally stabilize the FE current. This chapter describes Hitachi’s efforts over the past 40-plus years to develop CFE technology and STEMs. We also introduce cutting-edge application data obtained with the latest CFE-STEMs (with and without an aberration corrector), highlight Hitachi’s contributions to FE technology development, and show how the knowledge gained has been passed from generation to generation at Hitachi. Additionally, we show how CFE SEM/ STEM technologies were established.

2. THE DAWN OF HITACHI ELECTRON MICROSCOPES (BY HIROMI INADA)

K. Kasai, one of the founders of the 37th Subcommittee of the Japanese Society for the Promotion of Science, with regard to electron microscopes, moved to Hitachi from one of the Japanese National Laboratories, Electrotechnical Laboratory in 1939 and began to develop electron microscopes by using the experience he gained working on cathode ray oscil- lography in Germany. His guiding principle was that one should work in a manufacturing company to be able to do innovative practical development. Kasai joined forces with B. Tadano at Hitachi and, in 1940, they developed the first Hitachi TEM (model HU-1) (Komoda 1996a). A second TEM with improved resolution (the HU-2) was developed and installed at Nagoya Imperial University (now Nagoya University) in 1943. Hitachi established a central research laboratory (HCRL) in Kokubunji, a suburb of Tokyo, in 1942 where it continued developing electron microscopes despite the wartime conditions.

2.1. Crewe STEM Shock and Field Emission Development 2.1.1. Trigger for Field Emission Microscopes A. V. Crewe reported the successful development of an SEM equipped with a CFE gun (CFEG) in 1964 (Crewe, 1964; Crewe et al., 1970a). This report greatly affected Hitachi researchers, who were seriously fighting to obtain the highest TEM resolution in the mid-1960s (Komoda 1996b). Crewe and his group were provident in terms of advantages of CFE and the progress of UHV technology, and they began pioneering development of an STEM equipped with a CFEG in the early 1960s. They 126 Hiromi Inada et al. achieved a resolution of 0.5 nm and successfully observed individual atoms of uranium and thorium, even though their microscope had a simple design and contained a single-electron magnetic lens with a CFEG. This breakthrough observation overturned the conventional wis- dom about electron microscopy at that time and revealed the power of FE technology. Moreover, they directly observed individual atoms with a STEM, not a TEM, whereas most scientists believed that only a TEM could achieve such high-resolution imaging (Crewe et al., 1968a,b; Crewe, Wall, and Langmore, 1970b). Hitachi started developing SEMs in the early 1960s and commercialized the first thermionic gun SEM (model HSM-2) in 1969. Conventional SEMs with a thermionic gun at that time had a resolution of 10 nm, which was much worse than that of TEMs (0.3 nm); hence, nobody compared performance between TEMs and SEMs. However, Crewe’s development of an SEM in 1964 made it possible to compare SEMs with TEMs using the same criteria. An SEM can be used to acquire time series of various types of signals (secondary, transmission electron, X-ray, and energy loss) from a specimen simultaneously by focusing on an electronically specific point and/ or area by imaging it with a narrow scanning electron beam. The development of the CFE-SEM meant the birth of a new scientific instrument capable of simultaneous nanometer-order analysis and imaging. Although some scientists (Eggenberger, Hart, and Libal, 1968; Siegel et al., 1968; Wiesner and Everhart, 1969) had started early FEG electron microscopes, none of the electron microscope vendors had succeeded in building a CFE-SEM. Accordingly, Hitachi was driven to quickly develop a CFE-SEM due to its importance. R. Ueda (Nagoya University) said in the preface of the first issue revival of the Journal of Electron Microscopy in 1975, ‘‘Crewe STEM was excellent and nobody had imagined before such achievement and his outstanding concept’’ (Ueda, 1975). This statement reflects the sensational effect the FE-SEM had at that time.

2.1.2. Field Emission Gun Development at Hitachi Hitachi launched a major project in 1969 to produce ultrahigh-resolution FE-SEMs and FE-TEMs. As a microscope manufacturer, Hitachi focused on ease of use and ease of production, as well as on imaging capability. In 1970, Hitachi invited Crewe to serve as a technical consultant for its development of a CFE-SEM. Field emission was discovered by R. W. Wood in the nineteenth century (Wood, 1897). Millikan and Lauritsen (1928) and E. W. Mu¨ ller (1937) studied the characteristics of this type of emission. FE patterns Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 127 were investigated (Gomer, 1961), and study of FE as a source of electrons began in 1955 (Butler, 1966; Dyke et al., 1953; Everhart, 1967; Martin, Trolan, and Dyke, 1960). FE electrons can be obtained by applying several thousand volts to the tip of a metal needle (FE tip) with a radius of less than 100 nm. The energy spread of the emitted electrons is narrow (0.2–0.3 eV) because the emitter runs at room temperature. FE electrons are emitted through the potential barrier of the surface from near the Fermi level because the barrier is thinned by the application of a negative electronic field with a strength of 107 V/cm to the metal surface, which is the tunnel effect. The current density J(A/cm2) is obtained using equation 1 from Fowler and Nordheim (Fowler and Nordheim, 1928), and the density strongly depends on the electric field applied to the FE tip and on the work function of the metal. () 2 5 3=2 F 6:83 10 f vðyÞ J ¼ 1:54 10 2 exp ; (4.1) ft2ðyÞ F where t2(y) and v(y) are elliptic functions nearly equal to 1, F is the electric field strength, and f is the cathode work function. The thermionic electron current density is obtained using the Richardson–Dushman equation. The CFE current density (104–106 A/cm2) is three orders of magnitude larger than that of the thermionic electrons (1–10 A/cm2). The FE source is ideally a point source, and the diameter of the virtual source ranges from 5 to 10 nm because of the small FE tip, which is 1/1000 the source size of the thermionic emission (1–10 m). Thus, changing the FE source can reduce the beam spot size; that is, it can improve SEM image resolution. An electron gun should be designed to have less spherical aberration and mechanical vibration. Crewe used the Butler electrostatic lens, which has less spherical aberration than his prototype design, for his CFE-STEM and thereby obtained high resolution. An FE electron gun with a Butler lens consists of an FE tip and a diode region for the extraction and acceleration electrodes (Figure 1). A. Tonomura at HCRL used a microcomputer to optimize the Butler lens shape and minimize the aberration (Shimoyama, Ichihashi, and Tamura, 1989; Tonomura and Komoda, 1973). Table 1 shows a comparison of the characteristics of emitters with FE and thermionic electron guns. Although applying the FE gun to electron microscopes was an obvious next step, it took many years to develop such a microscope that was practical. This was due to the instability of the FE current and the difficulty of achieving an UHV of the order of 10 8 Pa. 128 Hiromi Inada et al.

FE tip

V 1 1st anode V 0

2nd anode

FIGURE 1 Schematic diagram of FE gun with Butler electrode lens.

TABLE 1 Characteristics of Emitters

Thermionic emission Field emission W<310> (Tungsten filament)

Source brightness 109 A/cm2 sr 5 105 A/cm2 sr Source size 5–10 nm 1–10 m Energy spread 0.3 (eV) 2 (eV) Temperature 300 K (R.T.) 2800 K Vacuum pressure <10–8 Pa <10–3 Pa Lifetime 1 year 50 hours

3. COLD FE-SEM STUDIES AND EXPANSION TO DIFFERENT FIELDS 3.1. CFE-STEM Development at HCRL In 1968, a group at HCRL organized by T. Komoda began developing a CFE-STEM (Komoda et al., 1972). The prototype CFE-STEM consisted of (i) a gun chamber evacuated to 1 10 8 Pa and separated from the microscope column by a differential aperture and (ii) a microscope column evacuated to 3 10 5 Pa. A Butler FE gun and two stages of electromagnetic lenses (Figure 2) produced a convergent beam on the Field emission gun Ion-pump (1) 2 ϫ 10−10 torr

Ion-pump (2) ϫ −8 Aperture 5 10 torr

Condenser lens (stigmator) Objective lens (deflector) Ion-pump (3) Specimen stage

Detector 1 ϫ 10−6 torr (P.M. tube)

FIGURE 2 (Left) Schematic diagram of FE-STEM developed at HCRL and (right) general view of Hitachi’s first FE-STEM. 130 Hiromi Inada et al. specimen when an acceleration voltage of 50 kV and an extraction voltage of 5 kV were applied. A tungsten <310>-oriented FE tip in NaOH electro- polished was used as an electron source. Komoda’s group developed a pulsed power supply to stabilize the polishing and thereby maintain the quality of the FE tip. They calculated the electron path and aberrations with a microcomputer to optimize the Butler lens to machine it more precisely and thus minimize spherical aberration (Tonomura, 1973). Ultimately, a brightness of 5 108 A/cm2 was obtained. A side-entry specimen stage holding the specimen, a movable aperture, and a cold finger were inserted into the 6.5-mm gap in the objective lens pole piece, which had a bore of 2 mm. A transmission electron detector consisting of a phosphor and a photomultiplier was placed at the bottom of the microscope column. A contrast aperture was inserted immediately above the detector to control the detection angle and to produce three kinds of contrast. Crewe and Wall (1970c) and Zeitler (Zeitler et al., 1970a,b) asserted that a bright-field (BF) STEM image is equivalent to a conventional TEM image by reciprocity. The use of a contrast aperture resulted in (i) a BF-STEM detection angle of less than 10 mrad without elastic scattered electrons, (ii) a phase contrast of less than 2 mrad, producing a small illumination angle TEM image, and (iii) a dark-field (DF) STEM detection angle of more than 10 mrad, enabling detection of elastic scattered electrons. Hitachi achieved a resolution of 1 nm with their instrument and observed BF and DF contrast reversal of evaporated Au particles, the equal-thickness fringe of MgO single crystals, and a microtome tissue section (Figure 3) that clearly indicates a double-cell membrane.

FIGURE 3 Tissue section of biological specimen showing double-layered structure of cell membrane. Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 131

3.2. CFE-SEM Development at Naka Works Another FE project had been started by the Naka Works. A team led by S. Saito had the goal of producing an FE-SEM capable of secondary electron (SE) detection. This goal was set because the market and target were clear and because no one had achieved very fine feature analysis (on the order of 3 nm) for biological studies. The resolution available with tungsten-filament SEMs (10 nm) was insufficient. However, most electron microscopes produced in the 1970s were for biology, and scientists were looking for impressively beautiful electron micrographs. Hence, these were obviously tough years for scanning microscopes, which produced rough pixel images that were unacceptable to most scientists. Saito was dispatched to Perkin-Elmer Ultek Inc. to learn about and investigate UHV technologies for practical use, and they introduced. In 1970, Hitachi Naka Works invited Crewe to serve as a consultant to Hitachi and assist with the development of a CFE-SEM for two years. The Naka engineers brought a drawing board to a conference room to help them concentrate on working closely with Crewe to design a new concept microscope (Figure 4). They eventually succeeded in developing the world’s first commercialized CFE-SEM, model HFS-2, in 1972 (Komoda and Saito, 1972b)(Figure 5). It had an aberration-optimized Butler lens, a variable acceleration voltage of 1–25 kV, and resolution of 3 nm. The microscope column was separated into three chambers by using differential pumping apertures, and the chambers were pumped with individual ion pumps. The CFEG maintained a vacuum pressure of 10 8 Pa even when the pressure in the specimen chamber was 10 3 Pa. For longer running time, two FE tips were inserted into the gun, which eliminated the need to vent the gun chamber. The column had a single stage condenser lens and an objective lens, enabling the illuminating electron beam

FIGURE 4 Photograph taken at meeting with Professor Crewe of Chicago University and Hitachi colleagues during his consultancy (1972). 132 Hiromi Inada et al.

FIGURE 5 Hitachi’s first commercial FE-SEM (model HFS-2). to be varied widely in size, resulting in a probe size of 2–20 nm and a probe current of 10 pA to 2 nA. This CFE-SEM has been installed in major biology research facilities. Collaborative work with K. Tanaka of Tottori University led to the development of instruments with much higher resolution. Since then the resolution and FE stability of CFE-SEMs have continued to be improved (Figure 6). Thanks to the development of an in-lens pole-piece design, CFE-SEMs now have much higher resolution (<1 nm), and a recent CFE-SEM (model S-5500), which has an environment-resistant design, has a resolution of 0.4 nm in SE image (Figure 7).

3.3. Studies of Field Emission Stability The biggest problem in applying CFE technology to microscopes is improving FE stability and operation. It is not an exaggeration to state that progress in CFE technology is equivalent to progress in UHV technology. The improvements made include automatic V0/V1 ratio control for the Butler lens, noise reduction for the electronics, and beam monitor incorpora- tion for user operation of the sophisticated FE technique. The FE current fluctuates, and instability of the emission current increases with the probe current. This instability is one of the problems that must be overcome for FE technology to be used practically. Figure 8 illustrates the general behavior of the FE current as a function of time after flashing. Flashing the FE tip causes gas molecules on the tip surface to desorb and smooths the surface. An extraction voltage (V1)of 2–5 kV is then applied to obtain a total emission current of 1–100 A. Immediately after flashing, the FE current decreases as a gas monolayer builds up on the emitter surface. Stable region, relatively flat emission Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 133

500 Å

T2-bacterio phage ; taken by ultra T2-bacterio phage ; taken by ultra high resolution SEM, model HFS-2 high resolution SEM, model UHS-t1 (Prof.A.V.Crewe, Chicago University, (Prof.K.Tanaka, Tottori University, U.S.A, 1972) Japan) (1988)

FIGURE 6 Secondary electron micrograph image comparison of T2 bacteriophage, recorded with HFS-2 (left, in 1972) and with prototype in-lens–type FE-SEM for Professor Tanaka at Tottori University (right, in 1988).

16 HSM-2 14 Thermionic electron SEMs 12

HSM-2A 10

6 HFS-2 (5 nm) SEM resolution (nm) 4 Field emission SEMs S-700 S-800 2 S-900 S-5000 S-5200 S-5500 0 1960 1970 1980 1990 2000 2010 Year

FIGURE 7 Progress in image resolution of Hitachi FE-SEMs. The resolution of an early thermionic gun is plotted for comparison. Spatial resolution improved to sub-nanometer order with introduction of model S-900 due to incorporating in-lens design optics. 134 Hiromi Inada et al.

Emitter destroyed E

I by vacuum arc

Current decreasing Current increasing due to adsorption due to ion bombardment Emission current

IMIN

Time Flashing Cyclical lifetime

FIGURE 8 General behavior of field emission current as a function of time after flashing (Shimoyama, Ichihashi, and Tamura, 1989). current flows; however, continuous absorbing, desorbing, and migration of gas molecules occurs on the emitter surface; this derives FE noise, which is very fast random current fluctuation. After the region is kept stable for several hours (<10 hours), the FE current rises with a large fluctuation, which possibly destroys the tip. The emission current decreases as a function of the work function of the absorbing gas in accordance with Eq. (4.1). H. Todokoro et al.(Todokoro, Saito, and Yamamoto, 1982) of HCRL investigated the fluctuations in the FE current with the vacuum pressure of the FE gun and experimentally found that current stability is affected by ion bombardment of the FE source. The experiments were done using a vacuum chamber equipped with two FE guns (A and B). Gun A had a single FE tip and was used to record the total emission current under various vacuum pressures. The relative fluctuation, DI/I,as a function of the product of the pressure and emission current (P I) is shown in Figure 9. It is proportional to the logarithm of P I above 7 10 12 PaA. This indicates that the fluctuation increase is caused by ion bombardment of the residual gas molecules. This fluctuation represents a function of P I, not of I. Below 7 10 12 PaA, the relative fluctuation is less than 1% and constant, probably due to migration of the absorbed gas molecules on the FE tip surface. The results of this experiment show that FE current fluctuation caused by ion bombardment increases when the product of pressure and emission current exceeds a threshold. Gun B had two emitters 5 mm apart that were supplied with individual high voltages simultaneously to verify the origin of the ions (Figure 10). Emitter I emitted a current ranging from 5 to 250 A, and emitter II emitted a constant current of 5 A. As Figure 11 shows, the emission fluctuation of emitter II was maintained despite the very close placement of the emitters Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 135

− 4 ϫ 10 8 Pa − 4.5 ϫ 10 7 Pa − 1 ϫ 10 6 Pa − 4.5 ϫ 10 6 Pa 10 (%) ∞ /I ∞ D I 5

0 − − − − − 10 13 10 12 10 11 10 10 10 9 P ϫ I∞(Pa.A)

FIGURE 9 Relative emission fluctuation as a function of product of gun pressure and total emission current. Reproduced with permission of Todokoro, Saito, and Yamamoto, 1982. and their sharing of the same ambient gas pressure. This shows that the bombarding ions are generated close to the emitters. H. Shimoyama described the history of improvements in emission current (Shimovama, Ichihashi, and Tamura, 1989). For example, the total emission current steadily increased year by year due to improvements in vacuum technology, surface treatment, and reproductive FE tips. For instance, the emission current obtained was only a few microamperes in the 1970s compared with 100 A in the 1980s. A small desktop FE-SEM (model S-310) (Figure 12, W400 D500 H500 mm, released in 1976) sparked the idea for a critical dimension (CD)-SEM. The CD-SEM used a simple optics CFEG and a single objective lens, and it had a resolution of 10 nm in TV rate scanning. The gun had a single-electrode design, and the acceleration voltage was the same as the extraction voltage (4–6 kV). This microscope had an emission current (20 A) 10 times higher than previous microscopes, resulting in improved spatial resolution of quickly scanned images. Accordingly, it was an important breakthrough for improving FE stability and emitting a higher FE current. The model S-310 microscope was suitable for nonconductive specimens, such as cloth and semiconductor devices without charge-up, because of its low acceleration voltage. It nevertheless did not meet customer needs at that time (the late 1970s) because most users apparently expected beautiful art-like electron microscope images. It was better 136 Hiromi Inada et al.

V Ј 1 V1

REC. REC.

Emitter Emitter I II

Anode

1cm

FIGURE 10 Experimental apparatus of Gun B. Two FE tips were placed face to face and had the same anode distance of 10 mm. Reproduced with permission of Todokoro, Saito, and Yamamoto, 1982.

Emitter I 1.1

1.0 5 mA m 10 mA 50 mA 100 mA 250 A 0.9 1 min

3 ϫ 10−7 Pa 1.1 Emitter II 1.0

5 mA5mA 5 mA5mA5mA 0.9

FIGURE 11 Emission current fluctuations for two simultaneously operating FE tips obtained using different emission currents. Emitter I had emission current of 5 A to 250 A, whereas emitter II was controlled to emit a constant 5 A. Reproduced with permission of Todokoro, Saito, and Yamamoto, 1982. Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 137

FIGURE 12 Overview of desktop S-310 FE-SEM, which is now displayed in the reception area of Hitachi High Technologies America. suited for the early 21st century when there was a boom in interest for small SEMs. The S-310 low-kilovolt microscope, on the other hand, gave an essential for semiconductor device in line dedicated CD-SEM.

4. EXPANSION OF HIGH-VOLTAGE TEMs AND STEMs 4.1. Holography Studies by Tonomura with FE-TEMs Researchers at HCRL had an idea for using FE guns on TEMs at the very beginning of CFE-STEM development. TEM resolution had improved drastically through the 1970s under the heat of competition, and the ultimate resolution had been reached with conventional TEM. Gabor invented holography in 1948 (Gabor, 1949) to compensate for spherical aberration. Although A. Tonomura et al. (1968) proved the practicability of electron holography in their pioneering work using Hibi’s pointed filament (Hibi, 1956) in 1968, the resolution of the reconstructed image was not satisfactory due to poor electron beam interference. Tonomura et al. developed, in 1972,withButlerlensFEsourceasagun applicable of acceleration voltage of 50 kV, for electron holography (Figure 13)(Tonomura and Komoda, 1973). They developed an 80-kV CFE-TEM with higher coherency in 1978 and obtained as many as 3000 lines of Fresnel fringes (Tonomura et al., 1979b)comparedwiththe 300 fringes obtained up until then, and thereby ensured the realization of practical electron holography. The lattice resolution was improved to 0.062 nm due to a chromatic aberration effect. The Aharonov–Bohm effect was proven experimentally in 1982 (Tonomura et al., 1979b, 1982, 1998)with 138 Hiromi Inada et al.

FIGURE 13 Overview of 50-kV FE-TEM (Tonomura, 1972).

the 80-kV CFE-TEM. They also studied the use of holography using a 1-MeV CFE-TEM (Kawasaki et al., 2000) atHitachi’s Advanced Research Laboratory and investigated the dynamics of vortices (magnetic-flux quanta) in high– transition temperature (Tc) superconductors. Using this 1-MeV CFE-TEM, they obtained 10,000 Fresnel fringes and a lattice resolution of 0.049 nm.

4.2. Multistage Acceleration CFEG for TEM Applications Many remarkable CFE-TEM experiments have been done with the 80-kV CFE-TEM at HCRL; however, it was difficult to exceed an acceleration voltage of 80 kV with the Butler lens electron gun because of discharge between the emitter and electrodes. Endo of HCRL and his group developed a multistage electrode gun that produced an acceleration voltage of 125 kV to improve coherency and resolution. The distance between the emitter and ground electrode in the multistage electrode gun was Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 139

Insulator housing

Field emission tip

Electrodes

Insulator

FIGURE 14 Cross-sectional image of FE gun with multistage acceleration tube (Tonomura, 1984).

approximately 10 times that in the Butler lens gun (Tonomura, 1984) (Figure 14). This new CFEG produced a total emission current of 100 A and a consequent probe current of 8 nA. This gun incorporated countermeasures against outside magnetic fields. Extra care was needed when mounting the FE tip because of its tall design. This multistage electron gun was used not only for electron holography studies (Tonomura et al., 1982) but also for 100-kV analytical CFE-TEM (Koreeda et al., 1990).

4.3. Commercialized Analytical CFE-TEM/STEM Tomita and Naka Works designers developed the first analytical CFE- TEM (model H-600FE) equipped with an energy-dispersive X-ray (EDX) spectrometer and an electron energy-loss spectrometer (EELS) analyzer. It was installed at Osaka University in 1986. This TEM used a multistage electrode gun to produce 100 kV. It had SE and two STEM detectors and a maximum specimen tilt angle of 60, and it had a lattice resolution of 0.14 nm and an STEM resolution of 1 nm (Koreeda et al., 1990)(Figure 15). An energy spread of 1 eV was obtained with a serial EELS designed by S. Isakozawa of the Naka Works. Electron microscope column Energy dispersive (FE-Gun) X-ray micro analysis

C-F discri

SEM. image SEM. det.

0 EDX. det. I P1 Def. coil P2

EELS. control EELS. det

STEM. det Slit Ratemeter

FIGURE 15 (Left) Side view 100-kV field emission analytical TEM/STEM (H-600FE) installed at Osaka University. (Right) Schematic diagram of H- 600FE with analytical tools (SEM, EDS, and EELS detectors) (Koreeda et al., 1990). Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 141

5. DEVELOPMENT OF 50-KV STEM IN 1970S (BY SHIGETO ISAKOZAWA) 5.1. Development of Hitachi’s 50kV Prototype CFE-STEM The basic design for Hitachi’s 50-kV CFE-STEM was developed by S. Nomura and H. Todokoro at HCRL. S. Saito, Y. Minamikawa, and Kakiuchi at Naka Works worked on the detailed designs and the production of a prototype. Table 2 shows the specifications of the FE-STEM prototype. Figure 16 shows a schematic diagram of the basic design. A CFE cathode is used for the electron source. Condenser and objective lenses demagnify the electron beam, which is accelerated to 50 kV to form a spot with a diameter of 0.3 nm on the specimen. To meet the requirements for high-resolution observation, the specimen stage was designed on the basis of a TEM specimen stage. Specimens have to be thin sections and are placed on a 3-mm diameter grid. Forward-scattering electrons are detected with an annular dark-field (ADF) detector. The electron intensity detected by the detector is displayed on a cathode ray tube (CRT). As the driving signals of the CRT are synchronized with the raster scan signals, the arrangement of the electron intensity signals forms a DF-STEM image. UHV technologies were not well established in the 1970s, so the reliability of CFE sources was sufficient only for obtaining several microamperes of emission current for only a few hours with a 50-kV acceleration voltage. Nevertheless, this was good enough to observe a single atom. Much work was undertaken by Nomura and Todokoro to improve

TABLE 2 Speciﬁcations of the prototype FE-STEM

Item Specifications

Resolution 0.3 nm Maximum acceleration 50 kV voltage Maximum magniﬁcation 10,000,000 Gun type Cold-ﬁeld emission Time for exchanging 10 minutes specimen Vacuum evacuation system Three-stage differential pumping system Ion pump (60 L/sec, 20 L/sec, 150 L/sec) Column structure Components connected with metal gaskets Electron optics Illuminating, objective, projection lenses 142 Hiromi Inada et al.

Field emission gun CRT for display

Probe forming lens

X ray detector Specimen

Electron detector(A)

Electron energy Slit loss spectrometer

Electron detector(B) (B)

FIGURE 16 Concept of FE-STEM by Nomura. stage stability, reduce noise from the scan coil current, reduce specimen contamination, and so on. They were then able to obtain high-resolution STEM images. They also invented new techniques—for example, a specimen exchange method to evacuate air with a sorption pump and then insert it into the UHV chamber. Development of the 50-kV CFE-STEM was completed in 1975. Figure 17 shows an overall view of the CFE-STEM developed by Hitachi. Note that an EELS was mounted on the bottom of the microscope column. The basic design concept enabled not only single-atom observation, but also element analysis with atomic spatial resolution.

5.2. Bright-Field Images Obtained with 50-kV CFE-STEM Figures 18 and 19 show lattice fringes of carbon graphite and single-crystal Au film acquired to confirm the mechanical and electrical stabilities and probe size of the FE-STEM prototype. As the reciprocity relationship between conventional TEM (CTEM) and STEM had been studied at HCRL, it was well known that crystal lattice fringes could be obtained with BF-STEM exactly like those obtained with CTEM. The BF-STEM images in Figures 18 and 19 clearly show that crystal lattices with spacings of 0.34 and 0.20 nm can be observed. This means that the electron beam spot size was 0.3 nm or less and that the instrument was stable enough to obtain atomic resolution. The image in Figure 19 is certainly the first example of observing lattice fringes with spacing of 0.2 nm using BF-STEM. Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 143

FIGURE 17 General view of FE-STEM.

3.4 Å

FIGURE 18 Lattice fringe of carbon graphite (bright-field image).

3nm

FIGURE 19 A 0.2-nm lattice fringe of single-crystal Au (bright-field image). 144 Hiromi Inada et al.

5.3. Single-Atom Observation The intensities of electrons elastically scattered from 2-nm-thick carbon and from thorium single atoms were calculated by Nomura. The acceleration voltage was assumed to be 100 kV, and the electron beam spot size was assumed to be 0.3 nm. Figure 20 shows the relationship between the intensities of the scattered electrons and the scattering angle. It shows that electrons scattered from a thorium atom are distributed at a higher angle than carbon atoms and that their intensity is higher even at a scattering angle of 150 mrad or higher. DF imaging using CTEM, which uses a narrower range of scattering angles (less than 10 or at most 30 mrad), cannot achieve efficient detection. DF-STEM imaging with an ADF detector (which covers from 20–200 mrad) can drastically improve detection efficiency. The calculated relationship between atom image contrast and electron probe diameter is plotted for three probe sizes in Figure 21. Image contrast is defined as Iz/Ic, where Ic is the signal intensity from the carbon atoms supporting the thin film, and Iz is the signal intensity from the summation of a single atom and its supporting carbon. Taking the statistical variation in the number of carbon atoms in the supporting film into consideration, we can observe a single atom if the image contrast is higher than 1.5 (broken line). The smaller the probe diameter, the higher the image contrast. In reality, a signal intensity of 10–12 A or more is needed to obtain images with a better signal to noise ratio (SNR). If the ratio of detected

0.20 )

0 0.15 (I/I

A thorium atom 0.10

0.05 Carbon film (15 C atoms) Scattered electrons intensities

0 0 50 100 150 (mrad.) Scattered angle

FIGURE 20 Intensity angular distribution of scattered electrons (calculated by Nomura). Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 145

4.0

3.0 Probe size

2.0 5Å Contrast 10Å 1.0

0 02040 60 80 100

Atomic number

FIGURE 21 Atom observation contrast dependence on probe size (calculation).

100 − I0=1 ϫ 10 10A 50 100 kV Wgun ( Å )

10 F W LaB6 LaB 5 E FE gun 6 gun Electorn probe diameter 1 106 107 108 109 (A/cm2.sr.) Brightness

10 FIGURE 22 Relationship between probe size and brightness of electron gun for 1 10 A probe current. electrons scattered by a single atom to the incident electrons is 1%, the probe current must be 10 10 A or larger. For a probe current of 10 10 A, the relationship between probe size and electron gun brightness is as plotted in Figure 22. It shows that an FE gun must have a probe diameter of 0.5 nm or less. Specimen contamination was a serious problem. Magnification must be set to several million times to obtain images of single atoms. The STEM images became increasingly brighter trial by trial and eventually became completely white due to specimen contamination. 146 Hiromi Inada et al.

An anti-contaminator for the TEM was introduced to reduce the residual gas pressure surrounding the specimen, but no significant improvements were obtained. When a crystal lattice image is taken with a TEM, the area of interest is illuminated with an electron beam several micrometers in diameter. The image is recorded on a sheet of film at several hundred thousand times magnification and then magnified even more with an optical enlarger when the image is printed on photographic paper. The final image must be displayed on a CRT in the case of STEM. At several million times magnification, which is necessary for atomic resolution observations, a very small area of interest (several tens of nanometers) is scanned with an extremely fine electron probe. Therefore, specimen contamination in STEM is much worse than in TEM as the size of the target area is smaller by a magnitude of four. The ultimate answer to this problem was to illuminate an electron beam on the surrounding area of interest (i.e., on a wider raster scan area) at a lower magnification and a higher density for a longer time (20–30 minutes) before observation. This preliminary irradiation thins the contaminants in an area wider than the area of interest, which reduces their effect on the imaging. The effects of this preliminary irradiation last for 10 to 20 minutes. Schematic diagrams of the STEM detecting conditions are shown in Figure 23. Condition A is equivalent to the usual conditions for DF image observation with CTEM using the tilted-beam illumination technique. Condition B1 corresponds to the maximum illumination solid angle with CTEM. Condition B2 corresponds to the large angle (up to 60 mrad) obtainable only with STEM. Photographs of a thin carbon film (2-nm thick) taken under each of these detection conditions are shown in Figure 24. High-contrast spots are almost negligible under condition B2 but they still remain under condition B1. These results suggest that high-contrast spots in carbon film images are due to the interference of scattered electron waves and that they can be removed only by increasing the detector solid angle to 60 mrad.

(A) (B1) (B2)

9mrad 9mrad 9mrad

18mrad 60 9mrad mrad 9mrad 9 mrad

18mrad

FIGURE 23 Schematic diagrams of STEM detecting conditions. Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 147

(A) (B1) (B2)

10 nm

FIGURE 24 Dark-field images of carbon film (2-nm thick) captured under respective detection conditions of Figure 23.

5nm

FIGURE 25 Dark-field image of carbon film approximately 2-nm thick.

The DF image of carbon obtained under condition B2, in which only the thickness contrast is observed, is shown in Figure 25. A droplet of 5 thorium nitrate in 10 molar CH3OH solution was placed on this film. The image was observed using STEM. As shown in Figure 26, bright spots and spot clusters were observed. The individual bright spots were nearly the same size as the spots making up the spot clusters. Single atoms of thorium made those spots (Todokoro, Nomura, and Komoda, 1977).

5.4. Development of the Electron Energy-Loss Spectrometer In the 1970s, electron probe microanalyzers were used to analyze the elements in small targets. However, they were unable to detect Na or lighter elements (C, O, N, etc.), and characteristic X-ray signals from light elements could not be detected. The efficiency of signal acquisition is defined as detected signal intensity divided by illuminating electron 148 Hiromi Inada et al.

5nm

FIGURE 26 Dark-field image of thorium atoms on carbon film. intensity. The amount of detected characteristic X-ray signals is limited by the fluorescence yield and the detector solid angle. With EELS, the inelastic scattering electrons that cause inner shell excitations are distributed over a smaller scattering angle and are directly detected by the spectrometer. EELS was thus expected to be suitable for atomic resolution analysis because of its highly efficient signal acquisition and unlimited ability to detect lighter elements. In 1976 Nomura and Todokoro developed an EELS that could be attached to an FE-STEM. It was a magnetic sector type with a trajectory radius of 11 cm. The target energy resolution was 0.5 eV at 50 kV. The detection system was the serial type, composed of a slit, a scintillator, and a photo-multiplying tube. The energy-loss spectra were recorded with a chart recorder. A photograph of the prototype EELS mounted under the FE-STEM column is shown in Figure 27. Although EELS research was well under way, this attachment of an EELS to an FE-STEM was one of the earliest, and the fundamentals of the analytical STEM concept were established by this work. Figure 28 shows the analytical energy-loss spectrum results for boron nitride particles with a diameter of 20 nm supported on thin carbon film. Figure 28a shows an STEM image taken when electrons without energy loss were used; it is a ‘‘no-loss image.’’ Figure 28b shows an energy-filtered STEM image taken when electrons with an energy loss of 190–210 eV were used. This loss corresponds to the energy loss of boron K-shell excitation. Figure 28c shows line analysis of 190–210 eV. Figure 28d shows the electron energy-loss spectrum taken when the electron probe was focused on a boron nitride particle. The K-shell excitation edges of boron (B), nitrogen (N), and carbon (C) from the supporting film are evident in the spectrum. Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 149

FIGURE 27 Developed EELS equipped with STEM.

Estimating that the probe diameter was 1 nm and that the diameter of the boron nitride was 20 nm, Nomura concluded that 7 10 21 g of boron and 9 10 21 gofnitrogenweredetected.

5.5. Further Development of 50-kV CFE-STEM Many research institutes and laboratories with electron microscopes attempted to duplicate Crewe’s observation of single atoms, but only HCRL was able to do so, using the FE-STEM developed by Komoda, Nomura, and Todokoro. This prototype STEM was equipped with the most advanced technologies at that time, such as a CFEG, producing a UHV, a highly stable electronic power supply, and electron optics with atomic resolution performance. Moreover, this FE-STEM had the Hitachi-made EELS and a nondispersive X-ray detector and thus was the origin of the present analytical STEM concept. HCRL contributed to the development of the most advanced technologies for electron microscopes, and Hitachi’s Naka Works was 150 Hiromi Inada et al.

(d) (a)

ϫ1000 B,K

(b) ϫ100

C,K Relative intensity

N,K ϫ10 200Å

(c)

0 300 600 Energy loss (eV)

FIGURE 28 Analytical results of boron nitride using EELS. (a) d ¼ 0 eV, no-loss image. (b) d ¼ 190–210 eV, energy-filtered image. (c) Line analysis of d ¼ 190–210 eV. (d) Energy loss spectrum. responsible for commercializing and producing them. Commercialization of a dedicated FE-STEM based on this concept was suspended after many discussions and meetings because the market was mostly demanding TEMs at that time.

6. HITACHI’S FIRST COMMERCIALIZED DEDICATED STEM (BY TAKAHITO HASHIMOTO) 6.1. 200-kV Analytical CFE-TEM, HF-2000 During the 1970s, Tonomura and his group at HCRL were competing to achieve the best TEM image resolution. They expected a breakthrough when they applied electron holography to a TEM. The spherical aberration of the TEM objective lens determines the image resolution, and electron holography is an imaging method that does not suffer the effects Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 151 of spherical aberration because it does not use a lens. They also developed an electron gun with high brightness, which is essential for obtaining high-quality holograms. The FE-TEM they developed in 1978 is shown in Figure 29. The result was improved TEM image resolution. They obtained the highest image resolution ever obtained until then: lattice fringes of Ni (220) with half spacing (0.062 nm). Using electron holography, Tonomura’s group investigated spherical aberration correction for high-resolution imaging (Tonomura et al., 1979a), observed magnetic domains (Tonomura, et al., 1980), observed magnetic fields (Matsuda et al., 1982), and investigated magnetization of recording media (Osakabe et al., 1983). Using phase-shift amplified-interference electron microscopy, they measured the thickness of the recording media (Endo et al., 1984). In 1986, they used electron holography to confirm the existence of the Aharonov–Bohm effect (Osakabe et al., 1986; Tonomura et al., 1986).

FIGURE 29 FE-TEM developed by Hitachi CRL. 152 Hiromi Inada et al.

Work on high-voltage FEG technologies was transferred to Hitachi’s Naka Works. In 1983 Hitachi delivered a custom-made 100-kV analytical FE-TEM (model H-600FE) (Kamino and Tomita, 1983). It had outstanding analytical performance, especially in terms of spatial resolution. Electron probes with a diameter of 5 nm or less were available. Their probe current for EDX or EELS analysis was 10 times larger than the LaB6 gun model, and the illumination angle for nano-probe electron diffraction was one-tenth that of the LaB6 gun model. Advancement in the performance of commercial 200-kV TEMs began to slow in the early 1980s because their resolution had almost reached the theoretical limit. The makers of 200-kV TEMs were in a dead heat. One approach to further improvement was to increase the acceleration voltage. From 1984 to 1986, JEOL Ltd. released a series of 400-kV TEMs (the JEM-4000 series). In 1986 Hitachi released a 300-kV TEM (model H-9000). The TEM competitors were still in a dead heat. Hitachi’s engineers decided to develop a new model with something so special that competitors would not be able to catch up. The key technology for the hoped-for breakthrough was the field emission gun (FEG). With the aim of enabling new applications of electron holography and practical nano- analysis, Hitachi launched a project to develop a new model. Two researchers, M. Ichihashi and H. Murakoshi, moved from HCRL to Naka Works to work on developing a mass-production 200-kV FEG. The result was the development of a 200-kV FE-TEM (model HF-2000) (Isakozawa et al., 1989). This model, shown in Figure 30, was an epoch- making instrument in electron microscopy. Its nano-probe illumination performance with enough (several hundred nanoamperes) beam current, smaller (<0.4 eV) energy spread, and better beam coherency enabled many applications that were previously impossible. Many universities, research institutes, and laboratories used this model to analyze precipi- tates in steel (Hashimoto et al., 1991), the concentration of elements on stacking faults (Kamino et al., 1992), the metal particles on catalysts, the electronic states of semiconductors, and the stoichiometry of insulator oxides. In addition to Tonomura’s group, D. Joy, L. Allard and E. Vo¨lkl made extensive holographic studies of ferroelectric domain walls (Zhang et al., 1993; Vo¨lkl et al., 1999), p-n junctions, and semiconductor device dopant profiles. Steeds at Bristol University obtained the first convergent- beam–coherent electron diffraction pattern (Vine et al., 1992). In 1992 the first 300-kV FE-TEM (Murakoshi, Ichihashi, and Kakibayashi, 1992) was developed by researchers at HCRL. Technologies developed for the prototype were transferred to the production model (HF-3000) (Figure 31). Philips introduced an FE-TEM in 1990 (the CM-20 FEG), and JEOL released a new FE-TEM in 1992 (the JEM-2010 F). Both models were equipped with a Schottky emission gun (SEG). Most of Hitachi’s TEMs Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 153

FIGURE 30 200-kV FE-TEM (HF-2000).

FIGURE 31 300-kV FE-TEM (HF-3000). 154 Hiromi Inada et al. used a high-angle takeoff EDX geometry with a takeoff angle of 68 and a detection solid angle of 0.033 sr. Competitors used a lower-angle takeoff geometry (20–25) and a solid angle of 0.13 sr. The higher the takeoff angle, the better the reproducibility of the EDX analysis results. The higher the takeoff angle, the shorter the X-ray path length in the specimen, resulting in reduced absorption in the specimen, especially for characteristic X-rays from light elements such as oxygen. This characteristic enables flexible positioning and orientation of the specimen, which ensures highly reproducible analysis. Although the probe current of the FEG is several times higher than that of the SEG, the analytical sensitivity of the total system was only half that of competitive models because the detection solid angle was one-quarter that of theirs. The HF-2000 thus started losing competitiveness. STEM performance was another weak point. An objective lens using a high-angle takeoff geometry must have a larger upper polepiece bore to enable the X-ray signal to pass through. Therefore, its prefield lens is relatively weaker. The guaranteed smallest probe size of the HF-2000 was a diameter of 1 nm. This was small enough for EDX analysis and STEM observation with a maximum magnification of several hundred thousand times but was too large to obtain atomic-resolution STEM images.

6.2. ‘‘Gate Viewer,’’ Trigger for Dedicated STEM The weakness of the HF-2000 led Hitachi to start developing a new model. Initially, the project leader, Isakozawa, planned to create a ‘‘super FE-TEM.’’ It would have the largest detection solid angle among all commercial TEM models and atomic-resolution STEM capability. In the mid-1990s, the design rule for semiconductor research and development was 250 to 180 nm. FE-SEMs were widely used for confirming process validity and identifying the causes of failures and defects. SEMs were used by individuals such as device researchers, materials scientists, and electronics engineers. Most FE-SEMs in those days already had sufficient resolution (less than several nanometers) even at an acceleration voltage of 1 kV or lower and had an autofocusing function. Specimens were prepared relatively easily with cleaving silicon tips. Semiconductor companies, of course, had TEMs. TEM image resolution had already reached the sub-nanometer range, but specimen preparation, instrument operation, and image interpretation were very difficult compared with those of SEMs. In many cases, only handfuls of ‘‘expert operators’’ were allowed to use TEMs, and the time required to obtain investigation results could range from several days to a full week. This was too slow for the decision- making stage for establishing the process for next-generation devices. The manager of Hitachi’s Marketing Division, M. Kato, suggested to the design team that the semiconductor industry wanted electron microscopes with TEM-like image resolution and SEM-like ease of use. He knew that Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 155 such an instrument would have to be newly developed and he called it ‘‘Gate Viewer.’’ The thickness of the gate oxide layer in large-scale integrated circuits was already only several tens of nanometers and would reach 2 nm by 2000 (Committee of Semiconductor Technology, 1999). To measure a scale of 2 nm, the image resolution must be better than one-tenth the scale: 0.2 nm. Due to the strong edge effect, SEs from the substrate and gate electrode make it impossible to observe the gate oxide layer, even if a small probe were achieved. Only a TEM or an FE-STEM could achieve such a small resolution. Compared with TEMs, STEMs are advantageous in terms of the precision of the magnification setting because the number of components that need tuning is smaller. Precise magnification enables accurate measurement of the dimensions in images. Hitachi had accumulated experience in critical dimension (CD)-measurement SEMs, and even general-purpose FE-SEMs had a CD-measurement function in those days. For STEMs, the preparation of a thin specimen seemed to be the bottleneck, as it was for TEMs, but the focused ion beam (FIB) instrument (model FB-2000) that had become prevalent enabled fabrication of thin specimens with a specific area and a specific thickness without exceptional skill (Ishitani et al., 1994). A dedicated FE-STEM has the potential to be such an instrument, fulfilling the concept of the Gate Viewer, and so it was chosen as the basis of the new model. Development of the Gate Viewer FE-STEM (model HD-2000) began in the fall of 1995. It featured several advantages: (i) atomic-resolution imaging, (ii) the highest EDX sensitivity, (iii) multisignal reception from specimen, (iv) an FIB-compatible specimen holder, and (v) easy operation. Figure 32 shows the configuration of the HD-2000. The FEG was basically the same as that of the HF-2000, and several improvements and modifications to the microscope had increased reliability and productivity. The illumination lens system, composed of double-stage condenser lenses and an objective lens, was newly designed. A condenser-objective objective lens with a small spherical aberration coefficient was used so that a 0.2-nm or smaller probe could be used and that an EDX detector with the largest detection solid angle among all TEM models could be accommodated. For seamless operation with lower magnification, a scan coil with a larger inner diameter was used. A projector lens was used to select the detection angle for BF-STEM and DF-STEM. An Everhart– Thornley SE detector, a BF-STEM detector, and a DF-STEM detector were included in the standard configuration. There were several auxiliary ports for mounting a charge-coupled device (CCD) camera to be used for observing electron diffraction patterns and ronchigrams and for mounting an EELS on the lower column. The high-resolution FE-SEM in late ’90s was controlled with a scan generator, an image processor, and a personal computer. A scan generator with a low electrical noise level was initially used. As with an SEM, 156 Hiromi Inada et al.

FE-gun

Condenser lens SE detector

Scan coil EDX detector*

Objective lens Specimen Scattered electron Display Projector lens

Retractabl SEM DF-STEM DF-STEM detector Retractabl Scan generator

Image sensor* Signal processor (CCD camera) Transmission BF-STEM electron Control unit detector EDX analyzer

FIGURE 32 Configuration of Gate Viewer FE-STEM (HD-2000) (with optional EDX and charge-coupled device (CCD) camera).

higher magnification was achieved by reducing the amplitude of the scan coil current. However, this resulted in electrical noise. Pinstripe patterns, which the engineers thought were the very first BF-STEM lattice fringes, were caused by the electrical noise. The fringe spacing remained constant when the magnification was changed from 2 to 5 million times. A steady step-by-step improvement process was necessary to obtain actual lattice fringes. At the earliest stage of development, the prototype had a TEM lower column and a CCD camera for observing the spot size and directly setting the illumination angle. A ‘‘diffraction-limited’’ probe (Spence and Zuo, 1992) can be formed by demagnifying the electron source image. The a optimum convergence angle, Opt, for obtaining the smallest probe, dmin, is calculated from only the spherical aberration coefficient and electron wavelength (l), as shown in Eqs. (4.2) and (4.3) (Crewe, 1985). Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 157

= 4l 1 4 a ¼ (4.2) Opt Cs

¼ : 1=4l3=4 dmin 0 43Cs (4.3) Figure 33 shows a TEM image of the smallest probe taken with an experimental TEM column in which the HD-2000 objective lens pole piece had been installed. The line profile of Figure 33 is shown in Figure 34.

Measured probe

0.42 nm

FIGURE 33 Smallest measured probe.

8 0.17 nm

FWHM

Intensity (a.u.) 4

0 −0.6 −0.4 −0.2 0.0 0.2 0.4 0.6 Distance (nm)

FIGURE 34 Line profile of Figure 33. 158 Hiromi Inada et al.

HD-2000 200 kV x 5000 k TE 6.00 nm

FIGURE 35 A BF-STEM image of thin single-crystal Au film taken with HD-2000.

¼ The simulated full-width at half maximum (FWHM) with Cs 1.3 mm and the l at 200 kV ¼ 0.00251 nm was 0.17 nm, which is in good agreement with the theoretical value of 0.16 nm. Figure 35 shows an example of a BF-STEM image of thin single-crystal Au film. The smaller the probe, the smaller the probe current. A probe current of several tens of picoamperes (pA) is sufficient for STEM and SEM imaging. The smallest probe was achieved with this amount of probe current. For practical EDX analysis, a probe current of several hundreds of pA is necessary. Several illumination modes had to be fixed. The illum- a ination modes were designed to have a constant illumination angle, Opt, and a variable spot size and probe current. When the illumination angle is kept constant, probe size d can be approximately calculated using Eq. (4), 1 where ds is the electron source size and M is the magnification of the electron source image on the specimen. The contribution of chromatic aberration is negligible. sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 2 d ffi d2 þ d (4.4) min M s

A virtual source image is strongly demagnified in high-resolution mode, but the number of emitted electrons passing through the objective aperture is limited. In emissive mode, the virtual source image is moder- ately demagnified, and a larger number of electrons pass through the Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 159

Virtual source

C1 lens C1 cross over C1 cross over

C2 lens

OBJ.AP. C2 cross over

OBJ lens

Specimen Emissive mode High resolution mode

FIGURE 36 Illumination modes. (Left) Emissive mode. (Right) High-resolution mode. objective aperture (OBJ. A.P.). Figure 36 illustrates the differences in the ray diagrams for illumination modes. If the illumination angle at the specimen is kept constant, the typical probe size and probe current are f0.2 nm and 20 pA for high-resolution mode and f0.5 nm and 200 pA for emissive mode. Figure 37 shows a comparison of images formed with various detector signals. The specimen was a silicon device (a hemispherical grained capacitor test element group [TEG]) with a layer structure: SiO2 base layer, thick poly-Si layer with 100-nmf hemispherical bumps on the top side surface, thin Si3N4 layer, amorphous Si layer, and SiO2 top layer. The SEM image in Figure 37a shows the surface of a sectioned specimen. The BF-STEM image (37b) and DF-STEM image (37c) show cross-sectional views. The BF-STEM image is equivalent to a TEM image because of the reciprocal relationship between BF-STEM and TEM. A strong diffraction contrast is evident in the poly-Si layer. This characteristic can be used to distinguish crystalline areas or even crystal grains, but it can also make it difficult to interpret what is seen in the image. The DF-STEM uses scattered electrons, a larger atomic number (Z), a heavier density (r), or greater thickness (t). The diffraction contrast is averaged and moderated because a DF-STEM detector acquires all related diffracted waves. Espe- cially with a larger scattering angle, the atomic number is the dominant factor affecting the contrast, so a DF-STEM image is often called a ‘‘Z-contrast image.’’ In image 37c, the substance with the largest density is 160 Hiromi Inada et al.

(a) (b) SiN a-Si

Poly-Si

100 nm SiO2

(semi-spherical grained capacitor TEG) Poly-Si

SiO2

FIGURE 37 Images of Si device. (a) SEM, (b) BF-STEM, and (c) DF-STEM.

Si3N4 and the smallest is SiO2. The Si3N4 layer covering the poly-Si bumps is clearly evident. The amorphous and poly-Si layers have a similar contrast. Therefore, we can obtain much information about a specimen from SEM and STEM images. A. Ninomiya, an industrial designer in the Hitachi Design Division, drew a conceptual image of the HD-2000. He took into account that higher-resolution electron microscopes are generally installed in a quiet room on a stable floor in a small stray magnetic field without acoustic noise. These conditions are completely different from those of a clean room used for semiconductor fabrication; these rooms have a continuous downflow, noise from air conditioners and suction pumps, floor vibration with a frequency close to the resonance frequency of the HD-2000 with antivibration mounts, and so on. He thus designed the electron gun and microscope column to be covered with a permalloy shield and the EDX detector, evacuation pipes, and cables to be contained in an enclosure. As shown in Figure 38, the HD-2000 had a clean and photogenic appearance. In fact, it won the Good Design Award from the Japan Industrial Design Promotion Organization in 1998. The HD-2000 was released in 1998 (Hashimoto et al., 1998). A production model was exhibited at the 50th annual meeting of the Japanese Society of Microscopy. Installation was done over a weekend, and the machine was in operation during the entire exhibition period, displaying BF-STEM images of evaporated Au particle lattice fringes. Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 161

FIGURE 38 Photograph of Gate Viewer FE-STEM (HD-2000).

6.3. Novel Functions for HD Series 6.3.1. Energy-Dispersive X-Ray An EDX detector with a solid angle of 0.3 sr was designed by S. Foote of Noran Instruments (now Thermo Fisher Scientific Inc.) soon after the objective lens design was completed. Achieving the highest EDX sensitivity is an extremely important development objective. This solid angle was 2.5 times larger than those of competitors and the largest among all commercial electron microscopes. The shielding parts were redesigned to eliminate iron and cobalt system peaks from the objective lens pole piece. The original shielding parts could stop only scattered electrons, not bremsstrahlung X-rays. Subsequently, several models of EDX systems (e.g., EDAX, Horiba, and Oxford systems) were released.

6.3.2. Diffraction The diffraction capabilities of FE-TEMs were emphasized as an advantage by the various competitors. Diffraction patterns can also be observed with a dedicated STEM. With a condenser-objective objective lens, a diffraction pattern is formed on the back focal plane and projected onto STEM detectors, as shown in Figure 39. In standard STEM observation or 162 Hiromi Inada et al.

(a) (b)

Incident beam

Specimen

Objective lens Transmission <= B.F.P. electron (diffraction pattern)

Simplified TEM Actual TEM

Objective lens Reduced Objective lens convergence Specimen post-field angle Transmission electron CBED pattern BF-STEM Simplified STEM Actual STEM CCD camera detector STEM nano diff. mode

FIGURE 39 Diffraction patterns formed in TEMs and STEMs. analytical mode, the convergence angle is set to achieve the smallest possible probe size for a given probe current. Figure 40 shows the calculated relationship between the probe size and convergence angle of an electron beam for a constant probe current l of 200 pA. The Cs of the objective lens was 1.3 mm, and at 200 kV was 0.00251 nm. The probe size was calculated using the root mean square method with quadrature addition of the image size of the electron source on the specimen, the size of the disk of confusion by diffraction, and the spherical aberration. Figure 40 shows that the optimum convergence angle for the smallest probe at a probe current of 200 pA was 8 mrad. The diffraction pattern under this setting was the convergent- beam electron-diffraction pattern, as shown in Figure 41a.Whenthe mode is ‘‘nano-diffraction’’ with a convergence angle of 1.5 mrad, the probe is larger than in the standard STEM mode. Nevertheless, the probe is still operational up to several hundred kX of magnification, and a diffraction pattern with mostly parallel illumination can be observed. Figure 41b shows the diffraction pattern for Au thin film with the nano- diffraction mode. This mode is set by changing the lens current while Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 163

1.0E + 01

1.0E + 00

Diffraction Spheerical aberr. Probe size (nm) - 1.0E 01 e- source image RMS

1.0E - 02 0.1 1 10 100 Convergence angle (mrad)

FIGURE 40 Convergence angle and probe size.

(a) (b)

Standard STEM (emissive) Nano diffraction mode

FIGURE 41 Comparison of diffraction patterns from Au film [001]. (a) standard STEM (emissive) mode with a 7 mrad; (b) nano-diffraction mode with a 2 mrad. keeping the specimen height and objective aperture fixed. First, the probe is raster-scanned to determine the field of view. Then, the probe is placed at the point of interest on the image, and an electron diffraction pattern is obtained with the CCD camera mounted at the bottom of the column. Figure 42 shows an example DF-STEM image of a dynamic random access memory (DRAM) and diffraction patterns from several points of the image with nano-diffraction mode. 164 Hiromi Inada et al.

Si crystal grain ONO layer

Insulator

Capacitor

Substrate

HD 2000 200 kv ϫ 60.Dk ZC 500 nm DF-STEM image

Insulator Substrate (amorphous SiO2) (Si single crystal)

FIGURE 42 DF-STEM image of Dynamic Random Access Memory (DRAM) and diffraction patterns for nano-diffraction mode.

6.3.3. HD-2300 SEG and Auto Functions The HD-2300 was released in 2003. It was equipped with a 200-kV SEG developed by S. Watanabe. If the brightness of electron guns is compared with the speed of a runner, the FEG is a sprinter and the SEG is a marathoner. The SEG is stable 24 hours a day, 7 days a week, 52 weeks a year during the lifetime of its zirconium-oxide–coated tungsten cathode. The FEG model (HD-2300C) was produced for performance-oriented customers. Automated focus adjustment and astigmatism-correction functions (Inada et al., 2004) were also developed for this model. These functions were generally very useful to a majority of users, though setting the optimum defocus amount for high-resolution STEM observation at several million times magnification was beyond its capability. Most observations were done at magnifications from 500,000 to 1,000,000 times. This range was Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 165 too high for an FE-SEM operated at a lower acceleration voltage but was within the coverage of the functions. Magnification calibration function for the STEM was developed, because semiconductor manufacturers needed high accuracy device size measurement and nanotechnology features ranging from a few nm to tens of nm in size (Inada et al., 2005). By using the combined technique of micro scale, metrology standard silicon grating specimen and silicon single crystal thin film, the microscope enables to provide the absolute magnification error of less than 2% for all magnification range for the STEM. These features (stable higher performance and easier operation) matched the needs of the semiconductor industry.

7. CUTTING-EDGE APPLICATIONS WITH CUSTOMIZED HD MODELS (BY TOSHIE YAGUCHI) 7.1. 120-kV FE UHV for Nanotubes For element-selective single-atom imaging, K. Suenaga et al. developed the HD-2000 UHV 120-kV CFEG-STEM equipped with an EELS in 2003. With its newly developed magnetic lens and the EELS, it had improved spatial resolution and sensitivity, enabling to identify individual atoms. They used this system with spectrum imaging to identify elements. They also detected a single gadolinium (Gd) atom in a carbon (C) nanohorn (Hashimoto et al., 2004). Figures 43a and b show a BF-STEM image and the corresponding elemental map of Gd and C, respectively. Elemental maps were created from electron energy-loss spectra recorded at 150 eV (Gd-N edge) and 300 eV (C- K edge) with 0.5-nm spatial resolution. The numbers in Figure 43b denote the number of Gd atoms, which was determined using the integrated EELS intensity normalized by the inelastic cross section. The map clearly reveals a single Gd atom at the tip of the single-wall carbon nanohorn. This was a pioneering study that identified individual atoms in nanostructures.

7.2. 200-kV FE Ultrahigh-Resolution STEM K. Kimoto et al.(2007a)developed a high-resolution dedicated STEM with high stability for acquisition of distortion-free STEM images and high SNR analysis in 2006. It was developed based on a 200-kV commercial instrument (the HD-2300 FE-STEM) to which many modifications were made. A narrow-gap objective lens was used. Two postspecimen lenses were used to control the detection angles for the BF and ADF detectors. The SEG was replaced with a CFEG, which had a narrow energy spread of sub-0.5 eV (Kimoto and Matsui, 2002; Kimoto et al., 2005). A narrow energy spread is 166 Hiromi Inada et al.

(a)

5nm

(b)

1 2

FIGURE 43 BF-STEM image of (a) gadolinium with single-wall carbon nanohorn and (b) corresponding chemical map for Gd (yellow) and C (red). (The number labels in the picture (b) indicate the number of Gd atoms.) (Courtesy of Dr. K. Suenaga, AIST.) advantageous for analyzing the fine structure of electron energy-loss spectra. To prevent thermal drift, a shield box was placed over the high-voltage generator. A heavy metallic cover was used for the specimen stage to reduce acoustic vibration. The column was covered with rubber sheets to reduce temperature fluctuation (Muller and Grazul, 2001). The microscope was installed in a room of an anti-seismic building at the National Institute for Materials Science. Temperature fluctuations in the room were within 0.2 C/hr. The stray magnetic field in the room was less than 0.2 mG near the STEM column. Noisy apparatuses were placed in a separate room. Figure 44 shows the STEM image drifts measured during a period of 61 minutes. The average drift speed was 0.12 nm/min. Due to the high stability, the atomic columns in the ADF images could be observed. More- over, a single europium (Eu) dopant atom in b-SiAlON phosphor and small atomic displacement of 10 pm were directly observed (Kimoto et al., 2009; Saito et al.,2009). Many sets of images were sequentially acquired with drift correction using a digital beam control unit (DigiScan, Gatan) and averaged using Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 167

60 −4 50

−3 30 40

20 −2

Position (nm) 10 −1

0 0 (start)

−10 1 23 Position (nm)

FIGURE 44 Measured STEM image drift during 61-minute observation. Numbers in graph denote elapsed time in minutes.

1nm Eu

2þ FIGURE 45 ADF STEM image of b-SiAlON:Eu . The upper inset in the solid-line rectangle shows single-scanning image obtained in conventional manner, resulting in severe quantum noise. The lower inset in dotted rectangle shows simulation result. White arrows show Eu atom positions. 168 Hiromi Inada et al. image acquisition and analysis software (DigitalMicrograph, Gatan). Final images were obtained with a high SNR. The ADF image revealed Eu dopant b in the continuous atomic channel in a –Si3N4,asshowninFigure 45. Using the STEM with EELS and localized inelastic scattering, Kimoto et al.(2007b)performed two-dimensional elemental analysis with atomic- column resolution for the first time. Figure 46 shows an example result. An atomic column of La, Mn, and O in layered manganite (La1.2Sr1.8Mn2O7) was visualized two-dimensionally.

(a) (b) (c)

0.5 nm

La, Sr Mn

(d) (e) (f)

5.2/9.2 1.2/13.0 3.5/10.2

FIGURE 46 Atomic-column imaging using STEM and EELS. (a) Enlarged ADF image of analyzed area overlapped with crystal structure. (b) Calculated incident probe shape for EELS and ADF observations. (c)–(f) Core-loss images of La-N4,5, O-K, Mn-L3,2, and La-M5,4. Colored circles in (c)–(f) indicate the corresponding positions of atomic columns. (Courtesy of Dr. K. Kimoto, NIMS.) Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 169

7.3. 3D Structural and Elemental Analysis Submicrometer-resolution three-dimensional (3D) structural analysis is becoming increasingly indispensable for structural evaluation and failure analysis as devices become more integrated and smaller. To meet this need, 3D characterization techniques have been developed that use an FIB system in combination with an STEM and TEM. In this approach, a specially designed FIB-STEM–compatible specimen-rotation holder is used from initial specimen preparation through final STEM characterization. The specimen is milled to a pillar shape and mounted on the tip of a needle stub loaded in the rotation mechanism of the specimen holder. Typical specimen dimensions for 3D structural and elemental analyses are less than 200 nm in diameter and 5–10 m in length. The specimen holder enables 360 rotation and Æ20 tilt even in an instrument with a narrow-gap, high-resolution pole piece (Kamino et al., 2004). Since the specimen can be rotated a complete 360, the incident electron beam, detectors, and spectrometers can view it without obstruction over the full rotation range. This enables multidirectional observation of the specimen. In addition, a complete set of images can be taken. Using this stack of images, nanometer-scale tomograms without missing zone can be reconstructed. EDX analysis has been used for 3D elemental distribution analysis. Since the specimen had a pillar shape, the projected specimen shape remained identical at any rotation angle; consequently, the tilt series of observations and analyses were not affected by differences in shape and thickness. The limited signals of X-ray maps have been enhanced by principal component analysis (PCA) (Watanabe and Williams, 2004)in combination with X-ray spectrum image acquisition. Application of PCA enhances the weak signals in elemental distribution maps acquired in an extremely short time. Therefore, elemental distributions can be acquired for even shorter acquisition times, thereby reducing the total acquisition time for a complete tilt-series measurement (Yaguchi et al., 2008). Nevertheless, it took more than 12 hours for complete acquisition of a tilt series of X-ray maps. Figure 47 shows the 3D-reconstructed Ti (image a) and As (image b) distribution around a tungsten contact plug in a Si semiconductor device from 37 Ti-K edge maps at rotation angles between 0 and 180 with 5 steps. Note that the As dopant distribution, which is critical for semiconductor performance, was obtained three-dimensionally for the first time. EELS was also applied as another method. The elemental mapping was performed with a STEM using a real-time EELS imaging system consisting of a three-window energy filter mounted beneath the STEM detectors. This unique EELS imaging system originally had a two-window energy filter when it was developed in 2000 (Kaji et al., 2001). It was modified to have a three-window energy filter in 2008. Since electrons in 170 Hiromi Inada et al.

(a) (b)

50 nm 50 nm

FIGURE 47 3D-reconstructed (a) Ti and (b) As distributions around tungsten contact plug. They were obtained from a tilt series of 37 Ti K and As K maps over a tilt range of 0–180 with a 5 step. Simultaneous iterative reconstruction technique was used for 3D reconstruction. (Reproduced with permission of Yaguchi, 2008.) the three energy-loss ranges are collected simultaneously, a real-time energy-filtered elemental map can be obtained in a shorter time with the modified system.

8. ABERRATION-CORRECTED CFE-STEM (BY KUNIYASU NAKAMURA)

The 16th International Microscopy Congress was held in Sapporo in September 2006. Emperor Akihito and Empress Michiko were special guests at the conference. Hitachi High-Technologies (HHT) Corp. exhibited an HD-2700 Cs-corrected STEM. Before the president of HHT introduced the exhibited instruments, he congratulated the royal couple on the birth of their first grandson, Prince Hisahito, who was born that morning. The HD-2700 performed well during the five-day exhibition, demonstrating sub-A˚ ngstrom image resolution. The combination of atomic resolution and analytical probe current had been realized.

8.1. Atomic-Level Characterization Instrument The HD-2700 is a second-generation, commercial-dedicated STEM and is a promising successor to the HD-2000 and HD-2300. It supports both a CFEG and an SEG electron source. The CFEG is suitable for high-end research work, such as atomic-resolution imaging with EELS/EDS analysis. The SEG is better for long-term use, such as device inspection in the semiconductor industry. Its spherical aberration-correcting optics was a joint Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 171 project of CEOS (Heidelberg, Germany) and Hitachi. In addition to this new technology, much effort was devoted to stabilizing the entire system. Theoretically, the factors that limit the performance of a microscope are spherical aberration and chromatic incoherence, including the energy spread of the electron source. Incorporating a spherical aberration corrector and a CFEG is a feasible way to push the instrument to the next limiting criteria. However, performance degradation easily occurs due to zeroth-order aberration, such as instability of the high-tension, lenses and deflectors, specimen vibration due to acoustic waves or a stray magnetic field, drift of the optical conditions and specimen due to thermal instability, and specimen contamination. The highest attainable performance is unobtainable until all zeroth-order aberrations are eliminated. Figure 48 shows the appearance of the HD-2700. The complete covering helps reduce thermal instability and prevents transmission of acoustic waves from the environment. An ion pump is attached close to the specimen, which greatly reduces specimen contamination. The electronics systems for the high-tension, lenses and deflectors is highly advanced. Ultimate performance is obtained with the help of a spherical aberration corrector.

Microscope column with whole cover Cold-FEG

Condenser aperture Condenser lens Operating console

Spherical aberration Ion pump corrector

Objective lens Specimen stage

FIGURE 48 Appearance (left) and several elements inside whole cover (right) of aberration-corrected HD-2700 CFEG-STEM. 172 Hiromi Inada et al.

8.2. Theoretical Consideration of Advantages of Aberration-Corrected CFE-STEM Aberration correction using a multipole element is a conventional technique for improving the performance of an electron microscope, and there are several advantages of using a corrector (Erni et al., 2009; Nellist et al., 2004). Two types of correctors are commercially available (Haider et al., 1998; Krivanek, Dellby, and Lipini, 1999). However, neither type can correct chromatic aberration in principle. Thus, a CFEG is the best electron source for obtaining the ultimate performance of an aberration-corrected STEM because of its narrow energy spread. For an aberration-corrected system, a wave aberration function that includes all higher-order aberrations should be considered to estimate the attainable resolution. The incoherent broadening effect caused by chromatic aberration (variation of defocus) should also be incorporated. The wave aberration function w(o) is denoted as follows: 1 1 1 1 wðoÞ¼ Re o 2ÁA þ ooÁC þ o 3ÁA þ o2oÁB þ o 4ÁA 2 1 2 1 3 2 2 4 3 1 2 2 3 1 5 3 2 4 þ o o ÁC3 þ o oÁS3 þ o ÁA4 þ o o ÁB4 þ o oÁD4 4 5 1 1 þ o 6ÁA þ o3o 3ÁC þ o4o 2ÁS þ o5oÁR þ .. (4.5) 6 5 6 5 5 5

o ¼ l Á u Á expði Á yÞ; (4.6) where o is the complex angle (o denotes the complex conjugate of o), u is the spatial frequency, y is the azimuthal angle, and Re represents the real part. The details of all aberration coefficients are available elsewhere (Mu¨ ller et al., 2006). The probe intensity profile in real space is given by IðrÞ¼cðrÞÁcðrÞ (4.7)

p cð Þ¼ Á 2 wðoÞ Á ðoÞ r FT exp i l A (4.8)

AðoÞ¼1 for o a; AðoÞ¼0 for o > a; (4.9) where FT represents Fourier transform operation. 0 For incorporating chromatic aberration, extended defocus C1 is introduced:

0 DE C ¼ C þ C Á ; (4.10) 1 1 C E Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 173

D where CC is the chromatic aberration coefficient, E is the variation in electron energy, and E is the primary electron energy. Each wave aberration corresponding to the extended defocus is integrated by weighting the Gaussian distribution of the energy variation. The probe intensity profile 0 is defined again as functions of r and C1 . ð "# 2 0 0 0 1 ðE DEÞ Iðr; C Þ¼ cðr; C ÞÁcðr; C ÞÁpffiffiffiffiffiffiffiffi exp dDE (4.11) 1 1 1 2ps 2s2

pffiffiffiffiffiffiffiffiffiffiffiffi1 s ¼ ÁDE0; (4.12) 2 2ln2 D where E0 is defined in terms of the FWHM of the electron source energy spread. Figure 49 shows the calculated d59 criterion probe size (Haider, D ¼ Uhlemann, and Zach, 2000) at 200 kV for a CFEG ( E0 0.3 eV) and an D ¼ SEG ( E0 0.8 eV) as a function of the convergence angle. The residual and intrinsic aberrations listed in Figure 49 are assumed to emulate an actual system. The minimum probe sizes for the CFEG and SEG meet at a convergence angle of 25 mrad. The smallest attainable probe is 0.08 nm for the CFEG and 0.1 nm for the SEG. For a CFEG, the sub-A˚ ngstrom probe size remains in the range of 20–35 mrad. This is a big advantage of a CFEG. If the axial brightness of the gun is assumed to be the same for a CFEG and the SEG—although a CFEG is brighter than an SEG—a two times larger probe current is available if a CFEG with a 0.1-nm probe is used at 35 mrad compared with that if a SEG with a 0.1-nm probe is used at 25 mrad.

8.3. Evaluation of Aberration-Corrected CFE-STEM 8.3.1. Information Transfer with High-Angle ADF-STEM image Figure 50 shows DF-STEM images of Si (110) single crystals for (image a) uncorrected and (image b) corrected states. The corresponding power spectra are shown in images c and d, respectively. The superimposed images were simulated using an extended multislice method (Nakamura et al., 1997). The well-known dumbbell structure is clearly seen, and the SNR was much improved after correction. For the corrected STEM image, the information limits of 0.083 nm (Si (533)) and 0.078 nm (Si (444)) were transferred in the power spectrum, as predicted from Figure 49. These results confirm that a CFE-STEM with an aberration corrector is now an essential tool that can be used to routinely obtain atomic-resolution images. 174 Hiromi Inada et al.

0.35

0.3 ΔEo = 0.3 eV ΔEo = 0.8 eV 0.25

0.2

0.15 probe size (nm) 59

d 0.1

0.05

0 0 102030405060 Convergence angle (mrad)

Symbol Amplitude Initial phase(deg) A1 1.04 nm −41.8 C1 0nm - A2 6.66 nm 30 B2 7.24 nm 65 A3 525.7 nm −72 C3 −3.23 mm - S3 213.1 nm 136.2 A4 8.31mm 23.8 A5 3.1mm 0 B4,D4,C5, S5, R5 Not included - CC 1.7mm -

FIGURE 49 Calculated d59 probe size as a function of convergence angle.

8.3.2. Low-Kilovolt Capability The aberration correction can damage the specimen because the current density of the probe is increased more than tenfold. Such damage could be avoided by reducing the electron dose and electron energy. The imaging capability of the HD-2700 with aberration correction and low-kilovolt operation was theoretically considered and tested. Figure 51 shows the calculated 2D probe profile at 80 kV. The parameters for this calculation are listed in the figure. The profile has a sharp peak at the center and tails caused by residual higher-order aberrations. The estimated resolution as d59 criterion was 0.11 nm. Figure 52 shows a DF-STEM image of an Si (110) single crystal and the corresponding power spectrum at 80-kV operation. The dumbbell structure is clearly evident, and an information limit of 0.105 nm (Si (333)) Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 175

(a)

(c)

(b)

(d) 444

335

004

0.136 nm

FIGURE 50 DF-STEM images of Si (110) single crystals for (a) uncorrected and (b) corrected states; images (c) and (d) are power spectra corresponding to images (a) and (b), respectively.

6e - 3

5e - 3

E : 80 keV 4e - 3 ΔE0 : 0.3 eV

C3 : 10 m m 3e - 3 C5 : 2 mm

A5 : 2 mm

Intensity (a.u.) 2e - 3

1e - 3

0e + 0 −0.5 −0.4 −0.3 −0.2 −0.1 0.0 0.1 0.2 0.3 0.4 0.5 Distance (nm)

FIGURE 51 Calculated 2D-probe profile at 80 kV and its line profile. 176 Hiromi Inada et al.

333

0.136 nm

FIGURE 52 DF-STEM image of Si (110) single crystal and corresponding power spectrum at 80-kV operation. was transferred. The feasibility of atomic-scale analysis at 80 kV was confirmed by this result.

8.4. Advanced Application Results with HD-2700C The customized HD-2700C CFE-STEM was installed at the Center for Functional Nanomaterials at Brookhaven National Laboratory (BNL) by a group led by Y. Zhu and J. Wall in 2007. This is like a story of transmi- gration because the HD-2700 is a descendant of the FE-STEM on which Crewe consulted, and it was installed in the laboratory in which works Joe Wall, one of Crewe’s co-researchers on the initial CFE-STEM development in the 1960s. One of the most impressive results obtained with the HD-2700C is described below (Inada et al., 2009; Zhu and Wall, 2008).

8.4.1. Stability of EELS In the Brookhaven system, the critical parts of the high-tension circuit were carefully identified, and an anti-electromagnetic field shield and floor-vibration damping system were attached to the high-tension generator. In addition to these efforts, BNL built a highly stabilized microscope room in the Center for Functional Nanomaterials building, which was completed in 2007. As a result, the fluctuations in high tension were reduced. Figure 53 shows a time chart of the zero-loss peak of 200-keV electrons for a 130-sec duration. The spectra were acquired using a spectrometer (Enfina ER, Gatan), and the energy dispersion was 0.05 eV/channel. This time chart indicates that the stability of the total system was 0.5 ppm for Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 177

(a)

0.2 0.1 0 −0.1 Energy (eV)

0 1020304050607080 90 100 110 120 130 Time (s) (b) 4 9.0 mA 2.0 ϫ 10 m 9.0 A 4.0 mA 4.0 mA 2.0 mA 1.0 mA 2.0 mA

ϫ 4 m 1.2 10 1.0 A 0.4 eV

4 ϫ 103

0 −1.0 −0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Energy (eV)

FIGURE 53 Time chart of zero-loss peak of 200-keV electrons for 130-sec duration. long-term acquisition. An energy resolution of 0.30–0.35 eV in terms of FWHM was demonstrated for 10-sec acquisition, and one of 0.4 eV was demonstrated even under typical electron-extraction conditions to get a probe current large enough for spectrum imaging.

8.4.2. Visualization and EELS Analysis of Single Uranium Atom Figure 54 shows (image a) a DF-STEM image of uranium (U) individual atoms and clumps of U on carbon support film, (image b) an image intensity profile along the ‘‘spectrum imaging’’ line showing a single atom profile of 0.08 nm, and (c) the EELS integrated intensity profile at the U-O4,5 edge (96–150 eV) after background subtraction. The probe convergence angle and collection angle into the spectrometer were 28 and 30 mrad, respectively. This result indicates that the corrected CFEG probe was small enough and had sufficient current density to detect single atoms. Although a uranium atom has a strong scattering amplitude and should be an ideal point scatterer, the corrected CFE-STEM has the capability to resolve other single atoms by imaging or spectroscopy. 178 Hiromi Inada et al.

(b) ϫ105 HAADF-STEM 120

(a) 0.08 nm 80 Intensity (arb. unit) Spectrum imaging 40 0 0.2 0.4 0.6 0.8 1.0 (nm) (c) ) − U-O4,5 mapping 1200

2nm Survey image 0.13 nm 800

Electron counts(e 400 0 0.2 0.4 0.6 0.8 1.0 (nm)

FIGURE 54 (a) DF-STEM image of uranium (U) individual atoms and clumps of U on carbon support film. (b) Image intensity profile along ‘‘spectrum imaging’’ line. (c) EELS-integrated intensity profile at U-O4,5 edge after background subtraction.

8.4.3. EELS Spectrum Mapping of Pd/Pt Core Shell Catalyst Figure 55a shows a DF-STEM image of a palladium (Pd) nano-catalyst (core) with a platinum (Pt) shell, images b and c show the electron energy-loss (EEL) spectrum for the particle indicated by the arrow in image a, image d shows a DF-STEM image of a particle, and images e–g show the corresponding spectrum mapping images of the particle of image d for Pd, Pt, and C, respectively. The core-shell structure is clearly distinguished by the elemental mapping images. The most remarkable result is that the distribution of a very small amount of Pt was detected using the Pt- M4,5 edge, which is the high-energy loss portion, such as 2200 eV. This is a key benefit of an aberration-corrected probe.

8.4.4. EELS Column-by-Column Mapping of (Ca2CoO3)0.62 (CoO2) Figure 56a shows a DF-STEM image of (Ca2CoO3)0.62 (CoO2)viewedalong the [010] direction; image b shows a survey image showing the area for spectrum mapping and drift compensation; image c shows a DF-STEM image and corresponding elemental maps for Ca, O, and Co, respectively; image d is an overlay of the Ca, O, and Co maps; and image e is the EEL spectrum of the area. The experimental conditions were 64–341 mrad for the DF-STEM collection angle, 0.5 eV/channel for the energy dispersion, (b) (c) (a) Carbon support 500 12

C-k 10 400 Pt−M4,5 8 300 Pd−M4,5 −

e 6 200 Carbon 4 support 100 2 0 0 20 nm 300 400 500 600 2000 2100 2200 2300 2400 eV eV 20370 e− 3780 e− 64400 e− (d) (e) (f) (g)

5nm

HAADF-STEM Pd mapping −2560 Pt mapping −6390 C mapping −5198

FIGURE 55 (a) DF-STEM image of palladium (Pd) nano-catalyst with (platinum) Pt shell; (b) and (c) EEL spectra for particle indicated by arrow in image (a); (d) DF-STEM image of the particle; and (e)–(g) corresponding spectrum mapping images of the particle in image (d) for Pd, Pt, and carbon, respectively. CaO CaO CoO CoO2 CoO2 (a) (c) Ca O Co

0.5 nm 1nm

(b) (d) (e) Spectrum image

30 Ca

ϫ 3 20 2.19 2.19 nm 10

Spatial drift ϫ −

e O 10 Co

0 300 400 500 600 700 800 900 1nm eV

Ca O Co 2nm ADF-STEM survey image

FIGURE 56 (a) DF-STEM image of (Ca2CoO3)0.62 (CoO2); (b) survey image for spectrum mapping and drift compensation; (c) DF-STEM image and corresponding elemental maps of calcium (Ca), oxygen (O), and cobalt (Co), respectively; (d) overlay of Ca, O, and Co maps; and (e) EEL spectrum of area. Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 181

70 70 pixels for the scanning area at a 0.032-nm pixel distance, and a 0.05- sec dwell time for each pixel. The atomic columns of each element are clearly evident in each element map, and the overlaid image reconstructs the elemental atomic columns without arrangement of atomic columns in accordance with corresponding crystal structure shown in Figure 56(a).

8.4.5. Single Atom Imaging Using Secondary Electrons Zhu et al. demonstrated high resolution secondary electron imaging. Figure 57 shows a pair of SEM and DF-STEM images of uranium individual uranium atoms and clumps on a carbon thin film (~2nm). Quantita- tive intensity analysis of DF-STEM reveals the areas marked by small circles are individual atoms. They have achieved 0.1 nm spatial resolution with SEM detector imaging with aberration-corrected CFE-STEM (Zhu et al., 2009).

9. CONCLUSION AND FUTURE PROSPECTIVE (BY HIROSHI KAKIBAYASHI)

Many of Hitachi’s undertakings in developing and manufacturing STEMs were introduced in this chapter. The fusion of TEM technology by Hitachi started before World War II, and the CFE technology introduced by Crewe played an important role in Hitachi’s development of FE-STEMs. These were not fortuities. The very strong enthusiasm of the researchers and designers at Hitachi met the challenge to develop the world’s highest-

(a) (b)

2nm

FIGURE 57 Imaging individual uranium atoms with secondary electrons. Simultaneous acquisition of the SEM image using secondary electrons and back scattered electrons (a) and the DF-STEM image using forward scattered electrons (b), of uranium oxide nano- crystals and individual uranium atoms (raw data). 182 Hiromi Inada et al. performance STEMs. Their enthusiasm enabled Hitachi to develop various types of STEMs, from the 50-kV STEM in 1975 to the latest HD-2700 dedicated STEM. Along the way, M. Haider and H. Rose developed Cs-correction technology and it was introduced into the HD-2700. The result was elemental and chemical-bonding–state mapping and high- resolution STEM image observation at an atomic order. These microscopes contributed to the fine structure analysis of nano-materials, nano-electronic devices, and nano-biomaterials in various fields. CFE- STEMs have become an essential analytical tool in many fields. In the future, STEMs will be needed that can feed back atomic-level information by higher throughput for material and device processes. Moreover, it will become necessary to implement simultaneous atomic observation and manipulation under process ambient conditions. This means that STEMs will progress from being analytical tools to atomic-controlling instruments. Hitachi will continue to research and develop the ultimate instrument.

ACKNOWLEDGMENTS We thank Masashi Watanabe of Lehigh University, Kazutomo Suenaga of the National Institute of Advanced Industrial Science and Technology (AIST), and Koji Kimoto of the National Institute for Materials Science (NIMS) for their invaluable advice and support. We also thank Yimei Zhu, Joe Wall, Lijun Wu, and Dong Su of Brookhaven National Laboratory for allowing the use of their invaluable STEM imaging and analysis data. In addition, we acknowledge Maximilian Haider, Peter Hartel, Heiko Mu¨ ller, Frank Kahl, and Thomas Riedel for their help in the development of the spherical aberration corrector. We acknowledge and appreciate the advice about historical field emission technologies offered by Shobu Saito, Hideo Todokoro, Mikio Ichihashi, and Masahiro Tomita. Finally, we thank our colleagues at Hitachi High-Technologies Corp., Hidehito Obayashi, Mitsugu Sato, Kazui Mizuno, Takeo Kamino, Yasumitsu Ueki, Mitsuru Konno, Yuya Suzuki, Tadao Furutsu, Kazutoshi Kaji, Satoru Fukuhara, Hisaya Murakoshi, Takeshi Kawasaki, Tetsuya Akashi, Yoshifumi Taniguchi, Koichiro Saito, and Kaname Takahashi for their support and fruitful discussions.

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The Work of Albert Victor Crewe on the Scanning Transmission Electron Microscope and Related Topics

A. V. Crewe

Contents 1. Introduction 2 2. Some Chicago Aberrations: A Personal Collection 6 Acknowledgments 13 3. Electron Microscope Studies: Achievements of the Crewe Lab 13 Introduction 14 Construction (Reference Group A) 16 Source Development (Reference Group B) 17 STEM Development and Atomic Images (Reference Group C) 18 The Field Emission SEM (Reference Group D) 19 Energy Loss and Radiation Damage (Reference Group E) 20 Secondary Electron Production (Reference Group F) 21 DNA Labeling (Reference Group G) 21 Nucleosomes (Reference Group H) 22 Attempts at Aberration Correction (Reference Group I) 23 Theoretical Electron Optics (Reference Group J) 27 Optimization and the Super-High-Resolution SEM (Reference Group K) 28 Image Processing (Reference Group L) 30

Enrico Fermi Institute and Department of Physics, University of Chicago, Chicago, USA

1 2 A. V. Crewe

Three-Dimensional Reconstruction (Reference Group M) 31 Hemoglobin Work (Reference Group N) 32 References (of Chicago Aberrations) 37 References (of DOE Report) 38

1. INTRODUCTION

Any book on the scanning electron microscope (STEM) must begin with Albert Crewe, even though the early scanning microscopes of Manfred von Ardenne and Andre´ Le´aute´ were, strictly speaking, scanning transmission instruments. Unfortunately, Crewe’s health does not permit him to write a new account of his STEM work and so, with his approval, I am reproducing two earlier accounts of his contributions to electron optics and in particular, to the STEM. The first of these, ‘‘Some Chicago Aberra- tions,’’ an expanded version of his lecture at the Microscopy Society of America meeting in 2002, was published in Microscopy and Microanalysis. The other, much longer document is a report submitted to the U.S. Department of Energy, written with Oscar H. Kapp; this gives a full account of Crewe’s work during the period 1964–1992. I readily recognize that there are passages in this report that Crewe might have altered if he had been able to prepare it for republication—but in the circumstances, it seems best to reproduce it as written. Crewe was born in Bradford, in Yorkshire, on February 18, 1927, the only son of Wilfred and Edith Crewe, and studied physics at the University of Liverpool, graduating in 1947. In 1949, he married Doreen Blunsdon; they have four children, Jennifer, Sarah, Elizabeth, and David. In 1949, he was awarded his Ph.D. for his thesis on synchrotron design and in 1955, Crewe was invited to the University of Chicago as a visiting research associate, to work on the Chicago synchrotron. In 1958, he moved to the nearby Argonne National Laboratory, where a large- particle accelerator was planned. He soon became director of the Particle Accelerator Division at Argonne. In 1961, Crewe was appointed director oftheentirelaboratoryandatthesametimebecameinterestedin electron microscope design. Unaware of the work of Charles Oatley and his students in the Cambridge University Engineering Department, Crewe designed a new type of scanning electron microscope (SEM), which was to become known as the STEM. In 1966, a workshop was organized at Argonne, the object of which was to design a high-voltage electron microscope; a one-million-volt instrument had been in The Work of Albert Victor Crewe on the STEM 3 operation in Toulouse since 1960 and high-voltage electron microscope projects had been launched in England and Japan. Scientists were invited from Europe and Japan as well as from the United States and a design was completed but the project did not receive funding. In 1967, Crewe decided to return to his post at the University of Chicago, where he had been granted full professorship in 1963, and the first results from his first prototype STEM, with a field emission gun, were published soon after. I still have a vivid memory of his description of the planned microscope at an Institute of Physics meeting on ‘‘Non-Conventional Electron Micros- copy’’ in Cambridge in 1965, where many of Oatley’s group were in the audience. The subsequent part of his story is told in the papers reproduced in this volume. His work has been honored by many awards, including the following:

FIGURE 1 The quadrupole-octupole corrector is shown mounted on to a vacuum flange. Seven rectangular iron yokes are visible, the four equally spaced quadupoles and three interleaving octupoles. The electron beam was contained inside a Pt/Ir tube (not visible). The corrector was approximately 15 cm in height. 4 A. V. Crewe

FIGURE 2 The sextupole corrector is shown partially assembled. The field lens is in the center of the photograph and the beam tube is visible. The sextupoles were wound in grooves machined into the outer surface of the beam tube. Various ion pumps and valves are visible.

Chicagoan of the Year in Science (Chicago Junior Chamber of Commerce, 1962) Honorary degrees from the University of Missouri (1967), Lake Forest College (1968), Elmhurst College (1969), and the University of Liverpool (2001) National Academy of Sciences (1972) Electron Microscopy Society of America’s Distinguished Service Award (1976) Albert A. Michelson Medal of the Franklin Institute (1977) William Wrather Distinguished Service Professor and later Emeritus (1978) Ernst Abbe Award (1979) NewYork Microscope Society (1979) The Work of Albert Victor Crewe on the STEM 5

M D

FIGURE 3 Schematic of mirror system. The optical path of the system comprises two major regions. In the first region, electrons from a thermal field emission source (A) travel along the optical axis and can be brought into a first focus at the first grid (C1) by a magnetic field which is produced by a solenoid (G). A defining aperture (B) is placed about midway between the source and the first specimen. After being focused on the first specimen, the electrons enter the second region and refocus at a second grid (C2). The distance between the first and second grids is approximately the same as that between the source and the first grid. Also in the second region is an electrostatic mirror field that is produced by assigning potentials to a series of copper discs (M). Properly adjusted, the mirror field can reflect and refocus electrons at the reverse side of the first grid. Two sets of scanning coils (D, E), one at each of the first and second regions, provide the deflection fields to scan the focused electrons across the grids. They can be operated independently or synchronously. A pair of stigmator coils (F) are installed to correct the astigmatism.

Duddell Medal, The Institute of Physics, London (1980) Honorary Fellow of the Royal Microscopical Society (1984) Honorary Fellow of the Electron Microscope Society of China (1989). —Peter W. Hawkes 6 A. V. Crewe

2. SOME CHICAGO ABERRATIONS: A PERSONAL COLLECTION* by A. V. Crewe The Enrico Fermi Institute and Department of Physics, University of Chicago The account that follows is almost entirely based on my own memory, which is not a very reliable instrument. However, some of the events can be documented and references will be given whenever possible. If no reference is given, it simply means that some details have become lost in the mists of memory. It is to be hoped that sins of omission will be forgiven because they are unintended. This account is not intended to be a review and is more accurately describable as a personal recollection. This is the story of the work in Chicago on the correction or reduction of lens aberrations. It was an effort that extended over a period of almost 40 years, although it was never the primary focus of the work of the laboratory. That was always the development and exploitation of the field emission electron gun, particularly in the STEM and the SEM. That program began in 1963 and within a year or two the properties of the field emission source were established to be good enough that the ultimate performance of these instruments was determined by the objective lenses, not the source. This in turn meant that the lenses and their aberrations became matters of primary interest because the size of the focused electron probe is determined by the magnitude of the spherical (Cs) and the chromatic aberration (Cc). Everyone is aware that there has been much recent progress on the problem of correcting the aberrations of magnetic lenses, some of it in Chicago, but much more in Germany and here in the United States by Krivanek. The problem is now essentially solved but we must not forget that all this progress has had precedents and that, in Newton’s words, ‘‘we stand on the shoulders of giants.’’ Expressing a purely personal opinion, there are three historic events that greatly impressed me and convinced me that correction was both necessary and possible. Two of these were from the same author. I refer, of course, to Otto Scherzer. It was he who, in 1936, established the salient characteristic of the aberrations— Cs is always negative and Cc is always positive (Scherzer, 1936). It was a monumental calculation and is justifiably memorialized as the Scherzer theorem. It occupies 37 pages in the version given in the book by El Kareh and El Kareh. He once told me that he was conscripted into the German army and chose to work as a nighttime telephone operator for the simple reason that there were no calls in the night and he could do his own work.

* Reproduced with permission from Microscopy and Microanalysis 8 (2002), Supplement 2, 24 and 10(4), 2004, 414–419, courtesy of Cambridge University Press, copyright Microscopy Society of America. The Work of Albert Victor Crewe on the STEM 7

He completed the final calculations in one night. I asked him why on earth he even attempted to put the result in the form of a sum of squares, which is the key step in the proof. He simply replied, ‘‘I thought it would work.’’ The fact that the aberrations are always present means that the resolution of a microscope is determined by the magnitude of these aberrations 1=4 together with the effects of diffraction. The optimum depends upon Cs 1=2 or Cc , depending upon which of the two aberrations is dominant. Since the two coefficients are always finite, the only way to make substantial improvements is to find a way to correct them, and that means that one must avoid one of the three assumptions in the theorem, namely cylindrical symmetry, absence of space charge, and time independence. In a second paper Scherzer found a way to build a corrector by abandoning cylindrical symmetry. It was in 1947, after WWII, that he proposed a system of electron optical elements that could, in principle, correct both Cs and Cc by using cylindrical lenses (Scherzer, 1947). There are many names associated with the subsequent development of the corrector problem, principally Scherzer in Darmstadt, of course, but also Seeliger and Mollenstedt. There were also very significant contributions from the Cambridge group, Burfoot, Archard, and Hawkes, but I am not going to attempt to give the complete history here. I only want to say that, for me, the final step was the work of Deltrap. He actually built a system of four quadrupoles and three octupoles and showed that it would indeed correct spherical aberration (Deltrap, 1964). Specifically he used the image of a rectangular grid to show that he could reverse the sign of the third-order aberrations. His equipment was much too large for use in a microscope, and he was concentrating on the problem of increasing the current in a probe rather than in improving the resolving power, but nevertheless the principle was established. All that remained was to miniaturize the system. The Chicago side of the story is a small one and it begins in 1966 at Argonne National Laboratory. At that time we were beginners in electron microscopy and we (Isaacson, Wall, and Johnson) were attempting to get a field emission tip to function as the source of electrons in the first STEM. There was not much encouragement to think that it could be made to work, nothing except the skill of those students. Nevertheless, one could hope. As I recall, we had managed to keep a field emission tip in operation for a few hours at a time and we had a resolution of 1 nm or so. Perfor- mance was not good but it seemed to be getting better day by day. It seemed logical therefore to take a long-term look at the future. In this respect, Zworykin’s book (Zworykin et al., 1945) impressed me greatly. It still does. The account of the aberration problem that he gave seemed conclusive and he demonstrated that there was no suitable way to avoid the issue. The next significant event was reading a lengthy paper by Septier in which he described the suggestion for a corrector that had been 8 A. V. Crewe made by Scherzer and particularly the translation of that suggestion into the modern terminology of quadrupoles and octupoles (Septier, 1966). This made a corrector very amenable because I had been involved in high-energy physics and especially various aspects of accelerator technology. Quadrupoles and systems of quadrupoles were then in heavy use for controlling and focusing external beams. I had already designed and built several of them, and even though they weighed thousands of times more than would be needed in electron microscopy, the principles were the same. Extending to octupoles was also no problem. Later still, Deltrap’s thesis was published and was very impressive. He had developed a great intuitive feeling for the optics of quadrupole systems and his demonstration that a system of quadrupoles and octupoles could be made to work was conclusive. I have a copy of his thesis that I treasure and my only regret is that the hand-drawn figures have completely faded away. It looked like a very good idea and the STEM was clearly the instrument of choice for a corrector because there is no need to worry about the off-axis aberrations. Whereas a conventional microscope must carry the image information through the lens and its corrector, the STEM only needs to correct a paraxial beam. (Actually, it was many years later that we proved this—working with Parker and Golladay (Crewe and Parker, 1976; Parker, Golladay, and Crewe, 1976). David Cohen was a physicist who had worked with me on the construction of the 12-GeV Argonne accelerator and as that project neared completion, he was looking for something else to do, something different, and he asked for any suggestions. The corrector problem seemed to be an appropriate one. He enlisted the help of Phil Meads, who was then with Wm. Brobeck and Associates, because Phil had developed a computer program for particle beam lines that could handle multipoles. Together they produced a set of guidelines for an aberration corrector that had four quadrupoles and three octupoles (Crewe, Cohen, and Meads, 1968). When applied to a 30-kV STEM, the whole thing looked feasible—except for the mechanical tolerances, which were expected to be around 0.1 mm for the position of the pole pieces. A few months later, while preparing to leave Argonne to return to the University of Chicago, Walter Mankawich appeared on the scene. He was a skilled engineer and machinist at the University of Chicago who could machine metal to micron tolerances. He also was looking for a challenge and he had heard that I was due to return there in a few months. We began a long journey together. His primary work was the design and construction of the STEM and associated equipment but he used every spare moment to build jigs and fixtures for the fabrication of a corrector. It seemed reasonable at that point to assume that he could build the system with a 1-mm tolerance and that we could make up the difference The Work of Albert Victor Crewe on the STEM 9 between 1 mm and 0.1 mm by adding trim coils to adjust the magnetic potential on the poles. A couple of stories about Walter are appropriate here. Every precision machinist has precision steel blocks in his toolbox, 1-inch cubes, etc. Walter did not trust the ones that were available commercially, and however expensive, he made his own. I once asked him to make a pair of slits to be placed in vacuum for an electron spectrometer. He made them to fractional wavelength tolerances using speculum metal that he made himself. He made the magnetic spectrometer also and when it was put together, I mentioned that I admired the way that the joint between the pieces was almost invisible. He apologized, tapped the metal with a leather mallet, and the joint disappeared. Vernon Beck took over the project when he joined the group. He changed the design somewhat and added many new features (Crewe and Beck, 1974), and we built an entirely new STEM to test the device because it was too large to fit into the vacuum chamber of the existing STEM. It also happened that both Michael Thomson and Harold Rose spent a year or so in our laboratory in those years, but although both of them had experience in the correction business, they had other interests and did not get involved in the work. Building a corrector was, obviously, a long-term effort and they were only here for a short time. Walter and Vernon completed the corrector in 1976 and all 40 pole pieces were within the 1-mm tolerance limit, but when we attempted to operate it we were not able to make it work (Beck, 1977). In spite of the 40 trim coils we could not find a suitable setting. The effective centers of the quadrupoles and octupoles could not be brought together. My own conclusion at the time—right or wrong—was that the iron of the pole pieces was not sufficiently uniform, but it is also possible that the many power supplies were not stable enough. In any case, no other student would take a chance on it and so the work on aberration correction stopped. The essence of the problem was that we had 40 knobs to turn and only the image to look at. It may be noted that image analysis was in its infancy at that time. I recall that we had a state-of-the-art Nova computer with a magnificent memory of 32 K—not M, but K! A Fourier transform took a very long time indeed. It would be fair to say that the corrector failed because commercially available technology was not yet good enough and neither was our own. We were ahead of our time. Parenthetically, we might note that we eventually obtained a computing system, thanks to the persuasive efforts of David Saltzman. It arrived too late for this corrector, but one of the first calculations he and I performed was the wave optical optimization of diffraction and aberrations, both the third order and the fifth. We obtained limiting resolutions = : 1=4l3=4 : 1 6l5=6 of 0 43Cs and 0 48C5 , respectively (Crewe and Salzman, 1982). 10 A. V. Crewe

The factor of 0.43 for the third-order case agrees with the identical value obtained by Mulvey some time previously (Haine, 1961), but the factor of 0. 48 for the fifth-order case was new. It is important because it can be used to calculate the size of the optimum aperture. One might note that some time later Koops published his work in Darmstadt (Koops, 1978). He also failed to make his system work, even though his technology was better than ours. It was shortly afterwards that Scherzer published what I believe to be his last paper (Scherzer, 1978). In that paper he pointed out that the only problem with making the corrector work was that there was not enough financial support. The principle was good but the money was insufficient. It is a sentiment that I can fervently echo. It is appropriate here to make a few comments on the side. Vernon Beck was an outstanding student and I am not sure that his academic records have ever been surpassed. For this reason, there was a feeling among my students that if he could not make the thing work, nobody could. This alone prevented any further attempts. In addition to this, there is a culture in the United States that looks down on failures of any kind. It is a ‘‘one strike’’ rule. If something is a failure nobody wants to try again, with the prospect of failing a second time. It was not a personal problem, but it was certainly an issue for the funding agency (in this case, the Department of Energy [DOE]). Having failed, I could not ask for more support because it might fail again. Perhaps this is also due to the fact that the students were all physicists who were working towards a Ph.D. Null results are not acceptable for a thesis. In fact, although the work on the corrector was 90% of his effort, Vernon’s thesis was on another subject altogether. In fact, this thesis is of some interest today because it was on the subject of left-right asymmetry in scattering from single atoms, a contrast technique that is now possible because of the success of correctors. The next significant event came while giving a series of lectures on electron optics to my research group, namely the theory of electron guns, round lenses, quadrupoles, and octupoles. After finishing the course one of the students (I think it was Marylin Listvan) pointed out that I had not mentioned sextupoles at all. This was enough to force me to calculate their properties. It is easy enough to write down the equations of motion and solve them in a series approximation. Of course, there was no first-order focusing and the second-order result was just what you would expect, a round beam would become a three-pointed star. The surprise came in the third-order result. It came as an astonishment that the third-order aberrations were cylindrically symmetric about the axis and of the opposite sign to those of round lenses (Crewe, 1980). (This result had also been noted by Hawkes but I was unaware of his result.) Any of my students will happily point out that my calculations are notoriously full of errors so they needed to be checked. David Kopf The Work of Albert Victor Crewe on the STEM 11 performed the check, using his own methods and he found no errors (Crewe and Kopf, 1980a,b). So now we were in the position of having a very simple device, a sextupole, that had very desirable third-order properties because it could conceivably correct the aberrations of a round lens, but on the other hand, it had very undesirable second-order properties, producing a pronounced three-pronged star. The problem then was to find an electron optical system that eliminates the second-order focusing effect while retaining the third-order aberrations. It is very embarrassing to say that it took a few months before the solution appeared. I am reminded here of a story that I recently heard. It is that Winston Churchill once said ‘‘Americans always do the right thing— after trying everything else!’’ In this case, it was only by recalling the use of field lenses in light optics that the solution appeared. These lenses are often used to image one aperture onto the next one in order to preserve the intensity of the light. In our case, we can place a non-rotating round lens (actually two identical but opposing lenses) between two sextupoles, imaging one onto the other. With a suitable physical or electrical rotation between the two sextupoles the desired result is obtained, the second-order focusing effect can be removed because one sextupole cancels out the other, but the two third- order terms add (Crewe, 1982). This was published in July 1981 and became the basis for other work. For example, Shao and Jiye later added to the concept by considering the fifth-order aberrations as well and devising a way to control them (Crewe and Jiye, 1985; Jiye, Shao, and Crewe, 1985; Shao and Crewe, 1987). One of the advantages of using sextupoles is that the required excitation is small enough that one can use air coils—no iron is needed. With some funding from the National Science Foundation (NSF) we began construction of a 200-kV STEM with a sextupole corrector. This voltage was chosen so that the chromatic aberration caused by the inherent energy spread of the electron beam would not limit the final probe size. Unfortunately, when the microscope was more than half finished, their enthusiasm ran out and funding was terminated. We had to abandon the project. All was not lost, however, because Chen and Mu, who worked in the lab for a year or two each, returned to China and proceeded to build the sextupole corrector and installed it on an SEM column. They demonstrated that the total aperture aberration could be made to change sign using the same method as Deltrap (Chen and Mu, 1990). So the whole thing was not lost. It is unfortunate that their work is not widely recognized. The third—and maybe final—attempt at correction occurred as a result of an academic exercise, namely an attempt to develop electron optics ‘‘ab initio,’’ in a simplified notation, starting with uniform fields, a purely personal exercise. Surprisingly, the uniform field is normally treated in monographs, perhaps because it is too simple, but it is curious that there 12 A. V. Crewe are many, many papers on the aberrations of various lenses, but none that I could recall that gave the aberration constants for uniform fields. Having considered the magnetic and the electrostatic fields separately, the next logical step was to consider a uniform magnetic field superimposed on a uniform electrostatic field with an arbitrary angle between them (Chen and Mu, 1990; Crewe, 1992a). The focusing properties are remarkably simple and the most interesting case was the magnetically focused electrostatic mirror. It turned out to have third-order aberrations with opposite sign to those of a round lens. It was then possible to design a system with zero aberrations. We built such a device (Crewe, 1992b), and Frank Tsai showed that he could indeed eliminate both chromatic and spherical aberration (Crewe et al., 2000; Tsai, 2000). He used the characteristics of the focused probe itself to do this. This concept remains a curiosity because it is difficult to design a useful optical system. The only one appears to be a STEM configuration. Once again we obtained DOE support for such a STEM system, and once again the funding was terminated when the project was incomplete. I have mentioned the funding problem several times now and I would like to add some comments on the subject. The first thing to be said is that the work in the early years was generously supported by the DOE and predecessors, but later we encountered a situation where there was no U.S. government agency that supported instrument development for its own sake. All agencies insisted upon having specific uses for any instrument project. Generalizations were not acceptable. It was useless to suggest that improved resolution is always needed and generates its own uses. Such statements were unacceptable. In the past I have even gone so far as to point out that Galileo could never have been funded if these rules are applied. He knew nothing of the moons of Jupiter before he took a look at the planet. Similarly, Hooke, who made the most fundamental discovery in biology—the cell—did not know that they were there until he looked. On the other hand, one could argue that it is entirely reasonable to insist on having a specific use in mind for a new instrument. In principle this sounds good, but there are some drawbacks. One of these is that the reviewers are drawn from the community of appropriate users. In my case this has meant that proposals are reviewed by biologists, who, in general, are not experts in microscope design or in solution to second-order differential equations. In fact, I have had at least two good proposals turned down because one of the reviewers gave a negative review but started out by saying, ‘‘ I know nothing about this subject but...’’ and then proceeded to prove it. Hardly a peer review! This attitude on the part of the agencies may be changing slowly because the NSF has recently supported one of our proposals for a low-voltage microscope. We can only applaud this change. The Work of Albert Victor Crewe on the STEM 13

In summary, the net result of our efforts in Chicago on the aberration problem is not very encouraging. We have had one technical failure (the quadrupole-octupole system), two funding failures (the sextupole and the STEM with the uniform field corrector), one vicarious success (the Chen–Mu sextupole system), and one real one (the magnetically focused mirror). But perhaps we did stimulate some ideas and help to keep the corrector business going.

ACKNOWLEDGMENTS

The author is deeply indebted to all the students, associates, and collaborators who are mentioned in the text, and perhaps to others if there have been any lapses of memory. At its peak, this laboratory was a hive of activity and all in it were self-starters. They needed only a few occasional suggestions and very little guidance in carrying out the work described above. Thanks are also due to the DOE and its predecessors who provided the funding for virtually all the research. Some of the later work was supported by the NSF and the Block fund of the University of Chicago. The author is also indebted to the NSF for the support of our current project.

3. ELECTRON MICROSCOPE STUDIES: ACHIEVEMENTS OF THE CREWE LAB*

Progress Report DOE ER60437-7, for Period 1 July 1964 to 1 June 1992. By A. V. Crewe and O. H. Kapp, The University of Chicago, The Enrico Fermi Institute and The Department of Physics.

ABSTRACT

This is a report covering the research performed in the Crewe laboratory between 1964 and 1992. Because of limitations of space we have provided relatively brief summaries of the major research directions of the facility during these years. A complete bibliography has been included and we have referenced groups of pertinent publications at the beginning of each section. This report summarizes our efforts to develop better electron microscopes and chronicles many of the experimental programs, in materials science and biology, that acted both as a stimulus to better microscope design and also as a testing ground for many instrumental innovations.

* Notice: This report was prepared as an account of work sponsored by the United States government. Neither the United States not the Department of Energy, nor any of their employees, nor any of their contractors, subcontractors, or their employees, makes any warranty, express or implied, or assumes any legal liability or responsbility for the accuracy, completeness, or usefulness of any information, apparatus, productor process disclosed, or represents that its use would not infringe privatcly owned rights. 14 A. V. Crewe

Introduction In the following pages we will attempt to chronicle the successes and the failures of the work of this laboratory from its inception in 1964 to the present. We will not try to cover all the administrative changes that have taken place, but merely note that the work began under the auspices of the Atomic Energy Commission (AEC), continued through the era of Energy Research and Development Administration (ERDA), and is now funded by the Department of Energy (DOE). These changes were not functionally important because for most of these years, in fact until the past year, the personnel in the program management in Washington were the same. It is impossible for us to tell the whole story in such a short space beause there is so much to tell. For example, our records indicate about 500 publications of various kinds, all the way from abstracts of presentations at the annual meetings of the microscope society to full-length papers to chapters of books. Our records do not indicate how many invited talks have been given, but that must number in the hundreds. As one example, for the purposes of writing this report, we counted the number of references to our work in the Citation Index in the past 12 years. We counted more than 1000 such references and this was only to full-length papers with my name as the first one on the paper. It does not count references to shorter publications or to papers with someone else’s name in first place. From this we can surmise that the total number of references to our work must be several thousand. We must conclude that our work has been appreciated. We should also say at the outset that the work in this laboratory has been done by a large number of people over a number of years. There have been about a dozen graduate students and about as many under- graduates, postgraduates, and other visitors. We will try to acknowledge all of them, but undoubtedly we will miss some. This research program began when the author was still the Director of Argonne National Laboratory, in 1963. At that time, I conceived a new principle for the construction of an electron microscope. The general idea was to focus a beam of electrons into a very small probe and then scan the probe across the specimen in a raster fashion. The electrons passing through the specimen would pass through an energy analyzer and selected electrons would be used to provide the contrast in the image. This concept was very new and it had the desirable property of separating the resolution (the probe size) from the contrast mechanism. However, it was completely unconventional. This type of microscope is now known as the STEM (scanning transmission electron microscope). At that time, we were unaware of the efforts in England to develop a scanning microscope that used a totally different contrast mechanism, the secondary electrons. This was a successful effort and that type of microscope came to be called the SEM (scanning electron microscope). Perhaps The Work of Albert Victor Crewe on the STEM 15 it was a good thing that we knew nothing of this work since we might well have been very discouraged by the resolution calculations made by this group. They had come to the conclusion that a resolution better than about 50 A˚ was not possible! In fact, we had dimly appreciated this problem, which is associated with the type of electron source that they were using and which was the only one known at the time. Indepen- dently, we had concluded that the source would be a problem and we had decided to use a field emission tip for our electron source, even though they did not really exist anywhere. After checking with experts in the laboratory that the idea really was a new one, a prototype version of such an instrument was designed and construction began. The mechanical parts were made in the Argonne shops while the electronics group designed and built the electronics. This was no easy task since the original concept involved the use of technology that was either not available at all or was only just coming into the marketplace. To take just one example, as we have pointed out above, the electron source was conceived to be a field emission source. While the phenomenon of field emission was well known, nobody had ever made an electron source based on this principle. The effect was still being used primarily for research into the effect itself. In fact, the vacuum levels that were needed for field emission were so far removed from the levels that could be maintained in a practical instrument that the experts in the field stated that it simply could not be done. We decided to produce the best possible vacuum and hope for the best. For the record, it is relevant here to present the objections and comments that were made about this proposal at the time. These adverse comments had no real effect since ‘‘peer review’’ had not then been conceived and I was the director of the laboratory and therefore had some freedom in the matter of decision making. The first objection wss the one given above, namely that field emission could not be made to work as a practical source of electrons. Specifically, we were told that the effect could only be seen when the vacuum level was in the 10 15 torr range, a vacuum level that required the whole instrument to be placed in liquid helium, and that the best that could be done in a working instrument would be in the 10 10 torr range, a difference of a factor of 105! The second objection was that no specimen could possibly tolerate a vacuum of 10 10, and even if they did survive, they would not represent the real object, the structures would be completely destroyed. The third objection was that we could never maintain even the vacuum level of 10 10 in a working instrument because we would have persistent vacuum leaks and in any case, the only vacuum equipment on the market were the pumps and a few types of feedthroughs, notably linear motions. No rotary motions or electrical feedthroughs could be bought. 16 A. V. Crewe

The fourth objection concerned the electronics. How could we possibly make the electronics stable enough to focus electrons into a spot a few A˚ in diameter? The fifth objection was in the matter of a suitable image display system. The only image displays at the time were TV, which was then notably inferior, and oscilloscopes, which once again were so far short of the image quality available on the glass plates and film used in conventional electron microscopy as to be laughable. These comments were made by the experts in the various fields and were obtained by the simple expedient of talking to them and asking for advice. In fact, the only encouragement that I ever received was from people who knew nothing about technology but simply wanted better microscopes and welcomed any attempt to provide them. There is absolutely no doubt that a proposal to construct this microscope would never have passed a peer review. The objections were good ones and there were few if any replies that I could give. Nevertheless, we proceeded with the project in the belief that all these problems could be solved. Nowadays, this type of proposal does not stand a chance. The peer review system, while having its merits, effectively prevents an innovator from even trying to do what he believes to be possible. The system does not allow for the situation where there are no peers. This comment is not meant to be arrogant, nor is it to be taken in the intellectual sense of the word, but simply to say that it is quite impossible for an innovator to set down on paper all the relevant thoughts that pass through his mind. Any rational discussion of these thoughts would occupy many days of talk and may not even lead to a definite conclusion. Peer review by mail does not allow the opportunity for discussion and without this discussion it can easily turn into a vehicle for anonymous and even defamatory carping. We are reminded here of the recent explanations of the award of the Nobel Prize in economics to Prof. Coase here at the University of Chicago. Many years ago he presented his views to the faculty. At the start of the discussions the vote was 20 to 1 against him. Stigler has pointed out that the vote would have been even worse if Coase had not been allowed to vote! At the end of a very long discussion, the vote was 21 to 0 in favor of his views. One wonders what might have happened if he had been forced to pass a peer review by mail in order to be able to proceed with his work. It would never have been done.

Construction (Reference Group A) When we began the design and construction of the instrument, nobody at Argonne (or indeed almost anywhere else) had experience with ultrahigh vacuum (UHV) engineering and we all had to learn. Although there were some commercially available vacuum seals, we were forced to The Work of Albert Victor Crewe on the STEM 17 design many custom versions. The vacuum vessel that was built at that time is still in use and has never suffered from vacuum leaks. It is now 27 years old and still viable. It may be of some interest that the person who did the welding of all the mechanical joints was Frank Meyer. A few years later he put his experience to good use and founded Meyer Tool and Engineering Co. here in Illinois. This company specializes in high- vacuum and cryogenic fabrication and is known throughout the United States. He does a great deal of work for Argonne and Fermilab. At the very least this project gave the design and fabrication groups at Argonne some much needed experience and led to the formation of an industrial company of some size and competence.

Source Development (Reference Group B) At Argonne I was joined by the first three graduate students who worked in my laboratory, Wall, Isaacson, and Johnson. Together, we decided to find out how to build and operate a field emission source. For this purpose we designed and built a special unit for the rapid testing of the sources and in the course of about two years we began to understand the process and how to obtain good performance. That work has continued to this day, because we still do not know enough. Nevertheless, we learned enough in those two years that we could operate a field emission source for a sufficient length of time to have a practical microscope. What we learned was that while the total emission current was far less in our kind of vacuum than that in a more perfect one, we could nevertheless obtain enough current for our needs. This was totally unpredicted. As our work proceeded, we were able to operate our tips for a day, then a week, then a month, and finally, for several years without having to change them. This meant that our sources were even more reliable than even the very best conventional source. This, again, was totally unexpected and so out of the ordinary that very few people believed our results. It was during this period that we were successful in obtaining our first images in the microscope. These images were unremarkable as regards the resolution but they did demonstrate that unusual contrast could be obtained. In fact, we published some images taken at various energy loss intervals. These images were the first to indicate the analytical power of the instrument and in that respect, it was the ancestor of many modern instruments. Shortly after this work I resigned as laboratory director in order to return to the University of Chicago and the instrument was taken to the University. At that time, the AEC (as it was then) decided to continue support of the project and placed it in the biological science area. This was an arbitrary decision since the research was applicable to many areas of science but until recently this has not been a problem. This is a very important point in view of the recent difficulties that we have 18 A. V. Crewe encountered. The attitude of the program managers was always that the work was of high quality and that it could be considered to be a success even if the work did not directly benefit the biological sciences. As a result of this, we have always felt free to pursue productive lines of thought even if the ultimate benefit was to the material sciences anywhere else. It would be a great loss if ideas generated in one area could not be followed through simply because of the narrow interests of the immediate source of funding. We have never been prevented from following our own noses but it appears now that this concept is not universally appreciated.

STEM Development and Atomic Images (Reference Group C) In the period after our return to the University, we made several improvements to the microscope, but found that we could not obtain a resolution better than about 10 A˚ and we then began some investigations into the theory of probe formation, an effort that continues today. We soon found that we had set our apertures incorrectly and when the aperture was properly adjusted, the resolution immediately improved to 5 A˚ . However, this was not the end of the story. We had been joined by Elmar Zeitler who was on a one-year leave of absence. He gave our group some seminars on electron scattering and it became evident to me from these talks that our electron collection system could be improved. This inspired the idea of the annular detector for the elastically scattered electrons, perhaps the most important of all the contrast mechanisms. After installing an annular detector, we found that our resolution was really 2.5 A˚ all the time, but we had not been able to see it because the contrast was too low. In turn, this led to other ideas, notably that the use of the annular detector together with the energy loss spectrometer allowed us to provide almost complete separation between the elastic and the inelastic electrons. This meant that we were in a position to measure the mass of particles in the microscope and to measure the atomic number. Two pieces of information and two unknowns to solve for, the classical mathematical situation. Since that time, mass measurement and ‘‘Z’’ contrast have become very important, the one in biology and the other more recently in materials science. As we began to take images of smaller and smaller particles, we found that we could even obtain excellent images of unstained single-stranded DNA. something that could not be done in a conventional microscope. This led me to do the first calculations on the possibility of obtaining images of isolated atoms. The calculations indicated that this was easily done for the case of heavy atoms. Accordingly we looked around for a suitable sample and we were fortunate to have the cooperation of Michael Beer from Johns Hopkins. He brought along a sample of a thorium compound that he had been experimenting with for the purpose of The Work of Albert Victor Crewe on the STEM 19 staining amino acids in DNA. All attempts to image the atoms in this or any other specimen had failed. We were able to obtain clear images on the very first attempt. Later, we continued to pursue this capability, pushing it as far as it would go. We imaged lighter and lighter atoms, finding a practical limit at about zinc. We also tried to answer our critics (and there were many) by taking several successive images of the same atoms. To this date, I do not believe that anyone can do this using a conventional microscope. The act of viewing destroys that area of the specimen. But we could indeed do it because we had carefully investigated the contamination problem and learned how to control it. These lessons are still not heeded by most microscope operators. The final demonstration of our capabilities was the production of movies showing the motion of atoms on the surface of the specimen, movies which involved taking hundreds of successive images of the same atoms. These movies were taken in 1974, long before anyone else could obtain even one image of an atom. Perhaps it is worthwhile to point out that very few people have been able to match this performance. The instrument that was put on the market by Vacuum Generators did not have a high enough resolution to be able to image single atoms until the introduction of their new machine just two years ago. Hitachi built a STEM for their research laboratory about 1980 and produced atom images, but they never introduced it to the market. Apart from that, the only microscopes that are able to image single atoms are the ones at Brookhaven (one of them is from Johns Hopkins) whose designs are heavily based on ours. The concept of the original instrument (now called the STEM) was taken up by only one company, Vacuum Generators of England. They simply took our basic design and turned it upside down for convenience. In all other respects, it was identical to ours. There is no doubt at all that they violated the patents that were taken out by the AEC. Although I pointed this out to the AEC, they did nothing about it and I was informed that no royalties were ever received. In light of the fact that they have sold instruments with a total value in the neighborhood of 50 million dollars, these royalties alone could easily have kept our laboratory operating.

The Field Emission SEM (Reference Group D) Shortly before we all returned to the University we decided to adapt the field emission source to the SEM microscopes then being developed in Cam- bridge, England. When we calculated the resolution that we might expect, we found that we did not need any magnetic lenses in order to be able to compete with the miscroscopes then being sold. We found that our electron gun could provide all the focusing power that we needed. Naturally, this approach would not have been adequate for a commercial instrument 20 A. V. Crewe because it could not provide the flexibility of operation that would be needed for general use. Nevertheless, we felt that there was a place for such a simplified approach. At the University we succeeded in making this device work and we took many images with it. In fact, we used no lenses at all except for the lens action of the gun itself and we were able to demonstrate a resolution equal to the very best that could be done anywhere. This concept was taken up by one of the engineers who worked on the project at Argonne. He teamed up with Vincent Coates and they formed their own company, Coates and Welter. They produced the very first commercial instruments that used field emission sources. Their microscope design was almost identical to the one that we worked on together. This company was eventually sold to American Optical and then later the technology was sold back to Coates, who founded Nanometrics, a company that still makes field emission microscopes, primarily for the semiconductor industry. One other company took up our ideas and developed instruments and that is Hitachi of Japan. They decided to take our advice that field emission sources were the only ones capable of allowing the production of high-resolution SEM instruments. They invested an enormous amount of effort into this program, with spectacular results. Violating no patents, because they were only interested in the SEM, which was not covered by those patents, they developed many high-performace instruments over the years. We have been reliably informed that they have sold more than 1700 microscopes with a total value that must be close to 500 million dollars. In view of this success, all the major manufacturers are now selling field emission microscopes, and it may not be long before all microscopes are of this kind. It would be safe to say that this laboratory is the source of a billion dollar industry.

Energy Loss and Radiation Damage (Reference Group E) When we decided to build the instrument described above, the development of the SEM was not our main concern. We were really more interested in the interaction of the electron beam with the specimen in the microscope. It was well known, of course, that the electron beam produces damage in the specimen and there had been demonstrations of the loss of information in the diffraction pattern obtained from crystalline specimens. Unfortu- nately, there was little if any numerical data, principally because the conventional TEM is not a quantitative device. Also, since we had installed a spectrometer on the STEM in the faith and belief that the information obtained from it would be useful for image contrast, we needed to know more about the processes involved. For these various reasons, we had installed a spectrometer of our own design on the simple SEM and we began a series of experiments to study The Work of Albert Victor Crewe on the STEM 21 the phenomena of energy loss and specimen damage. The work was done by a succession of graduate students, among them were Isaacson, Johnson, Lin, and Ramamurti. Since one of our primary targets was DNA, the first specimens that were studied were the four bases and later we included some amino acids. In each case, we could measure the rate of destruction of the specimen in terms of mass loss, loss of the energy loss peaks, and loss of diffraction information. Using all this knowledge, we could put together a more or less complete picture of the damage process. We could even connect these processes in a general way with the known radiation chemistry (the G- values). It is interesting that this picture was complete enough that Unwin returned to Cambridge from a conference in Europe where I presented these results and then used them to design his famous experiment (with Henderson) on the imaging of the purple membrane. In fact, our series of experiments were conclusive enough that the numbers that were produced are the ones still in use today. They provide the basis for taking the steps needed to avoid radiation damage.

Secondary Electron Production (Reference Group F) One other significant experiment that was done with this setup was an investigation into the origin of secondary electrons. This work was done by Voreades and is not generally known in the community. He chose to publish his results in a rather obscure journal. Since the results were used for his thesis he was entirely in control of the publication of his data. In fact, the data was very important and eventually led me to reconsider the resolution limits of the SEM and this investigation had far-reaching effects. Acting on my suggestion, Voreades set up an arrangement for detecting the secondary electrons in time coincidence with the energy loss electrons. In this way he could show that all regions of the energy loss spectrum contributed to the secondary electron current and that the electrons came from a very thin region near the surface. In carbon, this corresponded to a layer only 20 A˚ thick. For heavier materials, the layer was even thinner. This result allowed us to generate a picture of the process whereby energy loss events produce secondary electrons that wander around in the body of the material and emerge from the surface if they have enough energy to escape. This is a simple model and it works.

DNA Labeling (Reference Group G) The story of the DNA labeling experiments is an interesting one with both good news and bad. It has relevance for the work now underway on the human genome project. 22 A. V. Crewe

When we demonstrated that single atoms could be seen in the microscope, one of the students (Langmore) decided to try to use this capability for visually sequencing strands of DNA. In the final version, the idea was to replicate single strands of DNA using thiolated bases. If this could be done selectively, the bases could then be labeled with mercury since the sulphur atom was exposed and reactive. The microscope would then be used to determine the spacing of the labels. The whole process could then be repeated by using different thiolated bases so that in the end there would be a great deal of redundancy in the data and sequencing should be virtually unambiguous. Langmore succeeded in doing all the wet chemistry and was able to demonstrate that the various steps were all feasible and the reactions were selective enough to ensure success. This feat alone deserves a medal! The difficulty came when the specimen was put into the microscope. What he observed was that the mercury atoms moved away from their original positions and the movement was large enough that the whole experiment was doomed. In fact, it was Langmore who first noted the movement of atoms on the surface of the carbon film, an observation that led us to investigate this whole phenomenon. That investigation took too long to be of any value in the sequencing attempt and Langmore eventually worked on a different specimen for his thesis. That series of experiments on the motion of single atoms indicated that the atoms were moving entirely due to their thermal energy, or rather the ratio of the thermal energy to the binding energy on the film. One can conclude from this that it may yet be possible to do the type of experiment that Langmore envisaged. What would be needed would be a system with a higher atom-carbon binding energy and to operate at low temperatures. Unfortunately, we have never had another student who wanted to try to develop this technology, but this does not invalidate the conclusion.

Nucleosomes (Reference Group H) The specimen that Langmore chose for his thesis was the nucleosome. He obtained very good images of them and used the data that had been obtained on the energy loss problem in a very clever way. He allowed the specimen to become damaged by the radiation and used the fact that the DNA was less sensitive to radiation damage than the protein. In this way he could determine the location of the DNA and he proposed a structure for the nucleosome. It is noteworthy what happened next. After graduation he went to the Medical Research Counsel (MRC) in Cambridge, England, where he wanted to continue the work on nucleosomes but was not allowed to since that problem was reserved for some of the ‘‘greats.’’ The structure of nucleosomes that was eventually published The Work of Albert Victor Crewe on the STEM 23 by the Cambridge group was virtually identical to Langmore’s structure, but his work only received a glancing reference!

Attempts at Aberration Correction (Reference Group I) It is well known that the limiting factors in the resolution of the electron microscope are the effects of the lens aberrations. This was fully recognized in the early days and the whole problem of lens aberrations has received a lot of attention over the years. The major contribution to this problem was made by Scherzer in 1936 when he showed that the aberration coefficients of the lenses are always of the same sign. The coefficient of spherical aberration is negative and that of chromatic aberration is positive. He was very careful to state the conditions under which this statement is true and the result is known today as Scherzer’s theorem. It applies to static fields with cylindrical symmetry and when the fields are defined by Laplace. This theorem implies that the aberration coefficients can never be zero, although this can only be inferred. Many people have confirmed his results (including me) and it can be assumed that he was absolutely correct. In effect, we showed that the only fields that can give zero coefficients are quite impractical because they involve fields that go to infinity at some point. It is natural then to try to design lenses with smaller aberrations. Many investigators have tried to do this over the past 50 years or so and it is reasonably safe to assume that everything that can be done in this direction has been done. In fact, the situation is a very difficult one. The coefficent of chromatic aberration is closely equal to the focal length of the lens and the only possiblilty is to reduce that number. Even if the required field strengths could be obtained, there would be no room for the specimen. In addition, the resolution limit defined by the chromatic aberration depends on the square root of the coefficient. This means that if we want to make an improvement of a factor of two, we need to improve the aberration coefficient by a factor of four. The situation is even worse when the limit of resolution is determined by the spherical aberration. This coefficient is, at best, about one half the focal length and the resolution depends on the fourth root of the coefficient so that an improvement by a factor of two means that the coefficient of spherical aberration must be reduced by a factor of sixteen. It can be said quite positively that no such factor can be found by design improvements. It was clear to Scherzer that the limit of resolution could only be reduced by using field distributions that avoided one or more of the constraints that he had identified. In 1946 he devised a system of multipole lenses that together could act as a correction system for a normal 24 A. V. Crewe lens. Over the course of the next few years he and his students investigated the optical requirements of this corrector and came to some alarm- ing conclusions. In particular, they discovered that the alignment requirements were very severe indeed. For example, all the 40 or so pole pieces needed to be placed in their proper positions within one tenth of a micron. This, of course, is mechanically impossible. Some encouragement was obtained when Deltrap published his thesis for work done in Cambridge. He constructed a model of the Scherzer corrector, scaling up the system to bench size in order to be able to test the functionality without, of course, being able to obtain high resolution. He simply wanted to show that the general principle would work. It did. In due time, the Scherzer group devised ways to overcome all the problems, at least on paper. Construction began around 1970 but the system never worked. Shortly before he died, Scherzer wrote that the primary reason for the failure was the inadequate financial support for the project. I personally believe that he was quite correct in this conclusion. At that time, this was the most ambitious electron optical project ever conceived but the manpower that he had available was very limited. Even while our group was still at Argonne, I began to think about the problem of aberration correction because it was certain that sooner or later we would run into the resolution problem. It seemed to me that the corrector design problem would be different for a STEM than for a conventional microscope and I asked David Cohen to look into the problem. He enlisted some outside help and eventually came up with a design that looked simpler than the Scherzer one. I redesigned the system to fit the parameters of our microscope and looked around for someone to build it. Here, we were fortunate that we had decided to return to the University of Chicago because there we found perhaps the only person in the country with the skills to fabricate the lenses to the tolerances that seemed necessary. That was Walter Mankowich. Over the course of the next few years he devised a method of making the pole pieces that seemed to be good enough. In fact, when the system was finally assembled, all 40 pole pieces were within 1 micron of their design positions, a truly remarkable feat. Naturally we knew that this would not be good enough for the system to work properly and we had incorporated trimming coils to provide the final adjustment. It turned out later that Scherzer had instituted essentially the same system of trimming coils. In the meantime, we had built a special microscope for testing the corrector. This was a 100-kV instrument that was put together by Vernon Beck, the graduate student who wanted to attempt the correction. Unfor- tunately, we could never make the corrector work. The reasons were varied, but the main reason was that the mechanical center of the multipole lenses was very different from the magnetic center and in addition, the magnetic center moved around with the excitation so that we could The Work of Albert Victor Crewe on the STEM 25 never align the system and maintain this alignment. After many heart- breaking attempts, we were forced to admit defeat. Until 1980, the Scherzer corrector was the only known method for correcting aberrations in the electron microscope, and as we have just pointed out, it could not be made to work. However, in that year we invented a new method that looked very promising. In some purely theoretical studies on the electron optical properties of the various multipole elements, we discovered that the third-order aberration of a sextupole lens is cylindrically symmetric and has the opposite sign of aberration from that ofaroundlens.Sometimelater,wedevisedasystemoflensesthatcanuse this effect to form a new type of corrector. The publication of the sextupole corrector paper attracted a great deal of attention and in the interval since then a number of people have checked the calculations using a variety of different methods. There is universal agreement that the calculations are correct and that a corrector can be made. We carefully investigated the construction problem and evaluated a few different options. One was to build a demonstration column that would simply confirm that the principle would work. Such a column would not have the high-resolution possibilities of a full-scale version. Another option was to build a system that would have the high-resolution capabilities. To do this, however, meant that we would have to build an entirely new piece of equipment. The reason for this is that correcting the spherical aberration alone would not give a convincing improvement in the case of a microscope operating at the voltage that we normally use (30 kV). Chromatic aberration becomes important surprisingly soon. We looked at this problem very carefully and in doing so we were forced to reevaluate the limiting factors in the resolution capabilities of all probe forming microscopes. As we will show later, this work led to some very significant improvements in other microscopes. In fact, the whole sextupole corrector project will have been worth the effort even if it never works! In the end, we proposed the construction of a 200-kV STEM with a sextupole correcting element. This proposal was made to the National Science Foundation (NSF) and was eventually funded, but not at the requested level. The entire system of lenses and corrector has been designed and everything has been fabricated with the exception of a few portions of the electronics. Some of the components have also been tested. Unfortunately, the support has not been adequate for completion of the project and progress has therefore been very slow in recent years. It should be noted that two of the visitors who worked in our laboratory, Er-gang Chen and Mu, returned to China and began working on the problem of the sextupole corrector. They chose to build a simpler version that they could use for demonstration purposes. Within the past year or so they have produced real evidence that the corrector works. They have 26 A. V. Crewe done this in the same way that Deltrap showed the original Scherzer corrector works. They will eventually try to obtain high resolution and we wish them well. Until this past summer, the sextupole corrector was still the only corrector that shows promise of allowing the design and construction of an electron microscope of super-high resolution in the normal voltage range for transmission microscopes. However, we have now invented a new method of aberration correction that seems to be much simpler. One of the electron optical elements that Scherzer did not take into account and which is excluded by his theorem is the electron mirror. It has been known for decades that the mirror has the property that the sign of both aberration coefficients are opposite to those in the normal round lens. The difficulty has always been that the first-order properties of the mirror are difficult to deal with and the mirror optics themselves have never been given much scrutiny. A discussion of the properties of the mirror can be found in old texts such as Zworykin, but one finds little encouragement there for the construction of a working system. Neverthe- less, there have been several investigations in the past and recently the mirror was revived by Rempfer, who proposed an idea for a corrector. It is reasonably certain that the Rempfer idea will not work, at least for high-resolution systems, for a variety of reasons that are too complicated for discussion here. We began our own investigation into mirrors quite recently and during the past summer, we developed some new ideas on a particular form of mirror that seems to have all the desirable properties without any of the ‘‘side effects.’’ We developed a complete proposal for a mirror corrector and have transmitted a proposal containing that idea to the NSF. This mirror corrector has the nice property that both spherical and chromatic aberration can be corrected. It is interesting that we cannot yet confirm the details of operation of the corrector using numerical calculations because there do not appear to be any numerical methods for the solution of this type of problem. The difficulty is the reversal of the direction of motion. It may be that there are some techniques that can be used but we have not yet found them. However, there is no doubt that correction can occur because the physics of the problem is very simple and the effect can easily be demonstrated on paper. At the moment we are developing an extension of the principle that might allow the design of a super-high-resolution SEM. In summary, we can say that in 1946 Scherzer invented a corrector. He worked on the problem for more than 35 years and was unable to make it work. We tried to make the same kind of corrector work and we also failed. In 1980 we invented a second type of corrector. That corrector is still under construction here and the Chinese have also begun to develop the idea. Our work has been impeded by lack of funds. In 1991 The Work of Albert Victor Crewe on the STEM 27 we invented a third corrector and this one seems to be simpler than the others and more likely to work. We believe that this general concept will lead to yet another corrector, this time for the SEM. Such a development would have significant applications in biology and also in the semiconductor industry. Finally, as we will see later, this whole problem area led us to a reassessment of the resolution problem and to general improvements in the microscope business that are still having ramifications today.

Theoretical Electron Optics (Reference Group J) Throughout this whole period we have been interested in the theoretical aspects of electron optics and although this is not our main activity, it has often been necessary to extend the field in order for us to proceed with the experimental work. As one example, we have performed countless calculations of the properties of magnetic lenses. The vast majority of these calculations have never been published because it was felt that they had no general interest to the community. Nevertheless, these calculations have given us a unique insight into the properties of lenses and that, in turn, has enabled us to design lenses for particular purposes with far more confidence than we could have done otherwise. It may be noted that lens design is as much an art as it is a science. It is surely possible to calculate the focal properties of any lens that one cares to specify. The problem is how to specify the lens in the first place. Only by having the experience can one guess at the most appropriate design for a particular purpose. In other words, one gets to know where not to look. Our interest in the problem of aberrations has also forced us into theoretical electron optics. Not only were we motivated to develop the theory for our correctors, but it was also necessary to investigate very carefully the foundations of Scherzer’s theorem. This led to some interesting, if academic, conclusions that had not been noticed previously. In fact, we were able to give convincing proof that zero aberrations could only be obtained with magnetic fields that have pathological parameters and cannot be made in practice. We have described some of our other efforts in theory above, but we can add here a brief description of our latest efforts. Over the course of twenty-five years or so, we have developed a set of what we might call ‘‘rules of thumb’’ for the design of lenses and electron guns. These rules seemed to work, but were not based on any known theory. We set out to develop that theory about two years ago. In a series of three papers we took a close look at the focal properties of weak lenses, both electrostatic and magnetic. We developed a theory and performed some careful calculations to verify the results. The calculations were not easy to perform for the simple reason that we were looking at the extreme 28 A. V. Crewe case of very weak lenses and this takes an inordinate amount of computer time. Fortunately, we have our own mainframe computer so that we could afford the time. The results were extremely interesting and have now been published. This allows anyone to obtain approximate values for the focal length and the aberration coefficients of both types of lens. These approximations are good enough that they also work even for stronger lenses. The most recent theoretical work was described above, namely the study of the motion of electrons in uniform fields. These calculations were described in the section on aberration correction. It might be suspected that this type of field had been thoroughly investigated in the past since it is an obvious problem. Indeed, this is true except for the fact that these were very early investigations by the pioneers in the field and the algebra was done long ago, long before the question of aberrations had been raised and before they had been well defined. Since we could not find an adequate treatment of the problem in the literature, it seemed worthwhile to write down the equations and solve them. We looked at four separate configurations of magnetic and electrostatic fields and obtained the solutions to the equations of motion. The interesting thing was that we found that two of the configurations had aberrations of the opposite sign to the normal ones. This raised several issues and suggested several applications of the ideas. Two of the applications are in the area of high-resolution microscopy and will be the subject of our renewal proposal. Other possibilities include improved mass spectromenters and improvements to ion microscopes.

Optimization and the Super-High-Resolution SEM (Reference Group K) The work that we did on the optimization of the parameters of scanning microscopes bears some discussion here. A few years ago it became apparent that the resolution of the SEM was stuck at about 15 A˚ . The microscopes from all the manufacturers had the same resolution and it appeared that nothing could be done about it. Many investigators were concerned with this issue and our involvement in it was motivated by a suggestion by a distinguished colleague that the only way to obtain better resolution would be to design and build a 200-kV SEM. This was obviously bad physics because it completely ignored the non-local nature of the production of secondary electrons. It should be noted that it may be unfair to criticize that colleague since we had considerably more experience with this problem. Not only hadwedonesomeresearchintheareaofsecondaryelectronproduction (see the discussion above), but we had some earlier experience with the The Work of Albert Victor Crewe on the STEM 29 theory of cloud chambers and bubble chambers and other detectors in high-energy physics. The general concept of non-local inelastic scattering is common knowledge in those areas, but not in microscopy, although any graduate student in physics ought to have some familiarity with the concept. In any case, it seemed important to perform an in-depth study of the physics of the resolution problem. We had already begun such a study for the STEM and it was a natural extension to consider the SEM. With regard to the STEM, the factors to be studied included spherical aberration, and diffraction. In our case, since we were considering the possibility of correcting the spherical aberration, the particular focus was on the effect of the fifth-order aperture aberration. These calculations were done using wave-optical analysis and represented the best that could be done with the problem. We had hoped that this study would put to rest forever the gross approximation to the resolution problem that occurred often in the literature, namely taking the r.m.s. of all these effects, an operation that is akin to taking the average of an apple, an orange, and a banana. Unfortunately, that statement still appears and we have given up correcting the authors. In the case of the SEM, which operates at lower voltages and where the contrast mechanism uses inelastic scattering, we must take into account the fact that these collisions can occur at large distances from the electrons. That is to say, the classical impact parameter can be several A˚ . Fortunately, we were well aware of this problem in microscopy since it is this fact that limits the use of inelastic scattering for high resolution images in the STEM. We have often demonstrated this by taking simultaneous images of atoms using elastic and inelastic electrons. At 35 kV, the effect is very obvious and we had already published the data. It was then only necessary to extend the argument to the case of the SEM. The conclusions of this study were that with proper optimization of the aperture in the SEM, using wave-optical calculations instead of gross averaging, the resolution should improve to about 5–7 A˚ , and that the best operating voltage would be about 5 kV. The only manufacturer who listened to these arguments and did something about it was Hitachi, although all of them had access to the information. Hitachi designed a special instrument, the 900 series, and it worked. Overnight, the best resolution of the SEM went from 15 A˚ to 7 A˚ . It took several years before the other manufacturers caught up with Hitachi, but today virtually all of them offer such an instrument, with the exception of the one U.S. manufacturer (Amray). We believe that this one contribution would justify the whole cost of our research support. 30 A. V. Crewe

Image Processing (Reference Group L) As a result of the measurements that we made on the radiation sensitivity of biological materials, we began to investigate the imaging properties of our microscope. The conventional electron microscope was very well suited for the study of molecules that could be crystallized into a regular array. This was the technique that was used by Unwin and Henderson in their study of the purple membrane. They were able to reconstruct the molecule from the information in the diffraction pattern, information that came from many different molecules. By using many different molecules to provide an image of just one, they could minimize the effects of radiation damage. We could not do this with the same degree of efficiency and therefore we would tend to produce more damage in the specimen. The reverse side of the coin is that the conventional microscope is not very good for taking images of molecules that cannot be formed into a regular array. Many molecules are such that they can only be imaged one at a time. On the other hand, the STEM can provide very good images of isolated molecules and the contrast is so good that excellent images can be obtained with far less dose than in the conventional TEM. We therefore began a program of imaging molecules and developing digital image processing techniques to take advantage of these capabilities. The first work in this area was by Golladay, who studied negatively stained myosin. By careful digital analysis of the image, working line by line and doing very careful averaging, he was able to locate the uranyl atoms with considerable precision. In doing so he introduced us to the general idea of digital image processing. Partially because of this success, we began to develop a more complete processing system using a Nova computer with the vast (for then) memory of 32 k. In order to avoid the repetitious work of programming, we developed an interactive image processing system where most of the operations could be done using a trackball and cursor. Later this capability was transferred to the IBM mainframe when we acquired one. We used the same philosophy and we have continued to add to its capabilities. The net result is a very powerful image processing system that can be operated by inexperienced operators. Virtually everything can be done without the need to do any programming at all. More recently, this program has been rewritten and transferred to a workstation environment since the capabilities of these devices are now at least as good as our original mainframe. The use of this system will be described in subsequent sections. The Work of Albert Victor Crewe on the STEM 31

Three-Dimensional Reconstruction (Reference Group M) The hemoglobin program in this laboratory initially concentrated on the use of low-dose images because this opened up several new possibilities. Specifically, by adding many such images together we could produce one image with good signal to noise ratio without sustaining the commensu- rate amount of damage. In this way we could produce the same quality of image that can be obtained from a conventional microscope when the specimen can be formed into two-dimensional ordered arrays. Unfortunately, this cannot be done with hemoglobin but our low-dose method is potentially just as good and the STEM can operate with lower doses than a conventional microscope. The images that were obtained by this method were very good indeed and furthermore, we were able to obtain images in three different projections, corresponding to three views taken 45 degrees apart. It was therefore natural to think of performing some kind of reconstruction in order to look at the molecule in 3D. Unfortunately, there was no existing algorithm to do this and there were many indications that it could not be done at all. All the algorithms for 3D reconstruction required many different views and indeed, required them to be taken at regular intervals. We simply could not do this; we had only three views but the images were of high quality. In considering this problem it seemed possible that other investigators had ignored some factors and that they were using other methods that had inherent inconsistencies. First of all, the only method that we could find that seemed to apply to our specimen was a form of algebraic reconstruction. In this method the first step is to add the three projections together into a 3D array. This step appeared to be wrong and indeed we felt that it could conceivably introduce significant errors. The reason for the suspicion is that any reasonable operation on the data ought to at least retain zeros. That is to say, if there is no mass apparent at a particular location in the image, then this is a valuable piece of information that must be maintained. The algebraic method does not do this. That first step can introduce mass into one of the projections from one of the others. We preferred to think of the problem in terms of probabilities. If a certain mass density is seen at a particular location in one of the projections, that mass has an equal probability of being found at any of the locations along the line of sight. The characteristic of probabilities is that they must be multiplied together, not added. Therefore our first step consists of multiplying the three projections together in order to find the starting point for the reconstruction. This automatically retains all zeros. The point that appeared to have been missed is that the biological material that we are using in the microscope has a uniform density. 32 A. V. Crewe

If there is any material at all at a particular point, then it has everywhere the same density. There either is or is not some material at a lattice point. The problem is then a Boolean one. We are looking for ones and zeros, with nothing in between. Given these two facts, our reconstruction problem reduces to utter simplicity. We multiply the probabilities in order to obtain a starting 3D matrix. We then take the maximum value and assume that there is indeed a one at that point. The value that existed at that point is then replaced with a one and the matrix is recalculated. Once again, the maximum value is replaced with a one and so on until the total mass discovered equals the total mass of the original object. Surprisingly, the method worked, although the first run took a great deal of computer time. Subsequently the method has been refined in order to increase the speed. The reconstructions obtained by this method and the various refinements that have been added since are the best that have ever been obtained of this molecule and the data matches that obtained subsequently elsewhere. All the evidence that we have indicates that this method does produce an accurate reconstruction.

Hemoglobin Work (Reference Group N) We have had a program of study into the structure and function of the invertebrate hemoglobins that has extented over the past thirteen years. The invertebrate hemoglobins represent a very broad range of respiratory proteins both in terms of structure and function. Their morphology can vary from single myoglobin-sized subunits to aggregates of several million molecular weight. In addition, their functional characteristics are complex and represent a variety of solutions to the need to deliver oxygen to tissue under profoundly different environmental conditions such as deep sea hydrothermal vents and oxygen-depleted polluted rivers. As a result of this complexity they represent attractive candidates for the study of structure-function relationships. Our primary approach has been to modify the protein using biochemical techniques, observe the attendant functional modifications, and relate this to the observed structure in the STEM. This work began when a graduate student of Serge Vinogradov (O. Kapp) traveled to our laboratory in the Fall of 1979 seeking STEM examination of his hemoglobin preparations. Kapp learned the use of the STEM from our then chief technician, Mitsuo Ohtsuki. Ultimately Kapp’s thesis included a large amount of work done in this laboratory. Following his thesis defense (Spring 1982) he joined our group as a research associate. When Dr. Ohtsuki returned to Japan in 1985, Kapp took over the operation of the microscopes and presently holds positions at the Enrico Fermi Institute and also an assistant professorship in the Biological Sciences Division. The Work of Albert Victor Crewe on the STEM 33

A particularly significant aspect of this work was the close working relationship of a biochemist with a top-notch electron microscope technician and physics-oriented graduate students. Generally, close proximity of the aforementioned groups is rare and the usual condition is one in which there are problems of communication due to everything from lack of physical proximity to differences in culture and language that are a natural part of the indoctrination of each discipline. Our research into 3D reconstruction techniques was stimulated by the desire of Kapp to obtain 3D structural data from STEM images of his preparations. Three of our Ph.D. students worked on this topic and a substantial number of publications resulted. In addition to obtaining 3D reconstuctions of several invertebrate hemoglobins, we were able to obtain reconstructions of depleted preparations in which one or more subunits were missing. With this team we were able to correlate the results of biochemical separation techniques such as column chromato- grahy and Sodium Dodecyl Sulfate (SDS) electrophoresis with data from the STEM and subsequently obtained differential reconstructions which mapped the location of the missing subunits by comparison with the 3D map of the native molecules. One of the earliest observations we made by STEM of the invertebrate hemoglobins was the greater-than-expected dimensions obtained for intact, native dodecamers. The annelid extracellular hemoglobins are composed of a bilayer of hexameric structures forming a complex with a diameter of about 300 A˚ and a height of about 200 A˚ . All previous reports of this group of proteins pointed towards dimensions of about 250 160 A˚ . Because the STEM has no post-specimen optics and images are taken in-focus our dimensions are likely to be more reliable because of the absence of phase contrast reversal effects at an unknown resolution limit. Our dimensions were corroborated by the dimensions obtained for wet crystals of Lumbricus hemoglobin by Prof. Warner Love. This along with other observations led us to believe that STEM of negatively stained specimens of these proteins provided useful and meaningful results. One of the first experiments we performed in the STEM was the examination of the alkaline dissociation products obtained by exposure of Lumbricus hemoglobin to pH 9 for 24 hours followed by column chromatography to separate the various size classes. In addition, these fractions were combined and re-exposed to neutral pH to generate a reassociated molecule. This observation led to experiments in which we would remove various subunits and observe the subsequent effect on the reassociation process. Subtle changes in the morphology of the reassociated molecule could be observed using false-color display. For example, a 5% difference in scattering cross-section of the 1/12th subunit, a difference not visible in grey-level display, was easily discerned using a color 34 A. V. Crewe palette of just 256 colors. We demonstrated that these subtle structural alterations were reproducible and were associated with dramatic changes in the functional characteristics of the molecule. For instance, the intact, native hemoglobin of Lumbricus displays a Hill coefficient n of about 4.3 at neutral pH. The reassociated molecule, however, provided values of about 2.3, the same value obtained for the putative 1/12th subunit. We also observed the dissociation-reassociation process described above for hemoglobin preparations that had been exposed to acid pH and then returned to neutral pH. The same functional alterations in the molecule were also observed. This work then led to the examination of preparations in which one or more subunits were removed before reassociation. One of the most striking results obtained with the STEM was the observation of hexagonal bilayers in preparations of Lumbricus hemoglobin in which the smallest heme-containing subunit (by SDS–PAGE) was completely absent. The location of this subunit was approximated by subtracting the 3D reconstruction of the native hemoglobin from the 3D reconstruction of the depleted preparation. The central hole in the molecule was nearly identical in size in the two preparations and it was thus assumed that the depletion about the outer periphery of the reassociated molecule was due to the absence of this particular subunit. Our research into 3D reconstruction techniques was stimulated by a desire to obtain 3D structural data from STEM images of various biological specimens. Three of our Ph.D. students worked on this topic and a substantial number of publications resulted. In addition to obtaining 3D reconstuctions of several invertebrate hemoglobins, we were able to obtain reconstructions of depleted preparations (described above) in which one or more subunits were missing. With this team we were able to correlate the results of biochemical separation techniques such as column chromato- grahy and SDS electrophoresis with data from the STEM and other techniques such as neutron scattering and low-angle x-ray scattering. One of the problems encountered in the reconstruction of non-periodic objects is the determination of the relative orientation in which the biological macromolecule appears on the substrate. We looked at this problem using multivariate statistical techniques and developed algorithms for classification of molecules into subgroups according to orientation. This work began with classification of orientation about a single rotation axis and then was extended to consider all orientations (two axes, the third being the electron beam path). The thesis of one student, Salzman, was based on work in this area. A particularly striking discovery in the hemoglobin work was the demonstration of the presence of a ‘‘bracelet’’ protein which is implicated in maintaining the integrity of the quaternary structures of these molecules. These subunits, which contain no heme prosthetic groups, form a The Work of Albert Victor Crewe on the STEM 35 bracelet, or torroid, upon which the remaining subunits bind. The hexagonal bilayer appearance of these giant proteins has an absolute requirement for the presence of these ‘‘linker’’ subunits. STEM examinations of preparations depleted of this subunit showed no hexagonal bilayer structures in the general field of view. Several ancillary techniques were developed as a result of the presence of an IBM 4381 super-mini mainframe in the lab starting in the early 1980s. This essentially unlimited access to reasonable fast (by present standards) computational power allowed us the luxury of algorithm exploration on a scale that would have not been attempted if we were required to pay for compute cycles. One example has already been mentioned, and that is our development of 3D reconstruction algorithms for use with STEM projection image data. Another example is the development of a technique to compute the stoichiometry of a multisubunit protein using a linear equations approach. We were able to demonstrate that the subunit stoichiometry of a protein can be calculated if the amino acid analysis is known for each isolated subunit and also the intact molecule. Expressed as a series of linear equations (subunit coefficients on the left hand and the intact molecule analysis on the right hand of the equation), a least squares solution will provide values for this overdeter- mined set of equations (20 amino acids ¼ 20 equations, usually in four or less unknowns) which agree very closely with known values and provide reasonable stoichiometries for the unknown ratios of the invertebrate hemoglobins. This problem is not trivial because the hemoglobins

TABLE I Reference Groups (Papers and Abstracts in Bibliography)

A. (Construction) Papers 26,27,38,42,39 Abstracts 36,34 B. (Field Papers 41,31 Emission) Abstracts 32,35,51 C. (STEM of Papers 37,43,46,65,66,67,68,70,73,74,90,98,105, Atoms) 107,110,124,160,203,211,215,238, 248,254 Abstracts 33,49,50,53,55,56,59,61,62,75,76,77,80, 81,91,92,93,96,99,100,101,129,132, 146,134,142,163,166,167,179,182,186, 193,197,217,244,252,260,261,263,265 D. (SEM) Papers 54,57,69,71,72,97 Abstracts 44,52,58,60,78,64,176 E. (Energy Loss) Papers 87,89,106,108,150,156,159,162,245

(continued) 36 A. V. Crewe

TABLE I (continued)

Abstracts 84,85,102,103,115,116,120,175,177,178, 180,191,195,144,171 F. (Secondaries) Papers 206 Abstracts 138 G. (DNA) Papers - Abstracts 82,83,104,133 H. (Nucleosomes) Papers - Abstracts 154,185,233

I. (Cs) Papers 198,286,302,319,280,367,372,373, 503,504 Abstracts 45,95,137,181,187,192,196,285,298,201 J. (Theory) Papers 208,209,214,221,239,272,279,283,287, 384,420,422,423,425,430, 431,432,437, 439,461 Abstracts 398,417,418,419,459,460 K. (Optimization) Papers 282,325,353,464 Abstracts 291,296,297,306 L. (Image Papers 308,322,448 Processing) Abstracts 313,294,316,320,326,329,365,381,385 M. (3D Papers 207,346,347,357,371,375,392,393,304, Reconstruction) 396,429,462,473,474 Abstracts 113,355,356,358,379,400,403 N. (Hemoglobin Papers 321,327,337,338,339,347,348,349,357, Work) 363,370,377,382,383,392,393,394, 396,404,411,421,427,429,443,444,466, 467,468,473,474,488,389,390,491,494, 495,498,503 Abstracts 300,307,312,314,318,324,334,356,358, 359,360,361,362,364,365,378,379,380, 381,386,387,395,413,414,415,426, 440,441,442,445,446,457,458,459,463, 464,465,479,480,481,482, 483,484,496,497

of many annelid hemoglobins contain approximately 200 copies of anywhere from 3 to 7 different subunits. Over the past several years we have been recognized as leaders in the field of invertebrate hemoglobin structure and function. In the past year The Work of Albert Victor Crewe on the STEM 37

Vinogradov and Kapp were the editors of a volume of papers that were the results of a meeting on the subject of the structure and function of the invertebrate respiratory proteins. In work closely allied to the study of protein structures we have developed a fairly extensive library of subroutines for examination of protein crystal structure and sequence alignment. Specifically, we rewrote the Needleman and Wunsch dynamic programming technique from FORTRAN into APL so that we could easily modify the interpreted code ‘‘on the fly.’’ This work has allowed us to more closely relate the results of STEM examination of biological macromolecules with the growing list of crystal structures contained in the Brookhaven database. Recently we have begun to simulate STEM images using the Brookhaven coordinates in an attempt to obtain a better understanding of radiation damage, orientation classification, and substrate effects in experimental STEM images.

REFERENCES (OF CHICAGO ABERRATIONS)

Beck, V. (1977). Aberration correction in the STEM. Optik 53, 241–255. Chen, E. G., and Mu, C. J. (1990). In ‘‘Proceedings of the International Symposium on Electron Microscopy, Beijing, China,’’ (Kuo, K. and Yao, J., eds.), p. 28–35. World Scientific, Singapore. Crewe, A. V., Cohen, D., and Meads, P. (1968). A multipole element for the correction of spherical aberration. Fourth European Conference on Electron Microscopy, p. 183 Rome. Crewe, A. V. (1980). The sextupole as corrector. Electron Microscopy 1, 36–37. Crewe, A. V. (1982). A system for the correction of axial aperture. Aberrations in electron lenses. Optik 60, 271–281. Crewe, A. V. (1992a). Electron motion in tuned fields, I. The algebra. Ultramicroscopy 41, 269–277. Crewe, A. V. (1992b). Electron motion in tuned fields, II. Some applications. Ultramicroscopy 41, 279–285. Crewe, A. V., and Kopf, D. A. (1980a). Limitations of sextupole correctors. Optik 56, 391–399. Crewe, A. V., and Kopf, K. (1980). A sextupole system for the correction of spherical aberration. Optik 55, 1–10. Crewe, A. V., and Parker, N. W. (1976). Correction of third-order aberrations in the scanning electron microscope. Optik 46(2), 183–194. Crewe, A. V., and Beck, V. (1974). A quadrupole-octupole corrector for a 100 KeV STEM. Proc. 32nd Ann. EMSA Mtg. pp. 426–427. Crewe, A. V., and Jiye, X. (1985). Correction of spherical and coma aberrations with a sextupole-round lens-sextupole system. Optik 69, 141–146. Crewe, A. V., and Salzman, D. A. (1982). On the optimum resolution of a corrected STEM. Ultramicroscopy 9, 373–378. Crewe, A. V., Ruan, S., Korda, P., and Tsai, F. (2000). Studies of a magnetically focused electrostatic mirror, I. Experimental test of first order properties. J. Microscopy 197, 110–117. Deltrap, J. H. M. (1964). ‘‘Correction of spherical aberration of electron lenses.’’ Thesis, Cambridge University, Pembroke College. 38 A. V. Crewe

Haine, M. E. (1961). ‘‘The Electron Microscope, The Present State of the Art.’’ p. 58. Inter- science, New York. Jiye, X., Shao, Z., and Crewe, A. V. (1985). The wave electron optical properties of a magnetic round lens corrected with sextupoles. Optik 70, 37–42. Koops, H. (1978). Erprobung eines chromatisch korrigierten elektronenmikroskopischen Objektives. Optik 52, 1–18. Parker, N. W., Golladay, S. D., and Crewe, A. V. (1976). A theoretical analysis of third-order aberration correction in the SEM and STEM. Proc. Scanning Electron Microscope Sympo- sium. IIT Research Institute, pp. 24–25. Scherzer, O. (1936). Some defects of electron lenses. Z. Physik 101, 593–603. Scherzer, O. (1947). Spha¨rische und chromatische Korrektur von Elektronen-Linsen. Optik 2, 114–132. Scherzer, O. (1978). Limitations for the resolving power of electron microscopes. Proc. 9th Int. Conf. Electron Microscopy Vol. III, pp. 123–129. Septier, A. (1966). The struggle to overcome spherical aberration in electron optics. Advances in Optical and Electron Microscopy 1, 204–274. Shao, Z., and Crewe, A. V. (1987). Spherical aberrations of multipoles. J. Appl. Physics 62, 1149–1153. Tsai, F. (2000). Studies of a magnetically focused electrostatic mirror, II. Aberration correction. J. Microscopy 197, 118–135. Zworykin, V. K., Morton, G. A., Ramberg, E. G., Hillier, J., and Vance, A. W. (1945). ‘‘Electron Optics and the Electron Microscope.’’ John Wiley & Sons, New York.

REFERENCES (OF DOE REPORT) 1951–59

1. Crewe, A. V., and Litherland, A. E. (1951). The Effects of Geometry on Scattering Distributions in the Wilson Cloud Chamber. J. Sci. Inst. 28, 182. 2. Crewe, A. V. (1951). The Multiple Scattering of m Mesons. Proc. Phys. Soc. 64, 660. 3. Crewe, A. V., and Evans, W. H. (1952). The Diffusion Cloud Chamber. Atomics 3, 221. 4. Crewe, A. V., and Ashmore, A. (1953). The Multiple Scattering of 7.5 Mev Deuterons in Metals. Proc. Phys. Soc. 66, 1172. 5. Crewe, A. V., Alston, M. H., Evans, W. H., Green, L. L., and Wilmott, J. G. (1954). The Scattering of 17.5 Mev Neutrons in Helium. Proc. Phys. Soc. 67, 657. 6. Crewe, A. V., Alston, H. M., and Evans, W. H. (1954). A Stereoscopic Reprojection Apparatus for Neutron Scattering Experiments. J. Sci. Inst. 31, 252. 7. Crewe, A. V., Alston, M. H., and Evans, W. H. (1954). Some Practical Aspects of Diffusion Cloud-Chamber Operation. Rev. Sci. Inst. 25, 547. 8. Crewe, A. V., and LeCouteur, K. J. (1955). The Extracted Proton Beam of the Liverpool Synchrocyclotron. Rev. Sci. Inst. 26, 725. 9. Crewe, A. V. (1955). Beam Extraction from the Liverpool Synchrocyclotron. Proc. Roy. Soc. 232, 242. 10. Crewe, A. V., and Kruse, U. E. (1956). Regenerative Beam Extraction on the Chicago Synchrocyclotron. Rev. Sci. Inst. 27, 5. 11. Crewe, A. V. (1956). Pion Production in the Pion-Nucleon Collisions at 240 Mev. Bull. Amer. Phys. Soc. 1, 276. 12. Crewe, A. V. (1956). Beam Extraction on Weak Focusing Machines. Proc. CERN Symposium 1, 141. 13. Crewe, A. V., Alston, M. H., Evans, W. H., and von Gierke, F. (1956). Positive Pion Production in Proton-Proton Collisions at 383 Mev. Proc. CERN Symposium 1, 334. The Work of Albert Victor Crewe on the STEM 39

14. Crewe, A. V., Alston, M. H., Evans, W. H., and von Gierke, F. (1956). A Study of Positive Pion Production in Proton-Proton Collision at 383 Mev. Proc. Phys. Soc. 69. 15. Crewe, A. V., and Cohen, Stanley (1957). Regenerative Action in High Energy Accelerators. Nuclear Instruments 1, 31. 16. Crewe, A. V., Kruse, U. E., Miller, R. H., and Pondrom, L. G. (1957). Search for Asymmetries in the Scattering and Decay of Pions. Phys. Rev. 108, 1531. þ 17. Crewe, A. V., Pondrom, L. G., and Kruse, U. E. (1958). p Production in Hydrogen by 450 Mev Protons. Bull. Amer. Phys. Soc. 3, 196. 18. Crewe, A. V. (1958). Magnetic Spectrometer for 1 Bev/c Momentum. Bull. Amer. Phys. Soc. 3, 333. 19. Crewe, A. V. (1958). Magnetic Spectrometer for 450 Mev Protons. Rev. Sci. Inst. 29, 880–884. 20. Crewe, A. V., Garwin, E., Ledley, B., Lillethun, E., March, R., and Marcowitz, S. (1959). þ Charge Independence in the Reactions r þ d ! p0 þ He3 and r þ d ! p þ H3 at 450 Mev. Phys. Rev. Letters 2, 269–270. 21. Crewe, A. V., Ledley, B., Lillethun, E., Marcowitz, S., and Pondrom, L. G. (1959). Elastic Proton-Deuteron Scattering at 450 Mev. Phys. Rev. 114, 1361–1365. 22. Crewe, A. V. (1959). Notes on External Particle Beams from the Argonne 12.5 Bev Synchrotron. Proc. Int’l Conf. on High-Energy Accelerators and Instrumentation CERN. 23. Crewe, A. V. (1959). The Argonne Zero Gradient Synchrotron (ZGS). Proc. Int’l Conf. on High-Energy Accelerators and Instrumentation CERN. 1960–65

24. Crewe, A. V., Ledley, B., Lillethun, E., Marcowitz, S., and Rey, C. (1960). Charge þ Independence in the Reactions r þ d! p0 þ He3 and r þ d ! p þ H3 at 450 Mev. Phys. Rev. 118, 1091. 25. Nambu, Y., and Jona-Lasinio, G. (1961). Dynamical Model of Elementary Particles Based on an Analogy with Superconductivity. II. Phys. Rev. 124, 246–254. 26. Crewe, A. V. (1963). A New Kind of Scanning Microscope. Journal de Microscopie 2, 369. 27. Crewe, A. V. (1964). Scanning Techniques for High Voltage Microscopes. Proc. AMU- ANL High Voltage Electron Microscope Mtg. Argonne National Laboratory, pp. 68–81. 28. Crewe, A. V., and Katz, J. J. (1964). ‘‘Nuclear Research USA.’’ Dover Publishers Inc, New York. 29. Crewe, A. V., and Katz, J. J. (1964). ‘‘Research USA.’’ McGraw Hill Book Co, New York. 30. Crewe, A. V., George, R. D., Ratner, L. G., and Teng, L. C. (1965). Experimental Area Beams from the ANL 12.5 Bev Proton Synchrotron. Bull. Amer. Phys. Soc. 10, 654. 31. Crewe, A. V. (1965). Some Effects of Quadrupole Misalignments in a Microscope. ‘‘AMU-ANL High Voltage Electron Microscope Newsletter, July 6.’’ 1966–69

32. Butler, J. W. (1966). Digital Computer Techniques in Electron Microscopy. Proc. 6th Int’l Congress for Electron Microscopy. Kyoto, p. 191. 33. Crewe, A. V. (1966). Energy Loss Techniques in a Scanning Microscope. Proc. 6th Int’l Congress for Electron Microscopy. Kyoto, p. 635. 34. Crewe, A. V. (1966). Experiments with Quadrupole Lenses in a Scanning Microscope. Proc. 6th Int’l Congress for Electron Microscopy. Kyoto, p. 627. 35. Crewe, A. V. (1966). Experiments with a Field Emission Electron Gun. Proc. 6th Int’l Congress for Electron Microscopy. Kyoto, p. 629. 36. Crewe, A. V. (1966). A Transmission Scanning Microscope. Proc. 6th Int’l Congress for Electron Microscopy. Kyoto, p. 631. 37. Crewe, A. V. (1966). Scanning Electron Microscopes: Is High Resolution Possible? Science 154, 729–738. 40 A. V. Crewe

38. Crewe, A. V., Butler, J. W., and Welter, L. M. (1967). A Computer-Controlled Electron Microscope System. Proc. Roy. Microsc. Soc. MICRO 67. 39. Crewe, A. V., Eggenberger, D. N., Welter, L. M., and Wall, J. (1967). Experiments with Quadrupole Lenses in a Scanning Microscope. J. Appl. Phys. 38, 4257–4266. 40. Crewe, A. V. (1967). Science and the War on .... Physics Today 20, 25–30. 41. Crewe, A. V., Eggenberger, D. N., Wall, J., and Welter, L. M. (1968). Electron Gun Using a Field Emission Source. Rev. Sci. Inst. 39, 576–583. 42. Lewis, R. N., Jung, E. A., Welter, L. M., Van Loon, L. S., and Chapman, G. L. (1968). Flexible High Voltage Supply for Experimental Electron Microscope. Rev. Sci. Instr. 39, 1522–1526. 43. Crewe, A. V., Wall, J., and Welter, L. M. (1968). A High Performance Transmission Scanning Microscope. Fourth European Regional Conference on Electron Microscopy. Rome, p. 7. 44. Crewe, A. V., Isaacson, M., and Johnson, D. (1968). A Simple Scanning Microscope of Medium Performance. Fourth European Regional Conference on Electron Microscopy. Rome, p. 75. 45. Crewe, A. V., Cohen, D., and Meads, P. (1968). A Multipole Element for the Correcting of Spherical Aberration. Fourth European Regional Conference on Electron Microscopy. Rome, p. 183. 46. Crewe, A. V., Wall, J., and Welter, L. M. (1968). A High-Resolution Scanning Transmis- sion Electron Microscope. J. Appl. Phys. 39, 5861–5868. 47. Welter, L. M., Stroud, A. N., and Resh, D. L. (1968). Scanning Energy Analyzing Microscopy of Chromosomes. Biophys. Soc. Abstracts, 12th Ann. Mtg. 48. MacKenzie, A. P. (1968). Freeze-Dehydration for the Scanning Electron Microscope. Biophys. Soc. Abstracts, 12th Ann. Mtg. p. A190. 49. Crewe, A. V., Wall, J., and Welter, L. M. (1968). A High Resolution Scanning Microscope. Proc. 26th Ann. EMSA Mtg. 50. Crewe, A. V. (1968). The Potentials of Scanning Microscopy. Proc. 26th Ann. EMSA Mtg. p. 352. 51. Crewe, A. V., and Isaacson, M. (1968). The Use of Field Emission Tips in a Scanning Microscope. Proc. 26th Ann. EMSA Mtg. 52. Crewe, A. V., Johnson, D., and Isaacson, M. (1968). An Electron Gun Scanning Microscope. Proc. 26th Ann. EMSA Mtg. 53. Thomson, M. G. R., and Crewe, A. V. (1968). The Possibility of Improving Scanning Microscope Images by After-Treatment. Proc. 26th Ann. EMSA Mtg. 54. Crewe, A. V., Isaacson, M., and Johnson, D. (1969). A Simple Scanning Electron Microscope. Rev. Sci. Inst. 40, 241–246. 55. Crewe, A. V. (1969). High Resolution Scanning Electron Microscopy. Proc. 2nd Ann. Scanning Microscope Symposium. IIT Research Institute, p. 11. 56. Crewe, A. V. (1969). Closing the Gap—A 5A˚ Scanning Microscope. Proc. 27th Ann. EMSA Mtg. p. 6. 57. Crewe, A. V., Isaacson, M., and Johnson, D. (1969). A High Performance Energy Analyzer for Use in Electron Scanning Microscopy. Proc. 27th Ann. EMSA Mtg. p. 14. 58. Crewe, A. V., Isaacson, M., and Johnson, D. (1969). Secondary Electron Detection in a Field Emission Scanning Microscope. Proc. 27th Ann. EMSA Mtg. p. 16. 59. Crewe, A. V., and Wall, J. (1969). Scanning Microscopy of Thin Biological Specimens. Proc. 27th Ann. EMSA Mtg. p. 48. 60. Johnson, D., and Morieraty, P. (1969). Examination of Schistosome Cercariae and Schis- tosomules by Scanning Electron Microscopy. Proc. 27th Ann. EMSA Mtg. p. 50. 61. Zeitler, E., and Thomson, M. G. R. (1969). The Theoretical Contrast in Scanning and Conventional High Resolution Microscopes. Proc. 27th Ann. EMSA Mtg. p. 170. The Work of Albert Victor Crewe on the STEM 41

62. Crewe, A. V., and Wall, J. (1969). High Resolution Scanning Microscopy. Proc. 27th Ann. EMSA Mtg. p. 172. 63. Stroud, A. N., Welter, L. M., Resh, D. A., Habeck, D. A., Crewe, A. V., and Wall, J. (1969). Scanning Electron Microscopy of Cells. Science 164, 830–832. 1970

64. Smith, J. V., Anderson, A. T., Newton, R. C., Olsen, E. J., Wyllie, P. J., Crewe, A. V., Isaacson, M. S., and Johnson, D. (1970). Petrologic History of the Moon Inferred from Petrography, Mineralogy and Petrogenesis of Apollo 11 Rocks. Proc. Apollo 11 Lunar Sci. Conf. 1, 897–925. 65. Crewe, A. V. (1970). High Resolution Scanning Microscopy of Biological Specimens. Berichte der Bunsen-Gesell. fur Phys. Chem. 74, 1181–1187. 66. Zeitler, E., and Thomson, M. G. R. (1970). Scanning Transmission Electron Microscopy. I. Optik 31, 258–280. 67. Zeitler, E., and Thomson, M. G. R. (1970). Scanning Transmission Electron Microscopy. II. Optik 31, 359–366. 68. Crewe, A. V., and Wall, J. (1970). Contrast in a High Resolution Scanning Electron Microscope. Optik 30, 461–474. 69. Crewe, A. V., Isaacson, M., and Johnson, D. (1970). Secondary Electron Detection in a Field Emission Scanning Microscope. Rev. Sci. Inst. 41, 20–24. 70. Crewe, A. V. (1970). The Current State of High Resolution Scanning Electron Micros- copy. Quarterly Reviews of Biophysics 3, 1–39. 71. Anderson, A. T., Crewe, A. V., Goldsmith, J. R., Moore, P. B., Newton, J. C., Olsen, E. J., Smith, J. V., and Wyllie, P. J. (1970). Petrologic History of the Moon Suggested by Petrography, Mineralogy and Crystallography. Science 167, 587. 72. Crewe, A. V., Isaacson, M., Johnson, D., and Smith, J. V. (1970). Scanning Electron Microscopy of Lunar Spherules. Geochimica et Cosmochimica Acta 34. 73. Crewe, A. V. (1970). Field Emission and an Electron Gun, Microscope Design Using Field Emission and Contrast Mechanisms in a High Resolution Scanning Microscope. ‘‘Lectures presented at Centre for Scientific Culture.’’ Erice, Sicily. 74. Crewe, A. V., Wall, J., and Langmore, J. (1970). Visibility of Single Atoms. Science 168, 1338–1340. 75. Crewe, A. V., Langmore, J., and Wall, J. (1970). High Resolution Capabilities in Scan- ning Microscopy. Proc. 7th Int’l Congress Electron Microscopy 1. Grenoble. 76. Crewe, A. V., and Wall, J. (1970). Contrast Methods in a High Resolution Scanning Microscope—the Use of Multiple Detectors. Proc. 7th Int’l Congress Electron Microscopy 1, 35. Grenoble. 77. Thompson, M. G. R., and Zeitler, E. (1970). Phase Contrast in the Scanning Transmission Electron Microscope. Proc. 7th Int’l Congress Electron Microscopy 1, 63. Grenoble. 78. Crewe, A. V., Isaacson, M., and Johnson, D. (1970). A Simple Multi-Purpose Scanning Microscope. Proc. 7th Int’l Congress Electron Microscopy 1, 209–210. Grenoble. 79. Crewe, A. V., Wall, J., and Langmore, J. (1970). High Resolution Scanning Electron Microscopy of Nucleic Acid Salts. Proc. 7th Int’l Congress Electron Microscopy 1, 467–468. Grenoble. 80. Crewe, A. V., Wall, J., and Langmore, J. (1970). Single Atom Visibility. Proc. 7th Int’l Congress Electron Microscopy 1, 485–486. Grenoble. 81. Crewe, A. V., Langmore, J., Wall, J., and Beer, M. (1970). Single Atom Contrast in a Scanning Microscope. Proc. 28th Ann. EMSA Mtg. pp. 250–251. 82. Wall, J., and Langmore, J. (1970). Studies of DNA Using a 5A˚ Scanning Microscope. Proc. 28th Ann. EMSA Mtg. pp. 252–253. 83. Langmore, J., and Wall, J. (1970). Conformational Study of tRNA, Using the High Resolution Scanning Electron Microscope. Proc. 28th Ann. EMSA Mtg. pp. 254–255. 42 A. V. Crewe

84. Crewe, A. V., Isaacson, M., and Johnson, D. (1970). Electron Beam Damage in Biological Molecules. Proc. 28th Ann. EMSA Mtg. pp. 264–265. 85. Crewe, A. V., Isaacson, M., and Johnson, D. (1970). The Energy Loss of 20 KEV Electrons in Biological Molecules. Proc. 28th Ann. EMSA Mtg. 86. Crewe, A. V., and Saxon, J. (1970). Interference Experiments with a Field Emission Source. Proc. 28th Ann. EMSA Mtg. 1971

87. Crewe, A. V., Isaacson, M., and Johnson, D. (1971). Electron Energy Loss Spectra of the Nucleic Acid Bases. Nature 231, 262–263. 88. Crewe, A. V. (1971). High Resolution Scanning Microscopy of Biological Specimens. Phil. Trans. Roy. Soc. 261, 61–70. London. 89. Crewe, A. V., Isaacson, M., and Johnson, D. (1971). A High Resolution Electron Spectrometer for Use in Transmission Scanning Electron Microscopy. Rev. Sci. Inst. 42, 411–420. 90. Crewe, A. V. (1971). A High Resolution Scanning Electron Microscope. Scientific American 224, 26–35. 91. Zeitler, E. (1971). Scanning Transmission Electron Microscopy. Proc. 4th Ann. SEM Symposium. IIT Research Institute, pp. 25–31. 92. Crewe, A. V. (1971). A 100 Kv Transmission Scanning Microscope. Proc. 29th Ann. EMSA Mtg. 93. Crewe, A. V., and Wall, J. (1971). Quantitative Analysis in High Resolution Transmis- sion Scanning Microscopy. Proc. 29th Ann. EMSA Mtg. 94. Crewe, A. V., and Beck, V. (1971). A New Color Display System for Scanning Transmis- sion Microscopes. Proc. 29th Ann. EMSA Mtg. 95. Crewe, A. V., and Saxon, J. (1971). Electron Holography and the Correction of Spherical Aberration. Proc. 29th Ann. EMSA Mtg. 96. Crewe, A. V. (1971). Production of Electron Probes a Few A˚ in Diameter. Proc. 7th Nat’l Conf. on Electron Probe Analysis. 97. Sheffield, J. B., Isaacson, M., and Johnson, D. (1971). Visualization of Surface Structures on Embryonic Cells with a Simple Electron Gun Scanning Microscope. Experimental Cell Research 64, 49–56. 98. Crewe, A. V. (1971). ‘‘High Intensity Electron Sources and Scanning Electron Microscopy’’,. Electron Microscopy in Material Science, pp. 162–207. Academic Press, New York. 1972

99. Crewe, A. V., and Retsky, M. (1972). Single Atom Visibility. Proc. 30th Ann. EMSA Mtg. 100. Crewe, A. V., and Zeitler, E. (1972). A 100 Kv High Resolution Scanning Transmission Microscope. Proc. 30th Ann. EMSA Mtg. 101. Crewe, A. V. (1972). Imaging of Single Atoms in Scanning Microscopy. Proc. 30th Ann. EMSA Mtg. 102. Crewe, A. V., Isaacson, M., and Johnson, D. (1972). Characteristic Energy Loss of Fast Electrons in Biological Materials. Biophys. Soc. Abstr. 16th Ann. Mtg. 48a. 103. Crewe, A. V., Isaacson, M., and Johnson, D. (1972). Implications of Electron Beam Damage in Electron Microscopy. Biophys. Soc. Abstr. 16th Ann. Mtg. 49a. 104. Crewe, A. V., Cozzarelli, N. R., Wall, J., and Langmore, J. P. (1972). Development of DNA Base-Specific Stains for Electron Microscopy. Biophys. Soc. Abstr. 16th Ann. Mtg. 52a. 1973

105. Crewe, A. V. (1973). Production of Electron Probes Using a Field Emission Source. In ‘‘Progress in Optics XI’’, (Wolf, E., ed.), pp. 223–246. Braunschweig, North Holland. The Work of Albert Victor Crewe on the STEM 43

106. Isaacson, M., Johnson, D., and Crewe, A. V. (1973). Electron Beam Excitation and Damage of Biological Molecules; Its Implications for Specimen Damage in Electron Microscopy. Rad. Res. 55, 205–224. 107. Langmore, J. P., and Wall, J. (1973). The Collection of Scattered Electrons in Dark Field Electron Microscopy. Optik 38, 335–350. 108. Crewe, A. V. (1973). Considerations of Specimen Damage for the Transmission Electron Microscope, Conventional Versus Scanning. J. Mol. Biol. 80, 315–325. 109. Johnson, D., and Isaacson, M. S. (1973). Cytosine Reflectance Measurements Using Electron Energy Loss Spectra and Synchrotron Radiation. Optics Communications 8, 406–408. 110. Wall, J. (1973). Development of the High Resolution Scanning Transmission Micro- scope. Scanning Electron Microscopy, pp. 158–163. 111. Langmore, J., Wall, J., Isaacson, M., and Crewe, A. V. (1973). Carbon Support Films for High Resolution Electron Microscopy. Proc. 31st Ann. EMSA Mtg. 76–77. 112. Wall, J., Langmore, J., Isaacson, M., and Crewe, A. V. (1973). Resolution Attainable with the Present Day STEM. Proc. 31st Ann. EMSA Mtg. pp. 230–231. 113. Isaacson, M., Langmore, J., Wall, J., and Crewe, A. V. (1973). Inelastic Scattering in Electron Microscopy. Proc. 31st Ann. EMSA Mtg. pp. 254–255. 114. Zeitler, E. (1973). Reconstruction of Objects from their Projections Using Orthogonal Functions. Proc. 31st Ann. EMSA Mtg. pp. 268–269. 115. Johnson, D. E., and Isaacson, M. (1973). Low Z Elemental Analysis Using Energy Loss Electrons in a Scanning Microscope. Proc. 31st Ann. EMSA Mtg. pp. 290–291. 116. Isaacson, M. (1973). Inelastic Scattering and Beam Damage of Biological Molecules. Proc. 31st Ann. EMSA Mtg. pp. 478–479. 117. Lamvik, M. K., Isaacson, M. S., and Crewe, A. V. (1973). Studies of Aggregates of Two Muscle Proteins with Scanning and Conventional Transmission Electron Microscopy. Proc. 31st Ann. EMSA Mtg. pp. 600–601. 118. Crewe, A. V., Langmore, J., Wall, J., and Isaacson, M. (1973). Scanning Transmission Electron Microscopy at High Resolution. Proc. 31st Ann. EMSA Mtg. 119. Beck, V. (1973). Slow Scan Display System for a Scanning Electron Microscope. Rev. Sci. Inst. 44, 1064–1066. 1974

120. Isaacson, M. (1974). Characteristic Energy Loss of Electrons in Biological Materials. Proc. 4th Int’l Conf. on YUV Phys. Hamburg. 121. Rose, H. (1974). Phase Contrast in Scanning Transmission Electron Microscopy. Optik 39, 416–436. 122. Wall, J., Isaacson, M., and Langmore, J. P. (1974). The Collection of Scattered Electrons in Dark Field Electron Microscopy. Optik 39, 359–374. 123. Roth, T. A. (1974). The Field Emitter: Electric Field Calculation. J. Applied Phys. 45, 4771–4773. 124. Crewe, A. V. (1974). Scanning Transmission Electron Microscopy. J. Microscopy 100, 247–259. 125. Crewe, A. V., and Groves, T. (1974). Thick Specimens in the CEM and STEM I. Contrast. J. Applied Phys. 45, 3662–3672. 126. Hainfeld, J., and Isaacson, M. (1974). Human Red Cell Studies Using Scanning Electron Microscope. Proc. 32nd Ann. EMSA Mtg. pp. 162–163. 127. Lamvik, M. K., Pullman, J. M., and Crewe, A. V. (1974). Unstained Biological Specimens in High Resolution Electron Microscopy. Proc. 32nd Ann. EMSA Mtg. pp. 234–235. 128. Panem, S., Lin, P. S. D., Crewe, A. V., and Kirsten, W. H. (1974). Scanning Electron Microscopy of Murine Oncornavirus Infected Cells. Proc. 32nd Ann. EMSA Mtg. pp. 252–253. 44 A. V. Crewe

129. Crewe, A. V. (1974). The High Resolution Scanning Transmission Electron Microscope. Proc. 32nd Ann. EMSA Mtg. pp. 316–317. 130. Zeitler, E., and Isaacson, M. (1974). Simplified Calculation of Phase Contrast for Finite Detector and Illumination Angles. Proc. 32nd Ann. EMSA Mtg. pp. 370–371. 131. Crewe, A. V., and Beck, V. (1974). Resolution Limits Imposed by Specimen–Electron Interactions. Proc. 32nd Ann. EMSA Mtg. pp. 372–373. 132. Isaacson, M., Langmore, J., and Rose, H. (1974). Measurements of the Non-Local Nature of the Inelastic Scattering of Electrons in Carbon. Proc. 32nd Ann. EMSA Mtg. pp. 374–375. 133. Langmore, J. P., and Crewe, A. V. (1974). Progress Towards the Sequencing of DNA by Electron Microscopy. Proc. 32nd Ann. EMSA Mtg. pp. 376–377. 134. Langmore, J. P., Isaacson, M. S., and Crewe, A. V. (1974). The Study of Single Heavy Atom Motion in the STEM. Proc. 32nd Ann. EMSA Mtg. pp. 378–379. 135. Munch, J., and Zeitler, E. (1974). Interference Experiments with a Field Emission Electron Microscope. Proc. 32nd Ann. EMSA Mtg. pp. 386–387. 136. Roth, T. A., Munch, J., and Zeitler, E. (1974). The Effect of Top-Anode Distance on Field Emission Current. Proc. 32nd Ann. EMSA Mtg. pp. 420–421. 137. Crewe, A. V., and Beck, V. (1974). A Quadrupole-Octupole Corrector for a 100 KEV STEM. Proc. 32nd Ann. EMSA Mtg. pp. 426–427. 138. Voreades, D., and Crewe, A. V. (1974). Correlation Between Inelastic Electron Scattering and Secondary Electron Production. Proc. 32nd Ann. EMSA Mtg. pp. 428–429. 139. Groves, T., and Crewe, A. V. (1974). The Use of Thick Specimens in a STEM. Proc. 32nd Ann. EMSA Mtg. pp. 550–551. 140. Lin, P. S. D. (1974). On Some Testing Specimens for High Resolution SEM. Proc. 32nd Ann. EMSA Mtg. pp. 556–557. 141. Lamvik, M. K., and Lin, S. D. (1974). A New Specimen Mounting System for Scanning Electron Microscopy. J. Microsc. 101, 329–331. 142. Crewe, A. V., Langmore, J., Isaacson, M., and Retsky, M. (1974). Understanding Single Atoms in the STEM. Proc. 8th Int’l Congress on Electron Microscopy. Canberra. 143. Crewe, A. V., Langmore, J., Lamvik, M., and Pullman, J. (1974). Scanning Electron Microscopy of Unstained and Selectively Stained Biological Specimens. Proc. 8th Int’l Congress on Electron Microscopy. Canberra. 144. Crewe, A. V., and Zeitler, E. (1974). Radiation Damage and Energy Analysis in the STEM. Proc. 8th Int’l Congress on Electron Microscopy. Canberra. 145. Zeitler, E., and Crewe, A. V. (1974). Design and Construction of a 1 MEV Scanning Microscope. Proc. 8th Int’l Congress on Electron Microscopy, pp. 40–41. Canberra. 146. Crewe, A. V. (1974). High Resolution Scanning Microscopy. Proc. 8th Int’l Congress on Electron Microscopy. Canberra. 147. Crewe, A. V., and Groves, T. (1974). The Use of Thick Specimens in the STEM and CEM. Proc. 8th Int’l Congress on Electron Microscopy. Canberra. 148. Crewe, A. V., and Lin, P. (1974). Experience with High Resolution Scanning Electron Microscopes with Field Emission Sources. Proc. 8th Int’l Congress on Electron Microscopy. Canberra. 149. Isaacson, M., and Langmore, J. P. (1974). Determination of the Non-Localization of the Inelastic Scattering of Electrons by Electron Microscopy. Optik 41, 92–96. 150. Lin, S. D. (1974). Electron Radiation Damage of Thin Films of Glycine, Diglycine, and Aromatic Amino Acids. Rad. Res. 59, 521–536. 151. Isaacson, M., and Langmore, J. (1974). The Preparation and Observation of Biological Specimens for the High Resolution Scanning Transmission Electron Microscope. Proc. 7th Ann. Scanning Electron Microscope Symposium. IIT Research Institute, pp. 19–26. 152. Beorchia, A., and Bonhomme, P. (1974). Experimental Studies of Some Dampings of Electron Microscope Phase Contrast Transfer Function. Optik 39, 437–442. The Work of Albert Victor Crewe on the STEM 45

1975

153. Shau-da-Lin, P., and Lamvik, M. K. (1975). High Resolution Scanning Electron Micro- scopy at the Subcellular Level. J. Microsc. 103, 249–257. 154. Langmore, J. P., and Wooley, J. C. (1975). Chromatin Architecture: Investigation of a Subunit of Chromatin by Dark Field Electron Microscopy. Proc. Nat’l Acad. Sci. USA 72, 2691–2695. 155. Crewe, A. V., Langmore, J., and Isaacson, M. (1975). Resolution and Contrast in the Scanning Transmission Electron Microscope. In ‘‘Physical Aspects of Electron Micros- copy and Microbeam Analysis,’’ (Siegel, B. and Beaman, D. R., eds.), pp. 47–62. John Wiley & Sons, New York. 156. Isaacson, M. S. (1975). Inelastic Scattering and Beam Damage of Biological Molecules. In ‘‘Physical Aspects of Electron Microscopy and Microbeam Analysis,’’ (Siegel, B. and Beaman, D. R., eds.), pp. 247–258. John Wiley & Sons, New York. 157. Lin, P. S. D., and Becker, R. P. (1975). Detection of Backscattered Electrons with High Resolution. Proc. of the Ann. Scanning Electron Microscopy Symposium. IIT Research Institute, pp. 61–70. 158. Groves, T., and Lamvik, M. K. (1975). The Measurement of Signal Intensities for Thick Specimens in the STEM. Proc. of the Ann. Scanning Electron Microscope Symposium. IIT Res. Institute, pp. 79–83. 159. Crewe, A. V., and Isaacson, M. (1975). Electron Microspectroscopy. Ann. Rev. of Biophys. and Bioeng. 4, 165–184. 160. Crewe, A. V. (1975). Electron Microscopes Using Field Emission Source. Surf. Sci. 48, 152–160. 161. Groves, T. (1975). Thick Specimens in the CEM and STEM. Ultramicroscopy 1, 15–31. 162. Isaacson, M., and Johnson, D. (1975). The Microanalysis of Light Elements Using Transmitted Energy Loss Electrons. Ultramicroscopy 1, 33–52. 163. Beck, V., and Crewe, A. V. (1975). High Resolution Imaging Properties of the STEM. Ultramicroscopy 1, 137–144. 164. Ohtsuki, M., and Zeitler, E. (1975). Minimal Beam Exposure with a Field Emission Source. Ultramicroscopy 1, 163–165. 165. Groves, T. (1975). Plural Scattering and Thick Specimens in Transmission Electron Microscopy. Ultramicroscopy 1, 170–172. 166. Crewe, A. V. (1975). High Resolution Scanning Microscopy—What Next? Proc. 33rd Ann. EMSA Mtg. pp. 18–19. 167. Parker, N. W., Isaacson, M., and Langmore, J. P. (1975). Studies of the Adsorption and Diffusion of Single Heavy Atoms on Light Element Substrates Using the STEM. Proc. 33rd Ann. EMSA Mtg. pp. 104–105. 168. Lin, P. S. D., and Lamvik, M. K. (1975). A Simple Transmission Stage for a Scanning Electron Microscope. Proc. 33rd Ann. EMSA Mtg. pp. 134–135. 169. Lin, P. S. D. (1975). On the Contrast of High Resolution Backscattered Electron Images. Proc. 33rd Ann. EMSA Mtg. pp. 206–207. 170. Lin, P. S. D., and Crewe, A. V. (1975). Simultaneous Study of Both the Surface and Interior Structure of Biological Specimens in a Scanning Electron Microscope. Proc. 33rd Ann. EMSA Mtg. pp. 208–209. 171. Johnson, D. E., and Isaacson, M. (1975). The Detection of Transmitted Energy Loss Electrons for Elemental Analysis. Proc. 33rd Ann. EMSA Mtg. pp. 236–237. 172. Lamvik, M. K., and Crewe, A. V. (1975). Mass Determination by Direct Measurement of Electron Scattering. Proc. 33rd Ann. EMSA Mtg. pp. 240–241. 173. Johnson, D. E. (1975). An Image Reconstruction System Applied to High Voltage Microscopy. Proc. 33rd Ann. EMSA Mtg. pp. 292–293. 46 A. V. Crewe

174. Lamvik, M. K., and Lin, S. D. (1975). An Improved Specimen Mount for Scanning Electron Microscopy. Proc. 33rd Ann. EMSA Mtg. pp. 132–133. 175. Hainfeld, H., and Isaacson, M. (1975). Electron Energy Loss in Membrane Components: Possibilities of Contrast and Identification in the STEM. Proc. 33rd Ann. EMSA Mtg. pp. 506–507. 176. Lamvik, M. K. (1975). Scanning Secondary Electron Microscopy of Myofibrils Using Transmission Techniques. Proc. 33rd Ann. EMSA Mtg. pp. 556–557. 177. Ramamurti, K., Crewe, A. V., and Isaacson, M. (1975). Low Temperature Beam Damage to Biological Specimens in the Electron Microscope. Proc. 33rd Ann. EMSA Mtg. pp. 608–609. 178. Isaacson, M., Langmore, J., Lamvik, M., Lin, P., Golladay, S., Hainfeld, J., Pullman, J., and Furlong, D. (1975). Mass Loss of Biological Molecules in the Electron Microscope. Proc. 33rd Ann. EMSA Mtg. pp. 676–677. 179. Crewe, A. V. (1975). Recent Developments and Future Prospects for STEM. Proc. EMAG Mtg. Bristol, pp. 1–2. 180. Ramamurti, K., Crewe, A. V., and Isaacson, M. (1975). Low Temperature Mass Loss of Thin Films of L-Phenylalanine and L-Tryptophan Upon Electron Irradiation—A Preliminary Report. Ultramicroscopy 1, 156–158. 1976

181. Parker, N. W., Golladay, S. D., and Crewe, A. V. (1976). A Theoretical Analysis of Third- Order Aberration Correction in the SEM and STEM. Proc. Scanning Electron Microscope Symposium. IIT Research Institute, pp. 37–44. 182. Crewe, A. V. (1976). Some Thoughts on the Future of the STEM. Proc. Sixth European Conf. on Electron Microscopy, pp. 24–25. 183. Isaacson, M. (1976). The Potential of the High Resolution Scanning Transmission Electron Microscope in the Biological and Materials Science: Fact and Fiction. Proc. Sixth European Conf. on Electron Microscopy, pp. 26–28. 184. Hainfeld, J., Isaacson, M., and Sogard, M. (1976). The Use of Microspectroscopy in the STEM for the Identification of Membrane Components. Proc. Sixth European Conf. on Electron Microscopy, pp. 30–31. 185. Langmore, J. (1976). Quantitative Studies of Unstained Biological Structures in the STEM. Proc. Sixth European Conf. on Electron Microscopy, pp. 31–33. 186. Crewe, A. V. (1976). Design Trends in the STEM. Proc. Sixth European Conf. on Electron Microscopy, pp. 65–66. 187. Crewe, A. V., Beck, V., and Parker, N. W. (1976). Correction of Third Order Aberration in the STEM. Proc. Sixth European Conf. on Electron Microscopy, pp. 322–323. 188. Crewe, A. V., and Zeitler, E. (1976). A One Million Volt Scanning Microscopy: Progress Report. Proc. Sixth European Conf. on Electron Microscopy, pp. 322–323. 189. Pullman, J., and Crewe, A. V. (1976). Darkfield Microscopy of Unstained Specimens at High Beam Dosages. Proc. 34th Ann. EMSA Mtg. pp. 380–381. 190. Lamvik, M. K. (1976). Electronmicroscopic Mass Determination Without a Densitome- ter: Quantitative Electron Microscopy Using Photographic Methods. Proc. 34th EMSA Mtg. pp. 380–381. 191. Johnson, D. E., and Isaacson, M. (1976). Elemental Analysis of Thin Films Using Inner Shell Electron Energy Losses. Proc. 34th Ann. EMSA Mtg. pp. 414–415. 192. Parker, N. W., Golladay, S. D., and Crewe, A. V. (1976). Third-Order Aberration Correction in the SEM and STEM. Proc. 34th Ann. EMSA Mtg. pp. 464–465. 193. Isaacson, M. S., Kopf, D., Parker, N. W., and Utlaut, M. (1976). Some Observations on the Adsorption of Heavy Atoms on Carbon Substrates. Proc. 34th Ann. EMSA Mtg. pp. 498–499. The Work of Albert Victor Crewe on the STEM 47

194. Parker, N. W., Utlaut, M., and Crewe, A. V. (1976). Evaporative Cooling of High Current Density Mini-lenses. Proc. 34th Ann. EMSA Mtg. pp. 536–537. 195. Ramamurti, K., Crewe, A. V., and Isaacson, M. S. (1976). Electron Beam Damage at Low Temperatures. Proc. 34th Ann. EMSA Mtg. pp. 554–555. 196. Beck, V., and Crewe, A. V. (1976). Progress in Aberration Correction in the STEM. Proc. 34th Ann. EMSA Mtg. pp. 578–579. 197. Isaacson, M. S., Kopf, D., Parker, N. W., and Utlaut, M. (1976). Observations of Surface Diffusion at the Atomic Level by Means of Microcinematography in the STEM. Proc. 34th Ann. EMSA Mtg. pp. 584–585. 198. Crewe, A. V., and Parker, N. W. (1976). Correction of Third Order Aberrations in the Scanning Electron Microscope. Optik 183–194. 199. Crewe, A. V. (1976). Very Low Voltage Electron Microscopy. Ultramicroscopy 1, 267–269. 200. Crewe, A. V., and Lin, P. S. D. (1976). The Use of Backscattered Electrons for Imaging Purposes in a Scanning Electron Microscope. Ultramicroscopy 1, 231–238. 201. Parker, N. W., Golladay, S. D., and Crewe, A. V. (1976). A Theoretical Analysis of Third- Order Aberration Correction in the SEM and STEM. Proc. 9th Ann. Scanning Electron Microscope Symposium. Part I IIT Research Institute. 202. Isaacson, M. S., and Parker, N. W. (1976). The Potential of Scanning Transmission Electron Microscopy for Studying Surface Phenomena on an Atomic Scale. Proc. 9th Ann. Scanning Electron Microscope Symposium. IIT Research Institute, pp. 345–352. Part II. 203. Isaacson, M. S., Langmore, J., Parker, N. W., Kopf, D., and Utlaut, M. (1976). The Study of the Adsorption and Diffusion of Heavy Atoms on Light Element Substrates by Means of the Atomic Resolution STEM. Ultramicroscopy 1, 359–376. 204. Lamvik, M. K., and Groves, T. (1976). Minimization of Dose as a Criterion for the Selection of Imaging Modes in Electron Microscopy of Amorphous Specimens. Ultramicroscopy 2, 69–75. 205. Isaacson, M. S., and Silcox, J. (1976). Report of a Workshop on Analytical Electron Microscopy. Ultramicroscopy 2, 89–104. 206. Voreades, D. (1976). Secondary Electron Emission from Thin Carbon Films. Surf. Sci. 60, 325–348. 207. Lerche, I., and Zeitler, E. (1976). Projections, Reconstructions and Orthogonal Functions. J. of Mathematical Analysis and Applications 56, 634–649. 1977

208. Crewe, A. V. (1977). Post-Specimen Optics in the STEM: I. General Considerations. Optik 47, 299–311. 209. Crewe, A. V. (1977). Post-Specimen Optics in the STEM: II. A Corrected Spectrometer. Optik 47, 371–380. 210. Marsh, G. E. (1977). Accelerator System for 1-MV STEM. Rev. Sci. Inst. 48, 841–843. 211. Isaacson, M., Kopf, D., Langmore, J., Parker, N. W., and Utlaut, M. (1977). Abstract: Atomic Resolution Observations of Heavy-Atom Adsorbates on Low-Atomic-Number Substrates. J. Vac. Sci. Tech. 14, 434. 212. Lamvik, M. K., and Langmore, J. P. (1977). Determination of Particle Mass Using Scanning Transmission Electron Microscopy. Scanning Electron Microscopy 1, 401–409. 213. Ohtsuki, M., and Zeitler, E. (1977). Young’s Experiment with Electrons. Ultramicroscopy 2, 147–148. 214. Crewe, A. V. (1977). Ideal Lenses and the Scherzer Theorem. Ultramicroscopy 2, 281–284. 215. Isaacson, M., Kopf, D., Utlaut, M., Parker, N. W., and Crewe, A. V. (1977). Direct Observation of Atomic Diffusion by Scanning Transmission Electron Microscopy. Proc. Nat’l Acad. Sci. 74, 1802–1806. 48 A. V. Crewe

216. Ohtsuki, M., White, S. L., Zeitler, E., Wellems, T. E., Fuller, S. D., Zwick, M., Makinen, M. W., and Siegler, P. B. (1977). Electron Microscopy of Fibers and Discs of Hemoglobin S Having Sixfold Symmetry. Proc. Nat’l Acad. Sci. 74, 5538–5542. 217. Isaacson, M., Kopf, D., and Utlaut, M. (1977). Progress in Single Atom Microscopy. Proc. 35th Ann. EMSA Mtg. pp. 78–79. 218. Isaacson, M., and Utlaut, M. (1977). Considerations on the Use of Electron and Photon Beams for Determining Micro-Chemical Environment. Proc. 35th Ann. EMSA Mtg. pp. 240–241. 219. Collins, M. L., and Parker, N. W. (1977). Preparation of Titanium Microgrids for Scanning Transmission Electron Microscopy. Proc. 35th Ann. EMSA Mtg. pp. 322–323. 220. Sogard, M., and Deutsch, D. (1977). Observation of Human Low Density Lipoproteins in the STEM. Proc. 35th Ann. EMSA Mtg. pp. 472–473. 1978

221. Crewe, A. V. (1978). Collision Broadening in Electron Beams. Optik 50, 205–212. 222. Isaacson, M., and Utlaut, M. (1978). A Comparison of Electron and Photon Beams for Determining Micro-Chemical Environment. Optik 50, 213–234. 223. Parker, N. W., Utlaut, M., and Isaacson, M. S. (1978). Design of Magnetic Spectrometers with Second-Order Aberrations Corrected. Optik 51, 333–351. 224. Isaacson, M., and Gomer, R. (1978). Extended Range Field Emission Spectroscopy. J. Appl. Phys. 15, 253–256. 225. Isaacson, M. (1978). All You Might Want to Know About ELS (But Are Afraid to Ask): A Tutorial. Scanning Electron Microscopy I, 763–776. 226. Orr, J., Toan, N., Ohtsuki, M., Wellems, T., and Haselkorn, R. (1978). Glutamine Synthetase from Cyanobacterium Anabaena-7120. Federation Proceeding 37, 1431. 227. Parker, N. W., Crewe, A. V., Isaacson, M. S., and Mankawich, W. (1978). A New Analytical Electron Microscope. 9th Int’l Congress on Electron Microscopy I, pp. 18–19. Toronto. 228. Kopf, D., Utlaut, M., Crewe, A. V., Isaacson, M., and Mankawich, W. (1978). Some Instrumental Aspects of Single Atom Microscopy. 9th Int’l Congress on Electron Micros- copy I, pp. 20–21. Toronto. 229. Parker, N. W., Utlaut, M., and Isaacson, M. (1978). Magnetic Spectrometers with Corrected Second-order Aberration. 9th Int’l Congress on Electron Microscopy I, pp. 20–21. Toronto. 230. Golloday, S. (1978). Analysis of STEM Micrographs of Thick Objects. 9th Int’l Congress on Electron Microscopy II, pp. 106–107. Toronto. 231. Furlong, D. (1978). Use of the STEM to Obtain Information from One Level of a Complex Biological Object. 9th Int’l Congress on Electron Microscopy II, pp. 108–189. Toronto. 232. Ohtsuki, M., and Sogard, M. (1978). Observation of Biological Macromolecules Using Various Electron Microscopic Methods. 9th Int’l Congress on Electron Microscopy II, pp. 170–171. Toronto. 233. Usala, S., Isaacson, M., and Martin, T. (1978). Scanning Transmission Electron Micros- copy of Unstained Nucleosomes. 9th Int’l Congress on Electron Microscopy II, pp. 170–171. Toronto. 234. Isaacson, M., Collins, M. L., and Listvan, M. (1978). Electron Beam Damage of Biomo- lecules Assessed by Energy Loss Spectroscopy. 9th Int’l Congress on Electron Microscopy III, pp. 61–69. Toronto. 235. Crewe, A. V. (1978). Is There a Future for the STEM? 9th Int’l Congress on Electron Microscopy III, pp. 197–204. Toronto. 236. Lamvik, M. K. (1978). Muscle Thick Filament Mass Measured by Electron Scattering. J. Mol. Biol. 122, 55–68. The Work of Albert Victor Crewe on the STEM 49

237. Hainfeld, J., and Isaacson, M. (1978). The Use of Electron Energy Loss Spectroscopy or Studying Membrance Architecture: A Preliminary Report. Ultramicroscopy 3, 87–95. 238. Crewe, A. V. (1978–79). Direct Imaging of Single Atoms and Molecules Using the STEM. Chemica Scripta 14, 17–20. 239. Crewe, A. V. (1978–79). Some Space Charge Effects in Electron Probe Devices. Optik 52, 337–346. 1979

240. Crewe, A. V. (1978–79). Direct Imaging of Single Atoms and Molecules Using the STEM. Chemica Scripta 14, 17–20. 241. Crewe, A. V. (1978–79). Some Space Charge Effects in Electron Probe Devices. Optik 52, 337–346. 242. Deutsch, D. G., and Sogard, M. (1979). Observation of Human Low Density Lipopro- teins in the STEM and FECTEM. Micron 10, 25–28. 243. Isaacson, M., Kopf, D., Ohtsuki, M., and Utlaut, M. (1979). Contamination as a Psycho- logical Problem (Letter to the Editor). Ultramicroscopy 4, 101–104. 244. Isaacson, M., Kopf, D., Ohtuski, M., and Utlaut, M. (1979). Atomic Imaging Using the Dark-Field Annular Detector in the STEM (Letter to the Editor). Ultramicroscopy 4, 101–104. 245. Isaacson, M. (1979). Electron Beam Induced Damage of Organic Solids: Implications for Analytical Electron Microscopy. Ultramicroscopy 4, 193–199. 246. Hwang, D. M., Utlaut, M., Isaacson, M. S., Solin, S. A., and Minarski, L. (1979). Electron 24, Energy Loss Studies of C8K. Bull. Amer. Phys. Soc. 372. 247. Ohtsuki, M., Isaacson, M. S., and Crewe, A. V. (1979). Dark Field Imaging of Biological Macromolecules with the Scanning Transmission Electron Microscope. Proc. Nat’l Acad. Sci. (USA) 76, 1228–1232. 248. Crewe, A. V. (1979). Development of High Resolution STEM and Its Future. J. Electron Microscopy 28, S9–S16. 249. Crewe, A. V. (1979). Direct Imaging of Atoms in Crystals and Molecules. ‘‘Nobel Symposium.’’ Stockholm. 250. Utlaut, M., Hwang, D., Isaacson, M., and Solin, S. (1979). Electron Energy Loss Spectroscopy of Potassium Intercalated Graphite. Proc. 14th Biennial Conf. on Carbon. University Park, p. 316. 251. Isaacson, M., Utlaut, M., and Ohtsuki, M. (1979). Scanning Transmission Electron Microscopy of Very Thin Evaporated Carbon Films. Proc. 14th Biennial Conf. on Carbon. University Park, pp. 103–104. 252. Isaacson, M., Utlaut, M., and Ohtsuki, M. (1979). Direct Observation of the Diffusion of Heavy Atoms on the Surface of Thin Amorphous Carbon Films. Proc. 14th Biennial Conf. on Carbon. University Park, pp. 105–106. 253. Ohtsuki, M., Isaacson, M. S., and Crewe, A. V. (1979). Preparation and Observation of Very Thin, Very Clean Substrates for Scanning Transmission Electron Microscopy. Scanning Electron Microscopy II, 375–382. 254. Isaacson, M., Ohtsuki, M., and Utlaut, M. (1979). Electron Microscopy of Individual Atoms. In ‘‘Introduction to Analytical Electron Microscopy’’, (Hren, J., Goldstein, J. I., and Joy, D. C., eds.) pp. 343–368. Plenum Publishing Co, New York. 255. White, S. L. (1979). Images of Unstained Hemoglobin from the STEM. Proc. 37th Ann. EMSA Mtg. pp. 18–19. 256. Isaacson, M. S., and Ohtsuki, M. (1979). Scanning Transmission Electron Microscopy and Electron Microdiffraction of Ferritin. Proc. 37th Ann. EMSA Mtg. pp. 20–21. 257. Golladay, S. D., and Lamvik, M. K. (1979). Platinum Labeling of Methionine in Tropo- myosin Paracrystals. Proc. 37th Ann. EMSA Mtg. pp. 26–27. 50 A. V. Crewe

258. Usala, S., Brownstein, B., Haselkorn, R., and Agrawal, H. O. (1979). Transcription Associated with Bacteriophage 06 Nucleocapsid. Proc. 37th Ann. EMSA Mtg. pp. 32–33. 259. Ohtsuki, M. (1979). Dark Field STEM Observation of Negatively Stained Tobacco Mosaic Virus (TMV): Effects of Radiation Damage. Proc. 37th Ann. EMSA Mtg. pp. 130–131. 260. Utlaut, M., Ohtsuki, M., and Isaacson, M. (1979). Observations of the Diffusion of Heavy Atoms on Thin Amorphous Carbon Films. Proc. 37th Ann. EMSA Mtg. pp. 466–467. 261. Utlaut, M. (1979). Measurements of Single Atom Diffusion at Different Temperatures. Proc. 37th Ann. EMSA Mtg. pp. 470–471. 262. Isaacson, M., Ohtsuki, M., and Utlaut, M. (1979). Can We Determine the Structure of Thin Amorphous Films Using Scanning Transmission Electron Microscopy? Proc. 37th Ann. EMSA Mtg. pp. 498–499. 263. Isaacson, M., and Utlaut, M. (1979). On the Chemical Identificaion of Individual Atoms. Proc 37th Ann. EMSA Mtg. pp. 524–525. 264. Listvan, M. (1979). Thin Polycrystalline Graphite Films for High Resolution Micros- copy. Proc. 37th Ann. EMSA Mtg. pp. 544–545. 265. Utlaut, M., and Isaacson, M. S. (1979). A Comparison of Measured Single Atom Dis- tributions with Random Distributions. Proc. 37th Ann. EMSA Mtg. pp. 546–547. 266. Crewe, A. V. (1979). Something Old, Something New for the STEM. Proc. 37th Ann. EMSA Mtg. pp. 560–563. 267. Sogard, M. R. (1979). Resolution in Electron Backscattering: A Monte Carlo Study. Proc. 37th Ann. EMSA Mtg. pp. 564–565. 268. Ohtsuki, M., and Isaacson, M. S. (1979). Simple Test Specimens for Measuring Lattice Resolution and Probe Size of a STEM. Proc. 37th Ann. EMSA Mtg. pp. 568–569. 269. Parker, N. W., Utlaut, M., and Isaacson, M. S. (1979). Design of Aberration-Corrected Magnetic Spectrometers. Proc. 37th Ann. EMSA Mtg. pp. 578–579. 270. Parker, N. W., Utlaut, M., Isaacson, M. S., and Crewe, A. V. (1979). A New Analytical Electron Microscope. Proc. 37th Ann. EMSA Mtg. pp. 588–589. 271. Becker, R. P., and Sogard, M. (1979). Visualization of Subsurface Structures in Cells and Tissues by Backscattered Electron Imaging. Scanning Electron Microscopy II, 835–870. 272. Crewe, A. V. (1979). Some Limitations on Electron Beam Lithography. J. Vac. Sci. Technol. 16, 255–259. 273. Isaacson, M. (1979). Scanning Transmission Electron Microscopy at Near-Atomic Reso- lution: The Present State of the Art. Ultramicroscopy 4, 365. 274. Crewe, A. V., Isaacson, M. S., and Zeitler, E. (1979). Progress in Scanning Transmission Electron Microscopy at the University of Chicago. In ‘‘Unconventional Electron Micros- copy for Molecular Structure Determination’’, (Hoppe, W. and Mason, R., eds.), pp. 23–48. Friedr. Vieweg & Sohn, Braunschweig. 275. Hwang, D. M., Utlaut, M., Isaacson, M. S., and Solin, S. A. (1979). Electron-Energy Loss Study of Stage-1 Potassium-Intercalated Graphite. Phys. Rev. Letters 43, 882–886. 276. Crewe, A. V. (1979–80). Electron Microscopy. In ‘‘Encyclopedia of Physics’’, (Lerner, A. V. and Trig, A. V., eds.). 1980

277. Crewe, A. V. (1979–80). Electron Microscopy. In ‘‘Encyclopedia of Physics’’, (Lerner, A. V. and Trig, A. V., eds.). 278. Crewe, A. V. (1980). Color Conversion in Electron Microscopy. Scanning 3, 176–181. 279. Crewe, A. V. (1980). Imaging in Scanning Microscopes. Ultramicroscopy 5, 131–138. 280. Crewe, A. V., and Kopf, D. A. (1980). Limitations of Sextupole Correctors. Optik 56, 391–399. 281. Isaacson, M., Utlaut, M., and Kopf, D. (1980). Computer Processing of Electron Micro- scope Images. In ‘‘Topics in Current Physics’’, (Hawkes, P. W., ed.). Springer-Verlag, New York. The Work of Albert Victor Crewe on the STEM 51

282. Ohtsuki, M., and Crewe, A. V. (1980). Optimal Imaging Techniques in the Scanning Transmission Electron Microscope: Applications to Biological Macromolecules. Proc. Nat’l Acad. Sci. (USA) 77, 4051–4054. 283. Crewe, A. V. (1980). The Physics of the High Resolution Scanning Microscope. Rep. Prog. Phys. 43, 621–639. 284. Hwang, D. M., Solin, S. A., Utlaut, M., and Isaacson, M. S. (1980). Electron Energy Loss Studies of Stage-2 Potassium Intercalated Graphite. Bull. of the APS 25, 336. 285. Crewe, A. V. (1980). The Sextupole as Corrector. Electron Microscopy I, 36–37. 286. Crewe, A. V., and Kopf, D. (1980). A Sextupole System for the Correction of Spherical Aberration. Optik 55, 1–10. 287. Crewe, A. V., and Kopf, D. (1980). The Use of Non-optimal Apertures in the STEM. Optik 55, 325–327. 288. Sogard, M. R. (1980). Backscattered Electron Energy Spectra for Thin Films from an Extension of the Everhart Theory. J. Appl. Phys. 51, 4412–4416. 289. Sogard, M. R. (1980). An Empirical Study of Electron Backscattering from Thin Films. J. Appl. Phys. 51, 4417–4425. 290. Hwang, D. M., Utlaut, M., Isaacson, M. S., and Solin, S. A. (1980). Dielectric Functions of Stage One Potassium Intercalated Graphite Single Crystals Determined by Electron Microscopy. Physica 99B, 435–440. 291. Crewe, A. V., and Ohtsuki, M. (1980). The Use of Optimal Scanning in the STEM. 7th European Congress on Electron Microscopy. Netherlands. 292. Crewe, A. V., Lin, P., and Schuler, J. (1980). 1 MeV STEM: A Progress Report. Proc. 38th Ann. EMSA Mtg. pp. 6–7. 293. Crewe, A. V., Kopf, D., Ohtsuki, M., and Usala, P. (1980). Further Uses of Color in Scanning Transmission Electron Microscopy. Proc 38th Ann. EMSA Mtg. 294. Kirkland, E. M., Freeman, W. T., Ohtsuki, M., and Siegel, B. M. (1980). Digital Image Processing of STEM Images of Tobacco Mosaic Virus (TMV). Proc. 38th Ann. EMSA Mtg. pp. 40–41. 295. Listvan, M., Crewe, A. V., and Mankawich, W. (1980). A Temperature-Controlled High Resolution Stage. Proc. 38th Ann. EMSA Mtg. pp. 58–59. 296. Crewe, A. V. (1980). Theory of Optimal Scanning in the STEM. Proc. 38th Ann. EMSA Mtg. pp. 60–61. 297. Ohtsuki, M., and Crewe, A. V. (1980). Application of the STEM Optimum Imaging Method on a Biological Macromolecule. Proc. 38th Ann. EMSA Mtg. pp. 62–63. 298. Crewe, A. V. (1980). A New Possibility for Correcting Cs. Proc. 38th Ann. EMSA Mtg. pp. 274–276. 299. Crewe, A. V., Mittleman, R. K., and Rosman, R. L. (1980). Preparation of Thin Graphite Supporting Films. Proc. 38th Ann. EMSA Mtg. pp. 410–411. 300. Kapp, O. H., Ohtsuki, M., and Vinogradov, S. N. (1980). Structural Investigation of Invertebrate Hemoglobins Using the STEM. Proc. 38th Ann. EMSA Mtg. pp. 676–677. 301. Utlaut, M. (1980). Direct Observation of the Behavior of Heavy Single Atoms on Amorphous Carbon Substrates. Phys. Rev. B 22, 4650–4660. 302. Crewe, A. V. (1980). Studies on Sextupole Correctors. Optik 57, 313–327. 303. Isaacson, M., and Ohtsuki, M. (1980). Scanning Transmission Electron Microscopy of Small Inhomogeneous Particles: Applications to Ferritin. Scanning Electron Microscopy I, 73–80. 304. Ohtsuki, M. (1980). Observation of Unstained Biological Macromolecules with the STEM. Ultramicroscopy 5, 317–323. 305. Ohtsuki, M. (1980). Test Specimens for a Dark Field STEM Imaging: Measuring Lattice Spacing and Electron Probe Size of a STEM and Testing Visibility of Atoms of Various Atomic Numbers. Ultramicroscopy 5, 325–332. 52 A. V. Crewe

1981

306. Crewe, A. V., and Ohtsuki, M. (1981). Optimal Scanning and Image Processing with the STEM. Ultramicroscopy 7, 13–18. 307. Kapp, O. H., Ohtsuki, M., and Vinogradov, S. N. (1981). Oxygen Binding Study and High Resolution Scanning Transmission Electron Microscopy (STEM) of Alkaline Dis- sociation Products of Lumbricus terrestris Hemoglobin. Proc. 7th Int’l Biophysics Congress and 3rd Pan-American Biochemistry Congress. Mexico City, p. 60. 308. Kirkland, E. J., Freeman, W. T., Ohtsuki, M., Isaacson, M. S., and Siegal, B. M. (1981). Computer Image Processing of STEM Images of Tobacco Mosaic Virus. Ultramicroscopy 6, 367–376. 309. Kopf, D. A. (1981). Measurement of the Intensity Distribution in a STEM Probe. Optik 59, 89–110. 310. Ohtsuki, M. (1981). Preparation. ‘‘Observation, and Imaging Methods for High Reso- lution Scanning Transmission Electron Microscopy of a Biological Specimen.’’ Waseda University, Tokyo, Japan. 311. Snipes, J., Ohtsuki, M., Vinogradov, S. N., and Crewe, A. V. (1981). Nucleation of Indium Atoms on an Amorphous Carbon Substrate. Proc. 39th Ann. EMSA Mtg. pp. 220–221. 312. Kapp, O. H., Ohtsuki, M., Vinogradov, S. N., and Crewe, A. V. (1981). Scanning Transmission Electron Microscopy (STEM) of Annelid Extracellular Hemoglobins. Proc. 39th Ann. EMSA Mtg. pp. 432–433. 313. Crewe, A. V., and Ohtsuki, M. (1981). Digital Processing of STEM Images. Proc. 39th Ann. EMSA Mtg. 314. Kapp, O. H., Ohtsuki, M., Robin, N., Vinogradov, S. N., and Crewe, A. V. (1981). Alkaline Dissociation Products of Lumbricus terrestris Hemoglobin Viewed with the STEM. Proc. 39th Ann. EMSA Mtg. pp. 434–435. 315. Parker, N. W., Utlaut, M., and Crewe, A. V. (1981). Results from a New Analytical Electron Microscope. Proc. 39th Ann. EMSA Mtg. pp. 378–379. 316. Kirkland, E. J., Freeman, W. T., Ohtsuki, M., Isaacson, M. S., and Siegel, B. M. (1981). Digital Image Processing of STEM Images of Tobacco Mosaic Virus. Proc. 39th Ann. EMSA Mtg. 317. Hwang, D. M., Utlaut, M., and Solin, S. A. (1981). Electron Energy Loss Studies of Potassium Intercalated Graphite. Synthetic Metals 3, 81–88. 318. Vinogradov, S. N., Kapp, O. H., and Ohtsuki, M. (1981). The Extracellular Hemoglobins and Chlorocruorins of Annelids. In ‘‘Electron Microscopy of Proteins’’, (Harris, J. R., ed.), Vol. 3, pp. 135–164. Academic Press Inc, London. 1982

319. Crewe, A. V. (1982). A System for the Correction of Axial Aperture Aberrations in Electron Lenses. Optik 60, 271–281. 320. Crewe, A. V., and Ohtsuki, M. (1982). A Digital Processor for Use with STEM Images. Proc. 10th Int’l Congress on Electron Microscopy. Hamburg, West Germany, pp. 531–532. 321. Ohtsuki, M., and Crewe, A. V. (1982). A Thirteenth Subunit Revealed in Lumbricus terrestris Hemoglobin in STEM Low Dose Images. Proc. 10th Int’l Congress on Electron Microscopy. Hamburg, West Germany, pp. 493–494. 322. Crewe, A. V., and Ohtsuki, M. (1982). A ‘‘Stand Alone’’ Image Processing System for STEM Images. Ultramicroscopy 9, 101–108. 323. Crewe, A. V., and Scaduto, F. (1982). A Gradient Field Spectrometer for STEM Use. Proc. 40th Ann. EMSA Mtg. 324. Ohtsuki, M., and Crewe, A. V. (1982). Observation of a Central Subunit in Annelid Extracellular Hemoglobin Using the STEM Digital Processing Techniques. Proc. 40th Ann. EMSA Mtg. pp. 704–705. The Work of Albert Victor Crewe on the STEM 53

325. Crewe, A. V., and Salzman, D. (1982). On the Optimum Resolution for a Corrected STEM. Ultramicroscopy 9, 373–378. 326. Crewe, A. V., and Ohtsuki, M. (1982). Digital Processing of STEM Images and False Color Displays. Hitachi Instrument News 11, 2–5. 327. Kapp, O. H., Vinogradov, S. N., Ohtsuki, M., and Crewe, A. V. (1982). Scanning Transmission Electron Microscopy of Extracellular Annelid Hemoglobins. Biochimica et Biophysica Acta 704, 546–548. 328. Golladay, S. D. (1982). Tropomyosin Structure and Intermolecular Contacts in Mg Paracrystals (Ph.D. Thesis). Biochimica et Biophysica Acta 705, 277–283. 329. Crewe, A. V. (1982). A Low Cost Image Analysis System for Scanning Transmission Electron Microscopy Low-Dose Images. Scanning Electron Microscopy IV, 1597–1601. 1983

330. Chen, E. G., Ohtsuki, M., and Crewe, A. V. (1983). Attractive and Repulsive Forces Between Clusters: A STEM Study of Platinum Clusters. Proc. 41st Ann. EMSA Mtg. pp. 334–335. 331. Ohtsuki, M., Schuler, J. J., Crewe, A. V., and Ichinokawa, T. (1983). Specimen Heating Stage with In situ Evaporator in UHV-STEM for the Observation and Characterization of Monolayered Superstructures. Proc. 41st Ann. EMSA Mtg. pp. 308–309. 332. Listvan, M. A. (1983). The Development of Order in a System of Metal Clusters. Proc. 41st Ann. EMSA Mtg. pp. 336–337. 333. Listvan, M. A., and Andres, R. P. (1983). Production and STEM Examination of Con- trolled-Size Clusters. Proc. 41st Ann. EMSA Mtg. pp. 338–339. 334. Kapp, O. H., Ohtsuki, M., Crewe, A. V., and Vinogradov, S. N. (1983). Observation of Reconstituted Lumbricus terrestris Hemoglobin with the STEM. Proc. 41st Ann. EMSA Mtg. pp. 594–655. 335. Vinogradov, S. N., Polidori, G., Van Gelderen, J., and Kapp, O. H. (1983). The Dissociation of the Extracellular Hemoglobin of. Nephtys incisa, Comp. Biochem. Physiol. 76B, 207–214. 336. Listvan, M. A. (1983). Direct Observations of Small Gold Clusters and In Situ Cluster Growth by STEM. J. Mol. Catalysis 20, 265–278. 337. Vinogradov, S. N., and Kapp, O. H. (1983). Removal of Sodium Dodecyl Sulfate from Proteins by Ion Retardation Chromatography. In ‘‘Part J of Methods in Enzymology’’, (Hirs, C. H. W. and Timasheff, S., eds.), pp. 259–263. Academic Press, New York. 338. Vinogradov, S. N., and Kapp, O. H. (1983). Dissociation and Reassociation of Earth- worm Hemoglobin. Life Chemistry Reports (Suppl. 1), 147–156. 339. Vinogradov, S. N., Kapp, O. H., Messerschmidt, U., Schwartz, E., and Pilz, I. (1983). Nephtys: An Unusual Extracelleular Annelid Hemoglobin with a Central Subunit. Life Chemistry Reports (Suppl.1), 197–202. 340. Crewe, A. V. (1983). High Resolution Scanning Transmission Electron Microscopy. Science 221, 325–330. 341. Hwang, D. M., Parker, N. W., Utlaut, M., and Crewe, A. V. (1983). Observation of Non- hexagonal Superlattices in High-stage Cesium Intercalated Graphite. Phys. Rev. B 27, 1458–1461. 342. Ohtsuki, M. (1983). A High Resolution STEM and its Application to the Observation of Biological Macromolecules. J. Cryst. Soc. Japan 25, 38–50. 343. Ohtsuki, M., and Crewe, A. V. (1983). Direct Observation of Biological Macromolecules with the Atomic Resolution Scanning Transmission Electron Microscope. Phys. Soc. of Japan 38, 550–556. 344. Kritchman, E. M. (1983). Ideal Second Stages in Tandem with Spherical Mirrors. Solar Energy 30, 481–482. 345. Crewe, A. V., and Ohtsuki, M. (1983). Evidence for a Central Substructure in a Lumbricus terrestris Hemoglobin Obtained with STEM Low-dose and Digital Processing Techni- ques. J. Ultrastructure Research 83, 312–318. 54 A. V. Crewe

1984

346. Crewe, A. V., and Crewe, D. A. (1984). Inexact Reconstructions from Projections. Ultramicroscopy 12, 293–298. 347. Crewe, A. V., Crewe, D. A., and Kapp, O. H. (1984). Inexact Three Dimensional Reconstruction of a Biological Macromolecule from a Restricted Number of Projections. Ultramicroscopy 13, 365–372. 348. Kapp, O. H., Polidori, G., Mainwaring, M., Crewe, A. V., and Vinogradov, S. N. (1984). The Reassociation of Lumbricus terrestris Hemoglobin Dissociated at Alkaline pH. J. Biol. Chem. 259, 628–639. 349. Kapp, O. H., and Crewe, A. V. (1984). The Size and Shape of the Extracellular Hemo- globin of. Tubifex tubifex, Biochimica et Biophys. Acta 789, 294–301. 350. Chen, E. G., Ohtsuki, M., and Crewe, A. V. (1984). A STEM Study of a Platinum Deposit on an Amorphous Carbon Film: The Effects of Contact Voltage in the Nucleation Process. Surf. Sci. 144, 465–476. 351. Lewis, R. S., and Ohtsuki, M. (1984). Interstellar Carbon Grains from the Murchison Meteorite: Electron Microscopy. Proc. 47th Annual Meteoretical Soc. Meeting, p. L-8. Albuquerque, NM. 352. Ohtsuki, M., Edelstein, C., and Scanu, A. M. (1984). Similarity in Structures of Canine High Density Lipoproteins Containing Either Apo A-I or Human Apo A-II as Assessed by a Scanning Transmission Electron Micrsocpy Digital Techniques. Arteriosclerosis 4, 566a. 353. Crewe, A. V. (1984). Optimisation of a STEM. Proc. Roy. Microsc. Soc. MICRO 84 19, S59. 354. Crewe, A. V. (1984). Very High Resolution: A Place for the STEM. Proc. 42nd Ann. EMSA Mtg. pp. 372–375. 355. Crewe, A. V., and Crewe, D. A. (1984). Inexact Reconstruction: A. The Basic Method. Proc. 42nd Ann. EMSA Mtg. pp. 634–635. 356. Kapp, O. H., Crewe, D. A., and Crewe, A. V. (1984). Inexact Reconstruction: B. Applica- tion to a Biological Object. Proc. 42nd Ann. EMSA Mtg. pp. 636–637. 357. Crewe, A. V., Crewe, D. A., and Kapp, O. H. (1984). Three Dimensional Reconstruction of Lumbricus terrestris Hemoglobin from a Restricted Number of STEM Projections. Biophysical Journal 45, 371A. 358. Crewe, D. A., Crewe, A. V., and Kapp, O. H. (1984). Inexact Three Dimensional Reconstruction of Lumbricus terrestris Hemoglobin. Proc. ASBC/AII Meeting, p. 1562. St. Louis. 359. Schwartz, C., Vinogradov, S. N., Kapp, O. H., Crewe, A. V., and Mainwaring, M. (1984). Molecular Size and Subunits of Myxicola chlorocruorin. Proc. ASBC/AII Meeting, p. 1562. St. Louis. 360. Kapp, O. H., Crewe, A. V., Mainwaring, M. G., Schwartz, C., and Vinogradov, S. N. (1984). The Size and Shape of the Hemoglobin of Macrobdella decora. Proc. ASBC/AII Meeting, p. 1578. St. Louis. 361. Mainwaring, M. G., Vinogradov, S. N., Kapp, O. H., and Crewe, A. V. (1984). Acid Dissociation and Reassociation of Lumbricus terrestris Hemoglobin. Proc. ASBC/AII Meeting, p. 1579. St. Louis. 362. Vinogradov, S. N., Kapp, O. H., Crewe, A. V., and Mainwaring, M. (1984). Studies of the Extracellular Hemoglobin of Amphitrita ornata. Proc. ASBC/AII Meeting, p. 1579. St. Louis. 363. Vinogradov, S. N., Van Gelderin, J. A., Polidori, G., and Kapp, O. H. (1984). Dissociation of the Extracellular Hemoglobin of. Nephytys, Comp. Biochem. Physiol. 76B, 207–214. 364. Kapp, O. H., Crewe, A. V., Mainwaring, M. G., and Vinogradov, S. N. (1984). The Size and Shape of the Hemoglobin of Macrobdella decora. Proc. 8th Intl. Biophys. Congr. p. 234. Bristol, England. The Work of Albert Victor Crewe on the STEM 55

365. McNamara, G. R., Kapp, O. H., Barrett, T. A., Mooney, P. E., Jiye, X., and Crewe, A. V. (1984). A New Image Processing System for STEM Images. Proc. 8th Intl. Biophys. Congr. p. 234. Bristol, England. 366. Crewe, A. V. (1984). An Introduction to the STEM. J. Ultrastr. Res. 88, 94–104. 367. Crewe, A. V. (1984). The Sextupole Corrector 1. Algebraic Calculations. Optik 69, 24–29. 368. Ohtsuki, M. (1984). ‘‘An Introduction to the 2.5A˚ STEM.’’ Enrico Fermi Institute, The University of Chicago. 369. Ohtsuki, M., and Schudy, E. (1984). Assessment of Morphology of Assymmetrical Biological Macromolecules with Digital Techniques. J. Ultrast. Res. 89, 303–308. 370. Kapp, O. H., and Crewe, A. V. (1984). Comparison of the Molecular Size and Shape of the Extracellular Hemoglobins of Tubifex tubifex and Lumbricus terrestris, Biochim. et Biophys. Acta 789, 294–301. 1985

371. Crewe, A. V., and Crewe, D. A. (1985). Inexact Reconstruction: Some Improvements. Ultramicroscopy 16, 33–40. 372. Crewe, A. V., and Jiye, X. (1985). Correction of Spherical and Coma Aberrations with a Sextupole-Round Lens-Sextupole System. Optik 69, 141–146. 373. Jiye, X., Shao, Z., and Crewe, A. V. (1985). The Wave Electron Optical Properties of a Magnetic Round Lens Corrected with Sextupoles. Optik 70, 37–42. 374. Shao, Z., and Jiye, X. (1985). Two Dimensional Phase Retrieval from Image and Diffrac- tion Patterns in Electron Microscopes. Optik 71, 15–22. 375. Jiye, X., and Shao, Z. (1985). Three Dimensional Algebraic Reconstruction from Three Mutually Orthogonal Projections. Optik 71, 143–148. 376. Crewe, A. V. (1985). Towards the Ultimate Scanning Microscope. Scanning Electron Microscopy II, 467–472. 377. Vinogradov, S. N., Standley, P. R., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1985). The Molecular Size of Myxicola infundibulum chlorocruorin and its Subunits. Biochimica et Biophysica Acta 828, 43–50. 378. Jiye, X., and Kapp, O. H. (1985). Classification of Projection Images by Multivariate Matrix Analysis. Proc. FASEB Mtg. p. 1096. Anaheim, CA. 379. Kapp, O. H., and Jiye, X. (1985). Three Dimensional Reconstruction of a Biological Object Using Multivariate Matrix Analysis. Proc. FASEB Mtg. p. 1096. Anaheim, CA. 380. Kapp, O. H., Crewe, D. A., Crewe, A. V., Mainwaring, M. G., and Vinogradov, S. N. (1985). Three Dimensional Reconstruction of the Hemoglobin of Lumbricus terrestris from STEM Projections. 13th Int’l Congress of Biochemistry. Amsterdam. 381. McNamara, G. R., Kapp, O. H., and Crewe, A. V. (1985). A New Image Processing System for STEM Images. Proc. 13th Int’l Congress of Biochemistry. The Netherlands. 382. Jiye, X., and Kapp, O. H. (1985). Classification of Projection Images by Multivariate Matrix Analysis. Optik 70, 101–108. 383. Kapp, O. H., and Jiye, X. (1985). Classification of Electron Microscopic Images by Multivariate Matrix Analysis. Optik 70, 146–151. 384. Jiye, X., and Shao, Z. (1985). A Study of an Inhomogeneous Gradient Magnetic Field Spectrometer with a Curvilinear Axis. Optik 71, 73–79. 385. Salzman, D., O’Connor, M., Smith, R., Goodman, A., and Breslau, D. (1985). A Unified Environment for Image Processing, Graphics, and Instrument Control. Proc. 1st Int’l Conf. on Computer Workstations. pp. 173–180. San Jose, CA. 386. Lamy, J., Sizaret, P. Y., Musmeci, T., Kapp, O. H., and Vinogradov, S. N. (1985). Has Annelid Erythrocruorin a 6-fold Axis of Symmetry? Int’l Symposium on Invertebrate Oxygen Carriers Tutzing, West Germany. 387. Kapp, O. H. (1985). Capabilities of the STEM: Present and Future. Frontiersin Science Symposium, 190th American Chemical Society. Chicago. 56 A. V. Crewe

388. Levi-Setti, R., Crow, G., Wang, Y. L., Parker, N. W., and Mittleman, R. K. (1985). High- Resolution Scanning-Ion-Microprobe Study of Graphite and its Intercalation Com- pounds. Phys. Rev. Let. 54, 2615–2618. 389. Crewe, A. V. (1985). Gravure in the Digital Age. Gravure Technical Assoc. Convention. Chicago. 390. Crewe, A. V. (1985). The Chicago Microscope Project. IBM-ACIS Conf. Santa Clara. 391. Crow, G., Wang, Y. L., Parker, N. W., Mittleman, R., Qian, X. W., and Solin, S. A. (1985). Applications of Microscopic Probes in the Study of Graphite Intercalation Compounds. ‘‘Synthetic Metals.’’ 1986

392. Jiye, X., and Kapp, O. H. (1986). Three Dimensional Algebraic Reconstruction from Projections of Multiple Grey Level Objects. Optik 72, 87–94. 393. Kapp, O. H., and Jiye, X. (1986). Eigenvalue Analysis for Classifying Projection Images of an Object with Arbitrary Orientation. Optik 73, 51–55. 394. Jiye, X., and Kapp, O. H. (1986). Eigenvalue and Eigenvector Analysis in Multivariate Matrix Theory for Classifying Projection Images. Optik 72, 143–148. 395. Kapp, O. H., and The High Resolution, S. T. E. M. (1986). Now and Later. NAS Conference on Recent Advances in Electron and Light Imaging in Biology and Medicine. New York. 396. Jiye, X., and Kapp, O. H. (1986). Supplement to Three Dimensional Algebraic Recon- struction from Projections of Multiple Grey Level Objects. Optik 73, 10–12. 397. Jiye, X. (1986). Advances in Electronics and Electron Physics, Suppl. 17. in Academic Press. 398. Crewe, A. V. (1986). Limits of the STEM. Proc. 44th Ann. EMSA Mtg. pp. 764–765. 399. Mu, C., and Wolbach, W. (1986). Particulate Carbon at the Cretaceous-Tertiary Bound- ary. Proc. 44th Ann. EMSA Mtg. pp. 782–783. 400. Mu, C., and Shao, Z. (1986). Inexact Reconstruction with Added Noise. Proc. 44th Ann. EMSA Mtg. pp. 184–185. 401. Mittleman, R. K., and Parker, N. W. (1986). SbC15 Intercalated Graphite. Proc. 44th Ann. EMSA Mtg. pp. 780–781. 402. Mittleman, R. K., and Parker, N. W. (1986). Micrograph Image Processing System. Proc. 44th Ann. EMSA Mtg. pp. 872–873. 403. Barrett, T. (1986). Reconstruction with Noisy Data. Proc. 44th Ann. EMSA Mtg. pp. 186–187. 404. Mainwaring, M. G., Lugo, S. D., Fingol, R. A., Kapp, O. H., and Vinogradov, S. N. (1986). The Dissociation of the Extracellular Hemoglobin of Lumbricus terrestris at Acid pH and its Reassociation at Neutral pH. J. Biol. Chem. 261, 10899–10908. 405. Crewe, A. V. (1986). What to Teach About the Physics of the SEM. ‘‘SEM Mtg.’’ New Orleans. 406. Crewe, A. V. (1986). Thoughts on Images and Objects. Institute for Imaging Science. Univ. of Chicago. 407. Crewe, A. V. (1986). High Resolution Scanning Transmission Electron Microscopy. Frontiers of Electron Microscopy in Materials Science. Argonne National Laboratory. 408. Crewe, A. V. (1986). Is There a Limit to the Resolving Power of the SEM? Proc. XIth Int’l Congress on Electron Microscopy III, pp. 2105–2108. Kyoto. 409. Crewe, A. V. (1986). A Totally New Computer-Controlled High Resolution SEM. 5th Ann. Symposium on Adv. in Electron Microscopy. Beaufort, NC. 410. Crewe, A. V. (1986). Scanning Electron Microscopy at the Atomic Level. Proc. 1st Int’l New Materials Conf. and Exhibition 1, pp. 43–45. Osaka. 411. Vinogradov, S. N., Lugo, S. D., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1986). Bracelet Protein: A Quaternary Structure Proposed for the Giant Extracellular Hemoglobin of Lumbricus terrestris. Proc. Nat’l Acad. Sci. Sciences (USA) 83, 8034–8038. The Work of Albert Victor Crewe on the STEM 57

412. Listvan, M. (1986). Determination of Time Dependence of Domain Growth by Direct Observation of Small Metal Clusters. Surf. Sci. 173, 294–309. 413. Lugo, S., Kapp, O. H., Mainwaring, M., and Vinogradov, S. N. (1985). The Dissociation of the Extracellular Hemoglobin of Lumbricus terrestris at Acid pH and its Reassociation at Neutral pH. 30th Ann. Mtg. Biophysical Society; Biophysical Journal 49, p. 543a. 414. Vinogradov, S. N., Lugo, S., Mainwaring, M., and Kapp, O. H. (1985). Bracelet Protein: A New Model of the Quaternary Structure of Lumbricus terrestris Hemoglobin. 30th Ann. Mtg. Biophysical Society; Biophysical Journal 49, 543a. San Francisco. 415. Lugo, S., Kapp, O. H., Mainwaring, M., and Vinogradov, S. N. (1985). The Dissociation of the Extracellular Hemoglobin of Lumbricus terrestris at Neutral pH. 30th Ann. Mtg. Biophysical Society; Biophysical Journal 49, p. 543a. San Francisco. 1987

416. Parker, N. W., Mittleman, R. K., and Crewe, A. V. (1987). New Scanning Transmission Electron Microscope Microanalytical System. Rev. Scientific Instruments 58, 174–182. 417. Shao, Z. (1987). Probe Size and 5th-Order Aberration. 45th Ann. EMSA Mtg. 134–135. 418. Shao, Z., and Crewe, A. V. (1987). Chromatic Aberration and Low-Voltage SEM. Proc. 45th Ann. EMSA Mtg. 546–547. 419. Shao, Z., and Crewe, A. V. (1987). Spherical Aberrations of Multipoles. J. Appl. Physics 62, 1149–1153. 420. Shao, Z. (1987). A Study on Multipole Systems as Correctors. Optik 75, 152–157. 421. Shao, Z., Mu, C., and Kapp, O. H. (1987). Inexact Reconstruction of Multiple Grey Level Objects from their Projections. Optik 76, 12–17. 422. Crewe, A. V. (1987). Optimisation of Small Electron Probes. Ultramicroscopy 23, 159–168. 423. Shao, Z., and Crewe, A. V. (1987). Chromatic Aberration Effects in Small Electron Probes. Ultramicroscopy 23, 169–174. 424. Mittleman, R. K., Parker, N. W., and Crewe, A. V. (1987). Electron Diffraction Study of 36, SbCl5 Intercalated Graphite. Phys. Rev. B 7590–7600. 425. Beck, V. D. (1987). A Wave Optical Treatment of Space Charge. Optik 76, 167–169. 426. Kapp, O. H., Mainwaring, M. G., Vinogradov, S. N., and Crewe, A. V. (1987). A Scanning Transmission Electron Microscopic Examination of the Hexagonal Bilayer Structures Formed by the Reassociation of Three Out of the Four Subunits of the Extracellular Hemoglobin of Lumbricus terrestris. Proc. FASEB Mtg. 46, p. 2266. Philadelphia. 427. Kapp, O. H., Mainwaring, M. G., Vinogradov, S. N., and Crewe, A. V. (1987). A Scanning Transmission Electron Microscopic Examination of the Hexagonal Bilayer Structures Formed by the Reassociation of Three Out of the Four Subunits of the Extracellular Hemoglobin of Lumbricus terresris. Proc. Nat’l Acad. of Sciences (USA) 84, 7532–7536. 428. Mittleman, R. K. (1987). Electron Diffraction Study of Rubidium-Intercalated Graphite Through the Deintercalation Process. Phys. Rev. B 36, 6001–6011. 1988

429. Mu, C., Shao, Z., and Kapp, O. H. (1988). Three Dimensional Reconstruction from Noisy Projections. Optik 79, 99. 430. Shao, Z., and Crewe, A. V. (1988). A Study on the Optimisation of Apertures in an Aberrated Probe Forming System. Optik 79, 105–110. 431. Shao, Z., Beck, V. D., and Crewe, A. V. (1988). A Study of Octupoles as Correctors. J. Applied Phys. 64, 1646–1651. 432. Shao, Z., and Crewe, A. V. (1988). Ideal Lenses and the Scherzer Theorem: A Supple- ment. Ultramicroscopy 26, 385. 433. Shao, Z. (1988). Toward High-Resolution Low-Voltage SEM. Proc. 46th Ann. EMSA Mtg. pp. 218–219. 58 A. V. Crewe

434. Crewe, A. V. (1988). Instrumentation for a Corrected STEM. Proc. 46th Ann. EMSA Mtg. pp. 652–653. 435. Crewe, A. V. (1988). Some Notes on the History of the STEM. Proc. 46th Ann. EMSA Mtg. pp. 234–235. 436. Mooney, P. E. (1988). A Compact, Digital, Optically Isolated Control and Support System for a Field Emission Source on the 200 Kv STEM. Proc. 46th Ann. EMSA Mtg. pp. 654–655. 437. Shao, Z. (1988). Correction of Spherical Aberrations in the Transmission Electron Microscope. Optik 80, 61–75. 438. Shao, Z. (1988). New Lens for a Low Voltage SEM. Rev. Scientific Instruments 59, 1985. 439. Shao, Z. (1988). On the 5th Order Aberration in a Sextupole Corrected System. Rev. Scientific Instruments 59, 2429–2437. 440. Kapp, O. H., Wall, J. S., and Vinogradov, S. N. (1988). Quaternary Structure and Molecular Weight of the Giant Extracellular Hemoglobins. ‘‘Asilomar Conference on Oxygen Binding Heme Proteins.’’ Pacific Grove, CA. 441. Vinogradov, S. N., and Kapp, O. H. (1988). Determination of the Stochiometry of Multi-subunit Proteins. Asilomar Conference on Oxygen Binding Heme Proteins. Pacific Grove, CA. 442. Kapp, O. H., Wall, J. S., and Vinogradov, S. N. (1988). Bracelet Structures Obtained From Invertebrate Hemoglobins. Proc. Protein Society. San Diego, CA. 443. Suzuki, T., Kapp, O. H., and Gotoh, T. (1988). Novel S-S Loops in the Giant Hemoglobin of Tylorrhynchus heterochaetus. J. Biol. Chem. 263, 18524–18529. 444. Mu, C., Shao, Z., and Kapp, O. H. (1988). Three Dimensional Reconstruction from Noisy Projections. Optik 79, 99. 445. Kapp, O. H., Wall, J. S., Mainwaring, M. G., and Vinogradov, S. N. (1988). The Molecu- lar Dimensions and Masses of Annelid Extracellular Hemoglobins and Chlorocruorins. 32nd Ann. Mtg. Biophys. Soc. Arizona. 446. Vinogradov, S. N., Mainwaring, MG., and Kapp, O. H. (1988). The Structure of the Annelid Extracellular Hemoglobins. 32nd Ann. Mtg. Biophys. Soc. Arizona. 447. Shao, Z., and Crewe, A. V. (1988). A Study on the Optimisation of Apertures in an Aberrated Probe Forming System. Optik 79, 105–110. 448. Shao, Z., Beck, V. D., and Crewe, A. V. (1988). A Study of Octupoles as Correctors. J. Applied Phys. 64, 1646–1651. 449. Shao, Z., and Crewe, A. V. (1988). Ideal Lenses and the Scherzer Theorem: A Supple- ment. Ultramicroscopy 26, 385. 450. Shao, Z. (1988). Toward High-Resolution Low-Voltage SEM. Proc. 46th Ann. EMSA Mtg. pp. 218–219. 451. Crewe, A. V. (1988). Instrumentation for a Corrected STEM. Proc. 46th Ann. EMSA Mtg. pp. 652–653. 452. Crewe, A. V. (1988). Some Notes on the History of the STEM. Proc. 46th Ann. EMSA Mtg. pp. 234–235. 453. Mooney, P. E. (1988). A Compact, Digital, Optically Isolated Control and Support System for a Field Emission Source on the 200 Kv STEM. Proc. 46th Ann. EMSA Mtg. pp. 654–655. 454. Shao, Z. (1988). Correction of Spherical Aberrations in the Transmission Electron Microscope. Optik 80, 61–75. 455. Shao, Z. (1988). New Lens for a Low Voltage SEM. Rev. Scientific Instruments 59, 1985. 456. Shao, Z. (1988). On the 5th Order Aberration in a Sextupole Corrected System. Rev. Scientific Instruments 59, 2429–2437. 457. Kapp, O. H., Wall, J. S., and Vinogradov, S. N. (1988). Quaternary Structure and Molecular Weight of the Giant Extracellular Hemoglobins. Asilomar Conference on Oxygen Binding Heme Proteins. Pacific Grove, CA. The Work of Albert Victor Crewe on the STEM 59

458. Vinogradov, S. N., and Kapp, O. H. (1988). Determination of the Stoechiometry of Multi-subunit Proteins. Asilomar Conference on Oxygen Binding Heme Proteins. Pacific Grove, CA. 459. Kapp, O. H., Wall, J. S., and Vinogradov, S. N. (1988). Bracelet Structures Obtained From Invertebrate Hemoglobins. Proc. Protein Society. San Diego, CA. 1989

460. Shao, Z. (1989). Extraction of Secondary Electrons in a Newly Proposed Immersion Lens. Rev. Scientific Instruments 60, 693–699. 461. Crewe, A. V., Clayton, D. L., Crewe, D. A., and Moscicka, K. (1989). User-Friendly Field Emission. Proc. 47th Annual EMSA Mtg. San Antonio, TX 462. Crewe, A. V. (1989). Small, Cool Lenses. Proc. 47th Annual EMSA Mtg. San Antonio, TX 463. Kapp, O. H., and Vinogradov, S. N. (1989). Direct Calculation of Subunit Stoichiometry from Amino Acid Compositions of Multimeric Proteins and Their Subunits. Interna- tional Symposium on Invertebrate Dioxygen Carriers. Leuven, Belgium. 464. Kapp, O. H., Wall, J. S., and Vinogradov, S. N. (1989). Comparison of the Three-Dimensional Reconstructions of the Hemoglobins of Lumbricus terrestris and Macrobdella decora from STEM data. International Symposium on Invertebrate Dioxygen Carriers. Leuven, Belgium. 465. Kapp, O. H., Schmuck, M., Pilz, I., and Vinogradov, S. N. (1989). Comparison of the Molecular Dimensions and Hydrodynamic Properties of Annelid Hemoglobins Obtained by STEM and SAXS. International Symposium on Invertebrate Dioxygen Carriers. Leuven, Belgium. 466. Kapp, O. H., and Vinogradov, S. N. (1989). Direct Calculation of Subunit Stoichiometry from Amino Acid Compositions of Multimeric Proteins and Their Subunits. Invertebrate Dioxygen Carriers, pp. 213–217. University of Leuven Press. 467. Kapp, O. H., Wall, J. S., and Vinogradov, S. N. (1989). Comparison of the Three- Dimensional Reconstructions of the Hemoglobins of Lumbricus terrestris and Macrobdella decora from STEM data. Invertebrate Dioxygen Carriers, pp. 229–233. University of Leuven Press. 468. Kapp, O. H., Schmuck, M., Pilz, I., and Vinogradov, S. N. (1989). Comparison of the Molecular Dimensions and Hydrodynamic Properties of Annelid Hemoglobins Obtained by STEM and SAXS. Invertebrate Dioxygen Carriers, pp. 219–223. University of Leuven Press. 469. Shao, Z., and Lin, P. S. D. (1989). A High Resolution Low Voltage Electron Optical System For Very Large Specimens. Rev. Sci. Instrum. 60, 3434. 470. Shao, Z., and Wu, X. D. (1989). Appl. Phys. Lett. 55, 2696. 1990

471. Crewe, A. V. (1990). Some thoughts on ultra-high Resolution. Int. Symp. Elec. Micro., Beijing, China, p. 1. World Scientific. 472. Crewe, A. V. (1990). Beyond One Angstrom. Proceedings of the XIIth International Congress for Electron Microscopy. 473. Kapp, O. H., Qabar, A. N., Bonner, M. C., Walz, D. A., Wall, J. S., Vinogradov, S. N., Stern, M. S., and Schmuck, M. (1990). Quaternary Structure of the Giant Extracellular Haemoglobin of the Leech. Macrobdella Decora, J. Mol. Biol. 213, 141. 474. Kapp, O. H., Qabar, A. N., and Vinogradov, S. N. (1990). Calculation of Subunit Stoichiometry of Large, Multisubunit Proteins from Amino Acid Compositions. Anal. Biochem. 184, 74–82. 475. Shao, Z., and Wu, X. D. (1990). Properties of a Four-Electrode Adjustable Electron Mirror as an Aberration Corrector. Rev. Sci. Instrum. 61, 1230. 476. Shao, Z., and Wu, X. D. (1990). A study on Hyperbolic Mirrors as Correctors. Optik 84, 51. 60 A. V. Crewe

477. Wu, X. D., and Shao, Z. (1990). A Qualitative Analysis of Chromatic Aberration of ‘‘Thick’’ Electrostatic Lenses. Optik 84, 66. 478. Ruan, S., and Kapp, O. H. (1990). Progress on the Sextupole-Corrected Sub-Angstrom Resolution Scanning Transmission Electron Microscope: An Abstract. 1990 Midwest SPIE Meeting, pp. 27–29. Rosemont, IL, Sept. 479. Zmola, C., and Kapp, O. H. (1990). Image Processing Utilizing an APL Interface: An Abstract. 1990 Midwest SPIE Meeting, pp. 27–29. Rosemont, IL, Sept. 480. Zmola, C., and Kapp, O. H. (1990). Networking of an Electron Microscope Laboratory Internally and to the Internet: An Abstract. 1990 Midwest SPIE Meeting, pp. 27–29 Rosemont, IL, Sept. 481. Ryan, M. J., and Kapp, O. H. (1990). Development of an Image Processing System on a Second Generation RISC Workstation: An Abstract1990 Midwest SPIE Meeting, Rosemont, IL, Sept. 27–29. 482. Kapp, O. H., and Vinogradov, S. N. (1990). The Structure of the Hemoglobin of Eudystilia Vancouverii: Evidence for Isolated Bracelet Protein. 10th International Biophys- ics Congress-Satellite Meeting. Whistler, British Columbia. 483. Vinogradov, S. N., Mainwaring, M., and Kapp, O. H. (1990). The Quaternary Structure of the Giant Hemoglobins and Chlorocruorins. 10th International Biophysics Congress- Satellite Meeting. Whistler, British Columbia. 484. Kapp, O. H., Mainwaring, M. G., Vinogradov, S. N., and Crewe, A. V. (1990). The Extracel- lular Hemoglobin of Amphitrite Ornata: Its Molecular Size, Subunits and Its Dissociation. 10th International Biophysics Congress-Satellite Meeting. Whistler, British Columbia. 1991

485. Crewe, A. V. (1991). An Algebraic approach to the symmetrical einzel lens. Optik 88, 118–125. 486. Crewe, A. V. (1991). A New Characterization of the Magnetic Lens. Optik 89, 70–74. 487. Ruan, S., and Kapp, O. H. (1991). Progress on the Sextupole-Corrected Sub-Angstrom Resolution Scanning Transmission Electron Microscope. SPIE Proceedings 1396, 298–310. 488. Zmola, C., and Kapp, O. H. (1991). Image Processing Utilizing an APL Interface. SPIE Proceedings 1396, 51–55. 489. Zmola, C., and Kapp, O. H. (1991). Networking of an Electron Microscope Laboratory Internally and to the Internet. SPIE Proceedings 1396, 331–334. 490. Ryan, M. J., and Kapp, O. H. (1991). Development of an Image Processing System on a Second Generation RISC Workstation. SPIE Proceedings 1396, 335–339. 491. Walz, D., Chin, J., Timkovich, R., Wall, J., Kapp, O., and Vinogradov, S. (1991). Hierar- chy of Globin Complexes: The Quaternary Structure of Eudistylia vancouverii. J. Mol. Biol. 222, 1109–1129. 492. Shao, Z., and Crewe, A. V. (1989). On the Resolution of the Low-Energy Electron Reflection Microscope. Ultramicroscopy 31, 199–204. 493. Shao, Z., and Wang, Y. L. (1990). On the Optimisation of Ion Micro-Probes. J. Vac. Sci. Technol. B. 8, 95–99. 494. Sharma, P. K., Qabar, A. N., Kapp, O. H., Wall, J. S., and Vinogradov, S. N. (1991). The principal subunit of earthworm hemoglobin is a dodecamer of heme containing chains. In ‘‘Structure and Function of Invertebrate Oxygen Binding Proteins: Proceed- ings Biophysical Society Satellite Symposium of the Xth International Biophysics Con- gress’’, (Vinogradov, S. N. and Kapp, O. H., eds.), pp. 65–72. Springer-Verlag. 495. Sharma, P. K., Qabar, A. N., Kapp, O. H., Wall, J. S., and Vinogradov, S. N. (1991). Studies of the Dissociation of Eudistylia Vancouverii chlorocruorin. In ‘‘Structure and Function of Invertebrate Oxygen Binding Proteins: Proceedings Biophysical Society Satellite The Work of Albert Victor Crewe on the STEM 61

Symposium of the Xth International Biophysics Congress’’, (Vinogradov, S. N. and Kapp, O. H., eds.), pp. 79–86. Springer-Verlag. 1992

496. Kapp, O. H., and Chen, C.-T. (1992). Reconstruction from Limited Projection Data. SPIE Proceedings on Biomedical Image Processing and Three-Dimensional Microscopy. San Jose, pp. 26–29 Feb. 9–14. 497. Chen, C.-T., Kapp, O. H., and Wong, W. H. (1992). Stochastic Reconstruction of Incom- plete Data Sets Using Gibbs Priors in Positron Emission Tomography. SPIE Proceedings on Biomedical Image Processing and Three-Dimensional Microscopy. San Jose, pp. 131–134 Feb. 9–14. 498. Kapp, O. H., and Ruan, S. (1992). Analysis of STEM images on a RISC workstation with an APL interface. Proc. SPIE 1778, 117–119. 499. Ruan, S., and Kapp, O. H. (1992). An Automatic Field Emission Tip Conditioning System. Review of Scientific Instruments 63, 4056–4060. 500. Ruan, S., and Kapp, O. H. (1991). Progress on the sextupole-corrected sub-angstrom resolution scanning transmission electron microscope. SPIE Proceedings 1396, 298–310. 501. Ruan, S., and Kapp, O. H. (1992). An Automatic Field Emission Tip Conditioning System for the sub-angstrom field-emission STEM. In ‘‘SPIE Proceedings’’, (Ruan, S. and Kapp, O., eds.), SPIE Proceedings 1778, pp. 103–106. 502. Ruan, S., and Kapp, O. H. (1993). Scan System for a Sextupole-corrected Scanning Transmission Electron Microscope. Review of Scientific Instruments 64, 667–671. 503. Vinogradov, S. N., Walz, D. A., Pohajdak, B., Moens, L., Kapp, O. H., Suzuki, T., and Trotman, C. N. (1993). Adventitious Variability? The Amino Acid Sequences of Non- vertebrate Globins, Comp. Biochem. Physiol. B 106, 7–26. 504. Crewe, A. V. (1991). The Three Element Electrostatic Lens. Optik 90, 151–157. 505. Crewe, A. V. (1992). Electron motion in Tuned Fields. I. The Algebra. Ultramicrscopy 41, 269–277. 506. Crewe, A. V. (1992). Electron motion in Tuned Fields. II. Some Applications. Ultramicro- scopy 41, 279–285. Chapter 2

A Review of the Cold-Field Electron Cathode

L. W. Swanson and G. A. Schwind

Contents 1. Introduction 63 2. Work Function 65 3. Energy Distribution 67 3.1. Theoretical Background 67 3.2. Analytical versus Numerical Results 68 3.3. Measured Values of the FWHM for the Tungsten Cold-Field Electron 72 4. Source Optics 79 5. Column Optics Using the Cold-Field Electron Source 82 6. Current Stability 86 6.1. High-Frequency Current Fluctuations 87 6.2. Long-Term Current Drift 88 6.3. Cold-Field Electron End-of-Life Mechanisms 94 7. Summary 97 Acknowledgments 98 References 98

1. INTRODUCTION

The room temperature field electron (FE) source, coined the cold-field electron (CFE) source to distinguish it from the high-temperature thermal-field electron (TFE) and Schottky electron (SE) emission modes of operation, is currently used in some transmission electron microscopy (TEM),

FEI Co. Hillsboro, Oregon, USA

63 64 L. W. Swanson and G. A. Schwind scanning transmission electron microscopy (STEM), and scanning electron microscopy (SEM) products. A review of the key source characteristics (e.g., energy spread, brightness, emitter geometry, life, and stability) of the CFE source needed for successful electron optical applications are given. The TFE and SE modes of operation (Swanson and Schwind, 2009) and the basic science of field emission have been covered by numerous reviews (Dyke and Dolan, 1956; Gomer, 1961; Good and Mu¨ ller, 1956; Hawkes and Kaspers, 1989; Modinos, 1984; Mu¨ ller, 1951; Murphy and Good, 1956; Swanson and Bell, 1973) and are not addressed herein. Many of the early post–World War II investigations of field emission phenomena came from three groups led by Professors Erwin Mu¨ ller (Pennsylvania State University), Robert Gomer (University of Chicago), and Walter Dyke (Linfield Research Institute, McMinnville, Oregon). One of the authors (L.W.S) was a part of the latter two groups. The Dyke group had an early emphasis on a variety of possible applications of CFE and TFE sources and formed Field Emission Corpo- ration in the early 1960s and was the first company to use the FE source in a commercial product. Another research group at the University of Chicago led by Professor Albert Crewe in the late 1960s was instrumental in the successful application of the CFE cathode to both SEM (Crewe, Wall, and Walter, 1968) and STEM (Crewe, Isaacson, and Johnson, 1969) applications. Subsequently a variety of companies (e.g., Coats and Welter, Hitachi, JEOL, Vacuum Generators, and others) used the CFE cathode for commercial SEM, TEM, and STEM applications. Although the W/ZrO SE source (Swanson and Schwind, 2009) has since become the primary FE source used in most commercial SEM and TEM instruments, the CFE source may have some advantages for the highest-resolution STEM, TEM, and EELS applications. Despite the fact that a great variety of refractory metals, metalloids, semiconductors, carbon nano tubes, and a variety of nano tips have been investigated for their FE properties, the tungsten CFE source with an apex radius in the 100- to 200-nm range has continued to be the choice for commercial applications using the CFE source. Among other requirements, for a FE source to be useful for electron optical applications it must have its dominant emission direction on the optical axis and be resistant to long-term current drift and high-frequency current fluctuations in practical vacuum environments. As discussed in more detail in Section 2, it is necessary to orient the tungsten crystal so that the low work function planes are situated on the emitter apex. Simultaneously the FE source must provide adequate values of the brightness and energy spread at the required beam current. In this review, attention will be focused on results obtained from the tungsten CFE source oriented with the <310> and <111> crystallographic directions along the source axis. Emission characteristics of A Review of the Cold-Field Electron Cathode 65 interest for TEM and SEM applications such as energy spread and brightness are discussed in Sections 3 and 4. A comparison between a CFE and SE source in a typical SEM column is given in Section 5 and Section 6 addresses current stability issues.

2. WORK FUNCTION

Figure 1 shows the emission pattern from a tungsten emitter fabricated from drawn polycrystalline wire. For most electron optical applications, the useful current is contained in a small solid angle centered on the emitter axis. Unfortunately, emitters electrochemically etched from drawn tungsten are always oriented with the high work function (110) plane perpendicular to the emitter axis, resulting in negligible emission for a small acceptance aperture centered on the emitter axis. Interestingly, as shown in Table 1, the variation in work function with crystal face has an approximate inverse relationship with the atomic plane density in accordance with the semi-quantitative theoretical expectations put forth by Smoluchowski (1941). This has implications for the high-frequency current fluctuations discussed in Section 6. In the early 1960s the Dyke group at the Linfield Research Institute developed a zone melting technique using seed crystals of various

100 211

110

FIGURE 1 Emission pattern of a clean W<110> oriented emitter with the major crystal planes noted. 66 L. W. Swanson and G. A. Schwind

TABLE 1 Work function (Measured at 77K and 300K) and ﬂicker noise values for various planes of tungsten

Atom Density %Flicker Noise Crystal Plane Work Function (eV) (nm–2) (0–1 kHz) at 500K* 310 4.25 0.08* 6.34 8.3 { 111 4.47 0.02 5.79 2.6 112 4.96 0.09* 8.19 2.4 { 100 4.63 0.02 10.03 1.1 { 110 5.25 0.02 14.18 0

* From Swanson and Crouser (1967) by averaging values from various FE methods. { From Strayer et al. (1973) using an FE-retarding potential method.

orientation to zone melt 2-mm diameter polycrystalline tungsten rod into an oriented single-crystal rod that could subsequently be electrochemically etched to a 0.12-mm diameter wire, from which single-crystal emitters of various orientations could be fabricated. This allowed oriented single-crystal FE sources to be created from almost all refractory materials, including the borides and carbides of refractory metals (Mackie and Hinrichs, 1988). Without the ability to control the emission angular distribution by reliably orienting one of the low work function (i.e., high emission current density) planes on the apex of the FE source, as is the case for many varieties of carbon nano tubes (Dean and Chalamala, 1999; DeJonge et al., 2004), the FE source would be of little value for electron optical applications. In some cases, localization of emission on the emitter axis was accomplished through the formation of nano bumps on the emitter apex or the formation of a pyramid-shaped source FE (as opposed to a hemispherical shape) with a near single-atom emitting area that relies on localizing the emission to the emitter apex by virtue of localizing the highest electric field (Binh and Garcia, 1992; Kuo et al., 2006; Mu¨ ller et al., 1993, Qian, Scheinfein, and Spence, 1993). None of these nano-tip sources has yet appeared on the commercial scene. From the list of the work function values for the major planes of tungsten given in Table 1 (Strayer et al., 1973; Swanson and Crouser, 1967), it is clear that to have one of the low work function planes located at the emitter apex the emitter axis orientation must be <310> or <111>. Historically, the W<310>-oriented emitter has been the emitter orientation used for most commercial applications because of its low value of work function. In a subsequent discussion it will be pointed out that the <111>-oriented tungsten may have some advantages despite its slightly higher work function value. A Review of the Cold-Field Electron Cathode 67

3. ENERGY DISTRIBUTION

3.1. Theoretical Background The original derivation by Young (1959) of the total energy distribution J(e) based on the Sommerfeld free-electron model for the CFE source is e=d e=pd JðeÞ¼JFNe =dð1 þ e Þ; (2.1) ¼ e ¼ where p kT/d, E-Ef is the emitted electron energy relative to the Fermi ¼ level Ef and JFN is the T 0 K current density given by the Fowler– Nordheim (FN) equation as 3 2 e F 3 1=2 JFN ¼ exp½4ð2me’ Þ vðyÞ=F 8p’htðyÞ2 6 2 1:54x10 F = ¼ exp½6:831x10 9’3 2vðyÞ=F A=m2 ; (2.2) ’tðyÞ2 where F and ’ are the electric field (in volts/meters) and work function (in electron volts), respectively, and

1=2 11 1=2 d ¼ heF=4pð2me’Þ tðyÞ¼9:760x10 F=’ tðyÞ ðÞeV : (2.3)

The variables v(y) and t(y) arise from the inclusion of the image potential in the derivation of the FN equation and are tabulated functions ¼ 3 pe 1/2 ’ ¼ –5 (Good and Mu¨ ller, 1956) of the variable y (e F/4 o) / 3.7495 10 F1/2/’. Accurate empirical relationships for v(y) and t(y) have been formulated by Forbes (2006). The temperature range over which the approximations in deriving Eq. (2.1) are valid is defined by p < 0.7. It can be shown that the full width at half maximum (FWHM) of the total energy distribution (TED) at T ¼ 0K is 0.69 d. In addition, the average initial transverse energy of the emitted electrons, d (Swanson and Bell, 1973) can be obtained directly from the TED. For a TED taken at 77K, the value of d can be obtained directly from the trailing edge of the TED from the slope of lnJ(e) versus e. In the case of a retarding energy analyzer where the integral of the TED curve is measured, d can be e obtained from the slope of ln[(I-Io)/Io] versus , where Io is the maximum collected current (Swanson and Crouser, 1967). The two most important parameters characterizing an FE source are ’ b b ¼ and , where F/Vo (Vo is the extraction voltage). If Eq. (2.2) is written in terms of the emitter I(V ) characteristics o " # aðbV Þ2 b’3=2vðyÞ I ¼ o ; 2 exp b (2.4) ’tðyÞ Vo 68 L. W. Swanson and G. A. Schwind the slope ’3=2 ð Þ ¼b s y mfn b (2.5)

2 ’3/2 b of the FN plot (i.e., ln(I/Vo ) vs. 1/Vo) can provide a value of / , where b is a function of emitter radius and cone angle and the emitter to anode spacing. Complicating matters further, the b factor is not constant over the emitting planes of the hemispherical emitter apex (Swanson and Crouser, 1967; Dyke et al., 1953a). Thus, if one uses an FE tube that deflects the beam over a probe aperture such that one can obtain emission from various off-axis crystal planes, a constant b factor cannot be assumed. In short, from the slope of the FN plot alone it is difficult to obtain an accurate value of b and ’. As shown by Young and Clark (1966) and implemented by others (de Jonge et al., 2004; Swanson and Crouser, 1967,), the values of both ’ and b can be obtained if the slope of the FN plot and the value of d obtained from the TED are both available. Combining Eqs. (2.3) and ’ (2.5), the following expression for e is obtained: ð Þ ’ ¼3mfndt y e (2.6) 2VosðyÞ where Vo is the extractor voltage at which the TED was obtained and s(y) ’ is the derivative dv(y)/d(1/Vo). The value of can be inserted in Eq. (2.5) to obtain the corresponding value of b.

3.2. Analytical versus Numerical Results The fundamental equation for current density can be expressed in terms of the normal energy W (Murphy and Good, 1956) ð p 1 ð ; Þ¼4 emekT ð Þ þ W Ef ; J F T 3 D W ln 1 exp dW (2.7) h 0 kT where D(W) and W are the tunneling coefficient and the electron energy normal to the surface, respectively. Similarly, the current density of electrons with total energy E is ð p ð Þ E ð Þ¼4 emef E ð Þ ; J E 3 D W dW (2.8) h 0 where f(E) is the Fermi–Dirac function. Eqs. (2.7) and (2.8) can be integrated numerically to provide values for the current density J(N) and FWHM(N). D(W) is found by numerically solving the one-dimensional Schro¨dinger equation for the following potential barrier: A Review of the Cold-Field Electron Cathode 69

7 j Y A Y 6 C Y B 5 Ef 4 V(z)(eV) 3

1 zc

0 012345 z (nm)

FIGURE 2 Diagram of the surface potential defined by the Eq. (9) image potential model. Incoming, reflected, and transmitted wave functions cA, cB, and cC, respectively, are indicated.

0 for z < zc ð Þ¼ þ ’ 2= pe > (2.9) V z Ef eFz e 16 0z for z zc as depicted in Figure 2. The image potential term in Eq. (2.9) is artificially set at 0 at zc. The wave function for the starting condition for the numerical integration for large positive z to the right of the potential barrier is given by c ¼ Cexp(ikz). For negative z to the left of the potential barrier, the wave function separates into an incident and reflected component as c ¼ Aexp(ikz) þ Bexp(–ikz). The tunneling coefficient follows as 2 C DðWÞ¼ (2.10) A

The complete analytical expression for the current density JTF for an emitter that includes the effect of a finite temperature can be obtained by integrating Eq. (2.1) as follows: ð 1 p ¼ ðeÞ e ¼ p ; JTFE J d JFN ðp Þ (2.11) 0 sin p where the dimensionless parameter p < 0.7. To obtain the analytical e expressions J( ) and JTFE, a variety of approximations must be made that include the well-known Wentzel-Kramers-Brillouin (WKB) approximation in the derivation of JFN. The degree to which these approximations affect the validity of Eqs. (2.1) and (2.7) was discussed previously (Bahm, 70 L. W. Swanson and G. A. Schwind

Schwind, and Swanson, 2008). They compared the above analytical expressions (A) with an accurate numerical (N) calculation of JTFE J (A) and J(e) throughout the CFE, TFE, and SE regimes. Figures 3 and 4 show the ratios of the FWHM(A)/FWHM(N) and J(A)/J(N) obtained from the numerical and analytical (i.e., Eqs. (2.1) and (2.11)) as a function of T for various work function and J(N) values in the low-temperature (i.e., p < 0.7) range. As observed earlier (Bahm, Schwind, and Swanson, 2008), the analytical and numerically calculated values for the FWHM agree within a few percent over the range of ’, J, and T normally encountered for the low-temperature CFE and the higher-temperature TFE emission regimes. In contrast, J(A)/J(N) in Figure 3 shows a significant variation (up to 30%) from unity over the temperature and work function range that Eq. (2.11) is expected to be valid. For the ¼ numerical calculations a value of Ef 12 eV was used for tungsten (Mattheiss, 1965). Interestingly, the Eq. (2.11) analytical expression for J(A) does not depend on Ef, whereas J(N) exhibits a strong dependence on Ef as shown in Figure 5. In contrast, the FWHM(N) values are relatively ¼ independent of over the range Ef 4 to 18 eV. Since the discrepancy occurs only between J(N) and J(A), we suspect the problem lies with the approximations associated with D(W) in formulating the analytical expression for JFN. Further studies are necessary to determine the exact cause of the discrepancy between J(N) and J(A).

1.50 j =4.0eV 1.45 j =4.5eV 1.40 j =5.0eV 1.35 1.30 J(A)/J(N) 1.25 1.20 Ratios 1.15 1.10 FWHM(A)/FWHM(N) 1.05 1.00 0.95 0.90 0 200 400 600 800 1000 1200 T (K)

FIGURE 3 Variation of analytical and numerically calculated current density and FWHM ratios versus T for J(N) ¼ 1 108 A/m2 and the indicated work function values. A Review of the Cold-Field Electron Cathode 71

1.30

1.25

1.20 J(A)/J(N)

1.15

1.10

1.05 Ratio 1.00 FWHM(A)/FWHM(N) 0.95 J(N) = 107A/m2 0.90 J(N) = 108A/m2 J(N) = 109A/m2 0.85

0.80 0 200 400 600 800 1000 1200 T (K)

FIGURE 4 Variation of analytical and numerically calculated current density and FWHM ratios versus T for ’ ¼ 4.5 eV and the indicated J(N) values.

1.5

J(A)/J(N) 1.4 FWHM(A)/FWHM(N)

1.3

1.2

1.1

Ratio 1.0

0.9

0.8

0.7

0.6 0 2 4 6810 12 14 16 18 20

Ef (eV)

FIGURE 5 Variation of analytical and numerically calculated current density and FWHM 9 ratios versus Ef for ’ ¼ 4.5 eV and F ¼ 4.5 10 V/m. J(A) and FWHM values are 8.12 8 2 10 A/m and 0.238 eV, respectively, and independent of Ef. 72 L. W. Swanson and G. A. Schwind

The calculated values of FWHM(N) in Figures 6 and 7 versus T for various values of ’ and J(N) show that the sensitivity of FWHM(N) to J(N) and ’ diminishes with increasing T. The results given in Table 2 for a CFE source operating at T ¼ 300 K show that the dependence of the FWHM(N) values on both work function and current density is minimal. Decreasing values of both ’ and/or J(N) result in a slightly lower value for the FWHM(N).

3.3. Measured Values of the FWHM for the Tungsten Cold-Field Electron Other than the fact that, according to Table 1, the (310) plane of clean tungsten possesses the lowest work function, it is not clear why historically it has persisted as the most favored orientation for most commercial applications. From the standpoint of crystallographic symmetry and the amplitude of intrinsic current fluctuations at low temperatures (to be discussed in section 6.1), a case can be made that the (111) plane might be the preferred plane for electron optical applications. The slight decrease in FWHM(N) with decreasing ’ at a fixed value of J(N), according to Table 2, will provide a minimal advantage for a low ’ CFE source. In addition, the vacuum environment encountered in most commercial

0.50

0.45 4.00 eV 4.50 eV 5.00 eV 0.40

0.35

0.30 FWHM(N) (eV)

0.25

0.20

0.15 0 200 400 600 800 1000 1200

T (K)

FIGURE 6 Variation of the FWHM(N) versus T for the indicated values of work function for J(N) ¼ 1 108 A/m2. A Review of the Cold-Field Electron Cathode 73

0.50 J(N) = 107 A/m2 0.45 J(N) = 108 A/m2 J(N) = 109 A/m2

0.40

0.35

0.30 FWHM (N) (eV) 0.25

0.20

0.15 0 200 400 600 800 1000 1200 T (K)

FIGURE 7 Variation of the FWHM(N) versus T for the indicated values of J(N)for’ ¼ 4.50 eV.

TABLE 2 Calculated values of the FWHM(N) and F for the indicate values of J and w at T ¼ 300 K

J (A/m2) ’ (eV) F(V/nm) FWHM(N) (eV) 1 108 4.0 3.368 0.207 1 108 4.5 4.004 0.222 1 108 5.0 4.680 0.239 1 107 4.5 3.532 0.206 1 108 4.5 4.004 0.222 1 109 4.5 4.616 0.244 applications is such that the CFE source very quickly receives an adsorbed layer of residual gases that alters the work function from the atomically clean surface. Measurement of the energy distribution for the W(310) and W(111) planes of the CFE source has little value for electron optical analysis without the simultaneous measurement of the cathode current density or the more easily measured current per solid angle, referred to as the angular current density (I0). As shown by Wiesner and Everhart (1974), for an FE source with apex radius r the following relationship between I0 and J(N) exists: 74 L. W. Swanson and G. A. Schwind

0 r 2 I ¼ JðNÞ (2.12) m ¼ a y where the angular magnification of the emitted electron m o/ is y a defined as the ratio of the initial launch angle to the final angle o with respect to the emitter axis. Implicit in Eq. (2.12) is the assumption that J and I0 are uniform over the solid angle defined by the beam acceptance aperture, as is usually the case for electron optical applications and TED measurements. Using a sphere-on-orthogonal-cone structure (Dyke et al., 1953a) to model the emitter shape and equipotential surfaces between the cathode and anode, Wiesner and Everhart (1973) calculated a variety of emitter parameters as a function of emitter shape. They found that 0.39

TABLE 3 Summary of emission parameters for W(111) and W(310) planes at T ¼ 77 K *

V b J(N) FWHM(E) FWHM Plane I0 (A/sr) ’ (eV) (volts) d (eV) (m–1) (A/m2) (eV) (N) (eV)

W(111) 0.61 4.47 1306 0.154 2.68 1.02x107 0.25 0.21 W(310) 0.54 4.34 1230 0.149 2.70 8.57x106 0.23 0.21

* From Swanson and Crouser (1967). The I0 values were derived from the given data. FWHM(E) and FWHM (N) are the experimental and calculated values, respectively. A Review of the Cold-Field Electron Cathode 75

’ where VR is the emitter to collector electrode bias potential and c is the effective work function of the collector electrode. An important advantage of the integral analyzer is the relative ease with which the TED and I0 can be simultaneously measured. Figure 8 shows a typical output from an integral analyzer where the experimental curve has been fit to the ’ theoretical curve with d, T, and c as the adjustable parameters. The Eq. (1) theoretical curve is also shown in Figure 8 using the indicated values for the adjustable parameters. From the value for d and the slope ’ b mFN of the FN plot, the work function and factor can be determined from Eqs. (5) and (6) for the particular crystal plane seen by the opening angle of the analyzer. Fitting the theoretical expression Eq. (1) to the experimental data in Figure 8 requires a temperature of 530 K, even though the measurement was carried out at 300 K. This anomaly is due to the well-known and well-studied effect (Boersch, 1954) caused by statistical mutual Coulomb interactions between electrons in the high current density region near the cathode surface and, to a lesser extent, by the finite resolution of the analyzer. This energy-broadening effect, which has been studied extensively both experimentally (Bell and Swanson, 1979;

I'=10mA/sr 1.0 T = 530 K d = 0.19 eV j c = 3.47 eV 0.8

0.6

Differential curve theory Integral curve theory 0.4

Normalized I or J(E) Integral curve experimental

0.2

0.0 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 Voltage

FIGURE 8 The normalized, experimental retarding potential current obtained from a W<111>-oriented emitter at 300K and I0 ¼ 10 A/sr is shown along with the best fit to the calculated curve using T, d, and ’c as adjustable parameters. The differential curve calculated from the experimentally derived ’ and F is also shown. 76 L. W. Swanson and G. A. Schwind

0.8 CFE b (mm−1) 0.7 (a) W<310> 1.30 (b) W<310> 1.90 (a) 0.6 (c) W<310> 1.02 (d) W<111> 1.14 (b) 0.5 (c)

0.4

FWHM (eV) 0.3 (d)

0.2

0.1

0 200 40 60 80 100 120 140 160 180 200 Angular intensity (mA/sr)

0 FIGURE 9 The experimental FWHM(E) values for the TED versus I obtained from the indicated planes of the tungsten CFE source at 300K. Values for ’ and b are given in Table 4.

Fransen et al., 1998; Kim et al., 1997; Troyon, 1988) and theoretically ( Jansen, 1990), will be discussed as it relates to W<310> and W<111> CFE sources. Figure 9 compares experimental data obtained in this laboratory for clean W<310> and W<111> oriented emitters with similar data obtained by Troyon (1988). Table 4 provides the respective values of ’, b, r, and m obtained from the TED and I(V) data. The m values, obtained from Eq. (12) using measure r from Figure 10, are in the range expected from trajectory simulations (Wiesner and Everhart, 1973) for a spherical apex emitter. As observed in previous investigations (Schwind, Magera, and Swanson, 2006; Swanson and Schwind, 2009), the FWHM(E) values increase beyond the respective intrinsic (i.e., calculated) values with increasing I0. Although the data are limited, the contribution of the energy broadening to the FWHM(E) values within Troyon’s and our datasets appears to diminish with increasing b. Because of differing source/anode geometries, comparison of the influence of b on the FWHM values between the two data sets is not possible. Nevertheless, the variation of the energy broadening with b within the two datasets is counter to that observed for the larger-radius SE emitter (Schwind, Magera, and Swanson, 2006). Figure 11 provides a more detailed analysis of the energy broadening for the W<310> and W<111> CFE sources at 300K where a convolution program (Bronsgeest et al., 2007) has been used to extract the energy- A Review of the Cold-Field Electron Cathode 77

(a)

Acc.V Magn 500 nm 10.0 kV 50 000 x 11/29/05 dE W310

(b)

det Landing E mag HFW WD 500 nm TLD 5.00 keV 70 000 x 2.15 mm 3.6 mm FEI NanoSEM 630

FIGURE 10 SEM photos of the W<310> (a) and W<111> (b) CFE sources used to obtain the data for Figures 8–10.

TABLE 4 Emission characteristics obtained from the indicated CFE sources at T¼300 K*

–1 Plane r (nm) ’e (eV) b (m ) m { W(310) — 4.40 1.30 — { W(310) — 4.40 1.90 — W(310) 160 4.32 1.02 0.66 W(111) 120 4.40 1.14 0.62

* Values of the apex radius r were determined from the Figure 11 SEM photos. { Obtained from Troyon (1988) where the value of ’ was derived from the given I(V) data. 78 L. W. Swanson and G. A. Schwind

(a) 0.35 (a) Exp. (a) (b) Intrinsic 0.30 (c) Boersch

0.25 (b)

0.20

0.15 (c) FWHM (eV)

0.10

0.05

0.00 0 2 4 6 8 10 12 14 16 Angular intensity (mA/sr)

(b) 0.6 (a) Exp. (b) Boersch 0.5 (c) Intrinsic (a)

0.4

0.3 (b)

FWHM (eV) (c) 0.2

0.1

0.0 020406080100 120 140 Angular intensity (mA/sr)

FIGURE 11 Relative contributions of the experimental, intrinsic, and Boersch FWHM values versus I0 for the W<111> (a) and W<310> (b) CFE sources at 300K. broadening Boersch effect from the experimental TED. The Boersch contribution is described by a two-parameter bell-shaped distribution. One of the fitting parameters is the FWHM(B) of the bell-shaped distribution. By fitting the convolution of the intrinsic energy distribution and the bell- A Review of the Cold-Field Electron Cathode 79 shaped distribution with its two fitting parameters to the experimental TED, the Boersch contribution could be adequately determined. The results are presented in Figure 10 in terms of the experimental FWHM(E), intrinsic FWHM(N) (i.e., calculated), and the Boersch FWHM(B) contributions. A comparison between the W<111> and W<310> results shows that the energy broadening as determined by the FWHM(B) values not only increases with I0, but is substantially larger for the W(310) source, which also possesses the smaller value of b. It has been shown for SE sources thatFWHMðBÞ/b jI0n (Schwind, Magera, and Swanson, 2006; Swanson and Schwind, 2009). However, the limited data reported here for the W<310> and W<111> CFE sources suggest that FWHMðBÞ/b jI0n. Since all formulations for the variation of b with the cathode/anode geometry give b / 1=rp (Swanson and Schwind, 2009), we conclude from Troyon’s (1988) results (given in Figure 9) and the more recent results for the W<310> and W<111> CFE sources (given in Figure 10) that the Boersch effect diminishes with decreasing source apex radius. Most theoretical and empirical formulations of the energy broadening for high current density FE sources (Knauer, 1981) or for beam crossovers ( Jansen, 1990) are of the form 0l ð Þ/ I : FWHM B k s (2.14) r Vo

It therefore appears that the exponent k in Eq. (2.14) may be negative for the CFE source. Further experimental and theoretical investigation will be required to complete our understanding of the role of the source radius in the Boersch effect for the CFE source.

4. SOURCE OPTICS

In addition to knowledge of the source energy spread and beam angular intensity as described previously, it is also important to simultaneously know the source reduced brightness Br to fully describe the source optical properties. Unfortunately, Br is not easily measured under the same experimental conditions that the FWHM(E) and I0 values are obtained. A practical definition of Br requires a proper definition of the virtual source size dv as seen from the extractor electrode at voltage Vo.IfI is uniform over the acceptance aperture subtending the source, Br is defined by the following relationship: 4I0 B ¼ (2.15) r p 2 dvVo

Bronsgeest et al. (2008) pointed out that a proper definition of dv must take into account the current density distribution in the source image 80 L. W. Swanson and G. A. Schwind

plane. A useful definition for dv that avoids assumptions regarding the intensity profile is the diameter containing a certain fraction FC of the current. For FC ¼ 0.5, various intensity profiles yield a common value for dv measured by scanning the beam across the knife edge and defining dv by the 25% to 75% edge resolution. In addition, the total probe size obtained by the addition of various aberrations, using the FC ¼ 0.5 definition as described by Barth and Kruit (1996), is free of assumptions regarding the spatial distributions of the various aberrations. The value of ¼ dv obtained for FC 0.5 is denoted as (Bronsgeest et al., 2008) 1=2 r ET dv50 ¼ 1:67 ; (2.16) m Vo where ET is the average initial transverse energy of the emitted electrons. ¼ For the CFE emission regime, ET d; thus, by combining Eqs. (2.15) and (2.16) the following expression for the practical brightness Br50 is obtained: 0 I m 2 B 50 ¼ 1:44 : (2.17) r d r

Eliminating m/r from Eq. (2.17) by use of Eq. (2.12) leads to JðNÞ B 50 ¼ 1:44 ¼ 1:44B ; (2.18) r pd r ¼ p where Br J(N)/ d is the familiar expression for the differential or axial brightness (Worster, 1969). As observed previously (Bronsgeest et al., 2008), the practical source brightness exceeds the axial brightness by 44%. For SEM and TEM applications, one is interested in a FE source that simultaneously provides a large value of Br50 and a small value of ’ b FWHM(E). If and Vo are known, Br50 can be calculated using Eq. (2.18). The latter is plotted versus FWHM(E) in Figure 12 for the W<310> and W<111> sources. Since the W<111> curve lies to the left in Figure 12, it is favored over the W<310> source based on the above criteria. The relatively large calculated values of Br50 in Figure 12 are primarily due to the small values of dv50, or, equivalently, the large values of J(N) for these CFE sources. Values of dv50 obtained from Eq. (2.16) and J b ’ (N) (obtained knowing , Vo, and )aregiveninTables 5 and 6.The < > < > average dv50 values are 2.90 and 2.29 nm for the W 310 and W 111 sources, respectively. The latter values of dv50 are an order of magnitude smaller than the typical dv50 values for the SE source (Swanson and Schwind, 2009). The mutual Coulomb interactions that cause the energy broadening also cause a random, non-focusable modification of the electron trajectories. This leads to a radial broadening dblur of the virtual source size and A Review of the Cold-Field Electron Cathode 81

5.E+09

5.E+09 − CFE b (mm 1) 4.E+09 (a)W<111> 1.14 (b) (b)W<310> 1.01 4.E+09

3.E+09 srV) 2 3.E+09 (A/m r

B 2.E+09

2.E+09 (a) 1.E+09

5.E+08

0.E+00 0.20 0.25 0.30 0.35 0.40 0.45 0.50 FWHM (eV)

FIGURE 12 Graph shows the source reduced brightness Br versus the corresponding FWHM(E) values for the two CFE sources.

TABLE 5 Values of various emission parameters for the W<310> CFE at 300 K*

dv50 FWHM(E) FWHM(B) I0 (A/sr) F (V/nm) J(N) (A/m2) d (eV) (nm) (eV) (eV) 7.5 3.840 1.38 108 0.190 2.76 0.357 0.199 19 4.040 3.22 108 0.201 2.90 0.380 0.247 44 4.242 6.93 108 0.211 2.98 0.427 0.271 62 4.340 9.94 108 0.216 2.98 0.412 0.236 116 4.545 19.5 108 0.227 2.89 0.507 0.369

* The relevant values of ’, b, r, and m are given in Table 4.

TABLE 6 Values of various emission parameters for the W<111> CFE at 300 K*

dv50 FWHM(E) FWHM(B) I0 (A/sr) F (V/nm) J(N) (A/m2) d (eV) (nm) (eV) (eV) 2 3.735 5.42 107 0.165 2.78 0.237 0.039 5 3.932 1.32 108 0.174 2.31 0.273 0.062 10 4.104 2.71 108 0181 2.28 0.301 0.080 15 4.206 4.02 108 0.186 2.29 0.319 0.093

* The relevant values of ’, b, r, and m are given in Table 4. 82 L. W. Swanson and G. A. Schwind

a concomitant decrease in Br50. Although dblur occurring at a beam crossover in an optical column has been extensively studied (Jansen, 1990; Kruit and Jansen 2009), the nonhomogeneous field accelerating the particles in the region near the emitter surface of the FE source renders it impossible to treat the radial broadening analytically. Nevertheless, it is 9 2 expected that the calculated values of Br50 above 1 10 A/m srV in Figure 12 will be substantially lowered due to the radial broadening that increases dv50 beyond the value calculated via Eq. (2.16). 5. COLUMN OPTICS USING THE COLD-FIELD ELECTRON SOURCE

In a focusing column the conservation of the source brightness Br down to the target leads to the following useful relations between the probe a current Ip, and the convergence half angle i, the virtual source size dp ¼ (dp Mdv50) and image (Vi), and the object (Vo) side voltages: p2 2 0 2 Vi Ip ¼ ViBrðaidpÞ ¼ pI ðMaiÞ (2.19) 4 Vo where Br is defined by (E. 15). The total beam size in the image plane is usually limited by a combination of lens chromatic aberration dc, spherical aberration ds, diffraction dl, coulomb interactions dblur, and virtual source size dv. The contributions of these terms to the total beam size in the image plane have been given by Barth and Kruit (1996) in terms of the beam size dt50 that contains 50% of the beam current as follows: 663:6 dl ¼ ðÞnm (2.20) 1=2a Vi i dV dc ¼ 600Cc aiðÞnm (2.21) Vi

¼ : 4 a3ð Þ ds 1 8x10 Cs i nm (2.22) hi1=2 : = : = 2=1:3 ¼ 4 þ 4 1 3 4 þ 2 2 þ 2 2 1 3 2 þ 2 ; dt50 dl ds M dv50 M dblur dc (2.23) where M is the overall column magnification. The image-side voltage Vi is in volts, the spherical and chromatic aberration coefficients (Cs and Cc, respectively) are in millimeters, and dV is the full width containing 50% of the particles (FW50) value in electron volts of the energy spread. The above formulation of the aberration contributions gives results that are comparable to the more rigorous wave optical approach. A Review of the Cold-Field Electron Cathode 83

The total chromatic Cct and spherical Cst aberration coefficients of a two-lens optical system are given by (Orloff, 1983) 3=2 2 Vi Cct ¼ Cci þ CcoM (2.24) V0 3=2 4 Vi Cst ¼ Csi þ CsoM ; (2.25) V0 where Cci and Csi are the image-side aberration coefficients of the objective lens and Cco and Cso are the object-side aberration coefficients of the gun lens. Sources with a large value of dv50 (e.g., an SE source with dv50 30 to 60 nm) require the column to be operated in a strongly demagnified mode. This, combined with a high Vo (e.g., 5 kV), results in such a small gun lens aberration contribution that it can typically be ignored or at most considered a minor contribution to the total column aberrations. To illustrate the latter and other contrasts between the CFE and SE sources, a comparison between the commonly used W<100>ZrO SE and the W<111> CFE sources will be made using aberration coefficients of a typical focusing column. Tables 7 and 8 give source parameters and the focusing column aberration coefficients used to calculate dt50 versus Ip for a high (30-kV) and low (1-kV) beam voltage. A computer program was used to calculate the a optimum values of M for a range of i values to minimize dt at each value a of i using Eqs. (2.19) to (2.25). The results of the latter calculations are

TABLE 7 Working distance and column aberrations used for the ﬁgure 13 analysis

Cso (mm) Cso (mm) Cci (mm) Csi (mm) Working Distance (mm) 20 75 0.58 0.49 1.0

TABLE 8 Source parameters used for the ﬁgure 13 analysis

W(111) W(100)ZrO I0 (A/sr) 15 200 Vo (V) 3693 4032 r (nm) 120 370 m 0.62 0.21 FW50 (eV) 0.214 0.37 dv50 (nm) 2.29 16.8 ’ (eV) 4.40 2.92 T (K) 300 1800 84 L. W. Swanson and G. A. Schwind

(a) 100 (b)

(a) WZrO SE (b) W(111) CFE

(a)

10 50 (nm) t d

(a) (b) 1 0.01 0.1 1 10 100 1000 10,000 100,000 1000,000 Ip (pA) (b) 100 (b) (a) WZrO SE (b) W(111) CFE (a)

10 50 (nm) t d (a) 1

(b)

0 0.01 0.1 1 10 100 1000 10,000 100,000 1000,000

Ip (pA)

FIGURE 13 Plots of the beam size dt50 versus beam current Ip for a typical SEM column for a beam voltage of 1 kV (a) and 30 kV (b). The W<111> CFE source is compared with a W(100)ZrO SE source. Source and column parameters are given in Tables 7 and 8. The dashed lines show the effect of eliminating the gun lens aberrations.

< given in Figure 13. Not surprisingly, for Ip 1000 pA the CFE source provides a lower value of dt than the SE source, although less so for the 30-kV beam voltage. The individual contributions to dt are detailed in A Review of the Cold-Field Electron Cathode 85

(a) 100 (a) Total (b) Diffraction (c) Chromatic (d) Spherical (c) 10 (e) Virtual source

(a) 1

d (nm) (d) (b)

0.1 (e)

0.01 0.1 1 10 100 1000 10,000 100,000 Ip (nA) (b) 100 (a) Total (b) Diffraction (c) Spherical (d) Chromatic 10 (e) Virtual source

d (nm) (a) (b) (c) 0.1 (e)

(d) 0.01 0.1 1 10 100 1000 10,000 100,000 Ip (nA)

FIGURE 14 Aberration contributions for the Figure 13 (a) dt versus Ip results for a beam voltage of 1 kV (a) and 30 kV (b).

< Figure 14 for the CFE source. For Ip 1000 pA diffraction, chromatic and ¼ spherical aberrations are the primary contributors to dt at Vi 1 kV, whereas ¼ at Vi 30 kV the primary contributions come from the spherical and 86 L. W. Swanson and G. A. Schwind

¼ diffraction terms. At Ip 1000 pA, all aberrations contribute more or less equally. For different values of Cst and Cct, the relative contributions will be altered. < The improvement in performance for Ip 1000 pA using the CFE source comes with the requirement for a larger value of M (Figure 15), which in turn leads to a larger contribution from the gun lens (see dashed curves in Figure 13) to the overall aberration load in accordance with Eqs. (2.24) and (2.25). As noted previously (Kruit, Bezuijen, and Barth, 2006), this is a consequence for any CFE that achieves a large value of Br 0 by virtue of a small value of dv as opposed to a large value of I (see Eq. (2.15)), which is the case for the SE source. The larger required value of M for the CFE source implies more mechanical stability and magnetic shielding requirements for the column. Not withstanding beam current stability issues, the CFE source is a superior source for applications requiring a high resolution or a highly coherent focused beam.

6. CURRENT STABILITY

For electron optical applications, current stability considerations are confined to a relatively small fraction of the total current contained in a 1 to 10 mr semi-angle centered on the optical axis of the source. Since the basic

(a) W(111) CFE (b) WZrO SE 1

M 0.1

(a) 0.01 (b)

0.001 0.1 1 10 100 1000 10,000 100,000

Ip (pA)

FIGURE 15 Graph shows the optimum column magnification M corresponding to the Figure 13 (a) dt versus Ip results. A Review of the Cold-Field Electron Cathode 87 mechanisms associated with long current drift and short-term current fluctuations differ, it is instructive to consider the current stability issues in terms of high-frequency fluctuations about a mean current and long- term current drift usually in the direction of reduced current. According to the FN Eq. (2.2), current fluctuations and drift are due to fluctuations or drift in ’ and/or b. Ignoring the pre-exponential term in Eq. (2.2), it can be shown that small current fluctuations dI are given by dI b’3=2 3b’1=2 ¼ dF ¼ d’; (2.26) I F2 2F where b ¼ 6.83 109 (V/m(eV)–3/2), dF ¼ Vdb. The workfunction fluctuations d’ originate from fluctuations in the adsorbed molecules or atoms dn in the emitting area Ap subtended by the acceptance aperture as follows: d’ ¼ cdn; (2.27) ¼ pm m where c 4 /Ap, where is the dipole particle per adsorbed particle. Thus, a change in ’ can come about only from a change in the adatom m b coverage n/Ap or . To change I due to a change in requires either a microscopic geometric change (atomic size within Ap) or a macroscopic geometric change in the emitter shape.

6.1. High-Frequency Current Fluctuations The following mechanisms can contribute to varying degrees to fluctuations in I: 1. With a specific pressure of residual gases, fluctuations in n can be caused by adsorption/desorption processes. 2. Fluctuations in m can occur due to thermal induced transitions between two bonding states of adsorbed molecule (Kleint, 1971). 3. Fluctuations in n will occur for mobile adsorbed molecules or atoms due to surface diffusion in and out of Ap (Gomer, 1973). It has been shown that mechanism 3 in the list above can occur even for an atomically clean surface due to mobile, self-adsorbed atoms on a particular crystal plane (Swanson, 1978). These intrinsic current fluctuations vary significantly with crystal plane as shown in Table 1 where the value of dI/I values are approximately inversely proportional to the surface atom density of the respective crystal plane. As an illustration of the highly anisotropic nature of this current fluctuation mechanism, dI/I (310)/dI/I(100) ¼ 7.5 at 500K (see Table 1) and increases to 67 at 900 K (Swanson, 1978). The temperature threshold where these fluctuations exceed the statistical shot-noise level is as low as 300 K for the (310) 88 L. W. Swanson and G. A. Schwind plane for a frequency bandwidth of 10 kHz (Swanson, 1978). Interest- ingly, the Table 1 summary of current fluctuations suggests a lower value for the W(111) versus the W(310) plane. To illustrate the extreme sensitivity of a CFE source to a small change ¼ 2 in ad atomdensity (n/Ap); Ap 19.6 nm for a typical optical column a ¼ ¼ ¼ application with o 10 mr, r 150 nm and m 0.6. For the W(111) plane, the latter value of Ap contains 114 substrate atoms; thus, one atom diffusing in or out of Ap represents a 1% change in n/Ap. Besocke and Wagner (1973, 1975) observed that the workfunction of the W(110) plane decreased monotonically with increasing concentration of adsorbed tungsten atoms on the W(110) terrace. According to Eq. (2.26), a 1% variation in ’ would cause a 28% variation in I for a CFE source with a workfunction of 4.5 eV and a typical applied field of 3.5 109 V/m. Similarly, a 1% variation in b would result in an 18% variation in I. Clearly, both ’ and b must be controlled to stabilize current from a CFE source. Investi- gations by Saitou (1977) and Todokoro Saitou, and Yamamoto, (1982) suggest that current fluctuation due to ion bombardment dominates > –12 when PxIt 7 10 PaA, whereas current fluctuations due to mobility of adsorbed gas molecules dominates at lower values of PxIt. As shown elsewhere (Swanson and Schwind, 2009), the magnitude of the current fluctuations for an SE source increases with decreasing emitter radius and acceptance aperture solid angle. This result is not unexpected since the rms amplitude of concentration fluctuations for a mobile –1/2 adsorbed layer is proportional to Ap (Swanson, 1978). However, as the radius approaches that of a single atom, as is the case for some versions of room temperature nano tips (Kuo et al., 2006), the lifetime with respect to atomic displacement appears to be exceedingly large. All fluctuations due to atomic motion eventually cease, and only shot noise from the statistics of the emission process remains as the CFE source temperature is reduced below room temperature.

6.2. Long-Term Current Drift If adsorption of foreign molecules from the gas phase or from electron- stimulated desorption (ESD) of neutrals and ions (Madey and Yates, 1971) from electrode surfaces were eliminated, then all adsorbate-induced noise and long-term drift related to workfunction change would be minimal. Such a result was approached by the Dyke group in 1960 (Martin, Trolan, and Dyke, 1960) where stable emission current with limited fluctuation and drift was obtained for more than 2500 hours for total currents in excess of 100 A. This result was obtained with a tungsten CFE source in a sealed-off tube with excellent vacuum (<10–10 Pa) and electrode surfaces that were carefully outgassed and configured so that all secondary electrons (including elastically scattered electrons) were contained. A periodic A Review of the Cold-Field Electron Cathode 89 brief thermal flash of the CFE source at 2000K to remove contaminants and smooth the emitter surface extended the stable emission to 12,000 hours without any indication of a limit on further operation. Field emission devices using a CFE source with electrode surfaces that cannot be properly outgassed and uncontained elastically scattered electrons typically display a current fall off similar to that shown in Figure 16. The current decay after thermal cleaning of the cathode is substantial during the first hour of operation, even though a remotely positioned vacuum gauge suggests an adequate base pressure. The gas flux F is denoted by

¼ : 24 sP ð = 2 Þ; F 2 64x10 = atoms m sec (2.28) ðMTÞ1 2 where the P, M, and s are the pressure in Pascals, the molecular weight of the gas, and the sticking coefficient, respectively. Assuming a monolayer of residual gas requires 1019 atoms/m2, for P ¼ 4.0 10 8 Pa and s ¼ 0.1, one obtains from Eq. (2.28) the order of 105 seconds (28 hours) to form a monolayer. Given that the current fall-off for curve (a) in the Figure 16 CFE source is much faster than would be expected based on adsorption of electronegative gases from the residual vacuum, one

1.0 m Ј m 0.9 It( A) I ( A/sr) Vo(volts) (a) 1530 3774 0.8 (b) 3 5 3367 (c) 15 30 3780 0.7

0.6

0.5

0.4 (c)

0.3

Angular intensity (normalized) 0.2 (b) 0.1 (a) 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Time (h) FIGURE 16 Normalized angular intensity versus time for a W<111> CFE source at 300 K at the indicated starting conditions (see inset). The pressure and acceptance aperture semi-angle were 4.0 10 8 Pa and 0.56 mr, respectively. Curve (c) was taken with the extractor voltage off between data points. 90 L. W. Swanson and G. A. Schwind concludes that a local pressure increase at the emitter occurs and is due to other causes. In view of the significant reduction in the rate of emission current decrease observed in curve (b) as the initial total current It is decreased from 15 to 3 A, it is clear that neutrals generated by the total emission current are largely responsible for the increase in local pressure. The latter is confirmed by comparing curves (a) and (c) of Figure 16, where the rate of decrease in emission current is reduced even more by switching the current on only briefly during the current measurement. a ¼ By use of a Faraday cup with a small acceptance aperture ( 1/2 0.56 mr), only a small fraction of the total current was measured in the Figure 16 results. This resulted in an extremely small emitting area (<0.1 nm2)seen by the Faraday cup and accounts for the relatively large amplitude of the current fluctuations. These results are generally in accordance with the early observations of the Dyke group (Martin, Trolan, and Dyke, 1960), who put forth the following mechanisms governing long-term current stability for a CFE source in a high-vacuum environment: 1. In most situations the emission current decreases due to adsorption of electronegative neutral gas generated by primary electrons impinging on the anode surface and, of equal importance, from elastically reflected electrons that impinge on other nearby grounded surfaces. 2. Ions formed by the primary electron beam that impinge on a clean emitter cause an increase in current due to cathode sputtering and localized increases in the b factor. 3. Because of the configuration of the electric field near the emitter apex, only ions formed in a narrow, pencil-like cylinder centered on the emitter axis are able to impinge on the emitting area. Since a sharp maximum in the cross section for electron impact ionization occurs at a few hundred volts, most of the ions impinging on the emitting area are formed near the emitter. More recent investigations have confirmed and clarified the above mechanisms. For example, it is now known that the ESD cross section for neutral generation exceeds the cross section for ion generation by 100 for many electronegative adsorbates on metal surfaces (Madey and Yates, 1971). Further investigation of the ion trajectories formed by ESD at the anode and by electron impact ionization of neutrals in the gas phase confirms that very few ions created in this space reach the emitting area of the CFE source (Vernickel and Welter, 1970). Only those ions formed near the emitter and in a cylindrical volume of diameter approximately three times the emitter radius and centered on the emitter axis impinge on the actual emitting area. These ions impinge on the cathode with energy of 10% of the interelectrode potential (Smith, 1984). A Review of the Cold-Field Electron Cathode 91

1 ϕ i eV m f m i p ( )( ) p ( )/ p ( ) 4.35(a) 0.60 (b) 4.34 1.62

(a)

0.1 (b) Angular intensity (normalized)

I II III

0.01 0 5 10 15 20 25 30 35 Time (h)

FIGURE 17 Normalized angular intensity versus time for a W<310> CFE source at 300 K. Acceptance aperture semi-angle was 5.0 mr. Initial values of the work functions and ratio of the final to initial FN plot slopes are indicated in the inset.

Figures 17 and 18 show life test data taken in this laboratory in a high- vacuum environment using a CFE source and a Faraday cup behind the anode with a small aperture that subtended a 5.0 mr half-angle at the emitter. Emitter radii were 150 nm, thereby limiting the probe currents in Figures 17 and 18 to an emitting area of 1.8 nm2. To demonstrate the 0 life cycle mechanisms, It and the accompanying values of I in Figures 17 and 18 are larger than commonly used in practical applications. Unlike the experiment described above by the Dyke group, no provision was made to either outgas the anode or contain the elastically scattered electrons. Therefore we expect an increase in local pressure at the emitter due to ESD from nearby surfaces where the primary and scattered electrons impinged. In addition to the information provided in the captions and insets for Figures 17 and 18, Table 9 provides additional emission parameters and results for the life tests in Figures 17 and 18. Curve (a) of Figure 17 is a typical life cycle I(t) curve for a CFE source at 300K in this environment. Following Yeong and Thong (2006), the life cycle is divided into three distinct regions as follows: after thermal cleaning is region I, where the total and probe currents decreases rapidly by one to two orders of magnitude; next is region II, where the rapid downward current drift stabilizes for a period of time depending on the base vacuum and the neutral flux at the emitter due to ESD by the primary and scattered electrons; and region III is defined by an increase in the amplitude of current fluctuations and an increasing rate in the upward current drift. 92 L. W. Swanson and G. A. Schwind

1 ϕ i eV m f m i p ( )( ) fn ( )/ fn ( ) T(K) (a) 4.14 1.50 300 (b) 4.08 1.23 820

(b) 0.1 Angular intensity (normalized)

(a)

0.01 0 1020304050607080 Time (h)

FIGURE 18 Normalized angular intensity versus time at two emitter temperatures for a W(310) CFE source. The acceptance aperture semi-angle was 5 mr. Initial values of the work function and ratio of the final and initial FN plot slopes are indicated in the inset.

Yeong and Thong (2006) concluded that mechanisms occurring in the various regions were as follows: Region I. Adsorption of electronegative gases leads to an increase in work function and adsorbate-induced flicker noise as describe in Section 6.1. Region II. Equilibrium coverage of adsorbed gases is reached and the emitting area undergoes occasional ion impact, leading to a mottled field emission pattern with occasional bright spots. Depending on the opening angle of the aperture, current fluctuation amplitudes of a few to several percent and occasional large current spikes are observed. The time duration of region II can be substantially extended as shown in curve (b) of Figure 17, presumably due to a reduction in ESD flux since the base pressure was similar to that of curve (a). Region III. An increase in both the current and the amplitude of current fluctuations occurs due to random formation of adsorbate/substrate nano-protrusions. If left unattended the current increase in region III rapidly reaches a critical point and triggers a catastrophic arc destroy- ing the emitter (Dolan, Dyke, and Trolan, 1953; Dyke et al., 1953b). Additional insight into the above mechanisms can be gained by noting the ratio of the final to initial FN plot slopes mfn (f)/mfn(i) (see Eq. (2.5)) given in the inset of Figure 17. The combination of ion bombardment and TABLE 9 Data for ﬁgures 17 and 18 long-term stability tests*

–1 –1 1 Test b (m ) It (A) I’ (A/sr) Tr (sr ) It (A) I’ (A/sr) Tr (sr ) Figure Temperature (K) Duration (hr) P (10–8 pa) Initial Initial Initial Initial Final Final Final Cathode Electron Cold-Field the of Review A 17a 300 15 3.5 0.962 100 2023 20.2 13.6 479 35 17b 300 29 3.5 1.871 150 530 3.5 9.1 41 4.5 18a 300 21 2.4 0.889 100 1880 18.8 0.3 28.2 94 18b 820 73 2.7 0.888 100 1479 14.8 2.4 157 27

0 0 * The transmission Tr is defined as the ratio of I /It and initial and final values of It, I , and Tr are given. 93 94 L. W. Swanson and G. A. Schwind formation of nano-protrusions in the emitting area occurring in region III of curve (a) leads to a decrease in ’ (due to sputtering of electronegative adsorbate molecules) and an increase in b (due to formation of nano- protrusions). The latter would result in a decrease in mfn(f )/mfn(i) as observed. In contrast, for curve (b) of Figure 17 region III was not reached during the 30 hours of operation; therefore the dominant mechanism in play was workfunction increase due to adsorption and a limited change in b due to ion impact. In this case, we expect an increase in mfn(f )/mfn(i) as observed in the inset in Figure 17. Further evidence of surface modification during operation in region III is given in Table 9, where the final ¼ 0 value of the transmission (Tr I /It) has increased for curve (a) of Figure 17 while remaining nearly unchanged for curve (b). In most cases, the equilibrium coverage y of adsorbed molecules follows an equation of state y(P,T), such that y decreases with increasing T at a constant P. This is illustrated in Figure 18, where a life test on a W<310> CFE source at 300K is compared with a similar test at 820K. The test for curve (a) carried out at T ¼ 300K was terminated after 21 hours before entering the unstable region III regime. In contrast, the test for curve (b) carried out at 820K was terminated after 73 hours and showed no sign of entering the unstable region III regime. The value of I0 in curve (b) in Figure 18 decreased to 11% of its starting value compared with 1.5% for curve (a). From the inset in Figure 18, the mfn(f )/mfn(i) ratio increased for both curves, thereby indicating that an increase in workfunction was the primary cause of the current change for both tests. At 820K the atomic displacements due to ion sputtering of the cathode are rapidly annealed, thereby minimizing localized protrusion growth as discussed by Yeong and Thong (2006). These results show that operation at an elevated temperature can not only maintain a higher I0, but also significantly extend the region II regime longevity. However, an increase in temperature to further reduce the gas coverage will increase surface mobility and lead to undesirable field-induced emitter shape change (Bettler and Charbonnier, 1960) and, according to Figures 6 and 7, will increase the intrinsic FWHM(N).

6.3. Cold-Field Electron End-of-Life Mechanisms In this section, a few other mechanisms are discussed that cause end of life for the CFE source when a catastrophic arc, such as may occur with extended operation in region III, does not occur. Use of the CFE source in a commercial SEM or TEM usually involves software that implements an automated thermal processing of the source at the transition between regions II and III to reduce the prospect of a catastrophic vacuum arc and to return the emitter state to the starting region I condition. This thermal processing requires heating the source to a temperature and time A Review of the Cold-Field Electron Cathode 95

sufficient to remove most adsorbed gases (e.g., CO, N2,H2,H2O) and anneal atomic displacements due to cathode sputter damage of the surface. Usually a 60-sec heating period in the range of 1600 to 1800Kis sufficient to remove most contaminants except the last traces of WOx (Bell, Swanson, and Crouser, 1969). Care must be taken not to exceed 1800K during thermal processing to prevent massive dulling of the b emitter and the concomitant decrease and increase in and Vo, respec- < > tively. Radius growth for a single-crystal W 100 source was measured experimentally (Swanson and Schwind, 2009) and when analyzed according to a surface tension–based radius-dulling model (Barbour et al., 1960, Dyke et al., 1960) gave the following relation: at E r4 r4 ¼ 2:18 105 exp d ðmm4Þ; (2.29) f i T kT a where ri,rf,Ed, and ( in radians) are the initial and final emitter radii, activation energy for diffusion, and emitter cone semi-angle, respectively. The two emitter shapes shown in Figure 19 were fabricated by different electrochemical etching processes (Melmed, 1991) and are typical of the shapes used in commercial applications of both CFE and SE sources. The emitter shape in Figure 19b, formed by an AC electrochemical etching procedure, has a cone semi-angle of 7 to 10 (measured at 1-k magnification) for an apex radius of 200 to 500 nm. In contrast, the emitter shape in Figure 19a, formed by a DC electrochemical etching procedure, has a minimum cone semi-angle of 3 to 4 at the emitter apex and increases with distance from the apex. For the latter emitter shape, the minimum apex radius achievable is typically 50 to 100 nm, depending on the etching parameters. The emitter shape depicted in Figure 19a is the most commonly used shape for CFE sources because of its smaller apex radius. According to Eq. (2.29), Figure 19a with its smaller initial value of a is a more desirable shape to minimize the dulling rate. Inserting the measured ¼ ¼ a ¼ value of Ed 2.78 eV (Swanson and Schwind, 2009), ri 100 nm and 3 into Eq. (2.29), the radius increases by 20% after 19 hours at T ¼ 1800K. Because of the exponential dependence of the dulling rate on temperature, increasing the temperature to 2000K reduces the latter time to 3.5 hours. If one assumes a 60-sec heating period at 1800K to restore the emitter to its original pre–region I status and a 25-hour operating interval between heating cycles, the CFE source lifetime with respect to a 20 % increase in radius would be 28,500 hours. However, a heating temperature of 2000Kwould reduce the latter lifetime value to 5,250 hrs. Another more subtle end-of-life mechanism for the tungsten CFE source is severe carbon contamination depicted in Figure 20 for a W<110> source. Carbon is not easily removed by heating at 2000K and increases over time by heating the source at elevated temperatures in 96 L. W. Swanson and G. A. Schwind

(a)

Magn 100 mm 350 x 8/7/2007 DC 128A

(b)

Acc.V Magn 100 mm 10.0 kV 350 x 6/12/06 CMC ETCH 67319

FIGURE 19 Typical emitter shapes (a) formed by d.c. and (b) a.c. etching techniques (Melmed 1991). the presence of partial pressures of almost any carbonaceous gas. As shown in Figure 20b, carbon contamination increases the high work function (100) region (see Figure 1) that eventually engulfs the (310) and (210) planes. For a W<310> oriented CFE source a perplexing symptom of severe carbon contamination would be a gradual reduction in the probe current without an accompanying reduction in total current. The only method of removing carbon contamination without seriously dulling the emitter is heating at 1200 to 1500K in a partial pressure of oxygen. An examination of the Figure 20b emission distribution suggests that a similar result may occur for a W<111> emitter orientation. A Review of the Cold-Field Electron Cathode 97

(a) (b) (310)

(210)

FIGURE 20 Field emission patters of a W<110> clean (a) and heavily carbon-contaminated (b) source. The location of the originally low work function (310)–(210) crystal planes are noted.

7. SUMMARY

This review has touched on both positive and negative attributes of the CFE source with respect to microscopy applications. On the positive side, the following features stand out: (i) FWHM values 0.3 eV and lower were measured for I0< 10 A/sr; however, the broadening of the FWHM due to the mutual Coulomb interactions becomes substantial for higher values of I0. (ii) Because of the relatively small values of the virtual source for a CFE emitter, a reduced brightness in the range of 109 A/m2 srV was measured for FWHM values in the range of 0.3 to 0.4 eV. (iii) For SEM and STEM applications, the CFE source can outperform the Schottky source in terms of resolution at small beam currents—this is especially true for low voltage SEM applications, although advances in aberration correctors may mitigate this advantage. (iv) In an ultra–high-vacuum environment where ESD is minimized, there is no fundamental limit to source lifetime. On the negative side are the following: (i) Current fluctuations and long- term drift remain a practical challenge due to ESD and practical limits on achievable vacuum. (ii) The latter problem can be dealt with by a periodic high-temperature flash at 1800K to restore the emitter to its clean and smooth surface condition; however, care must be taken to minimize emitter dulling. (iii) The small virtual source size of the CFE source leads to a higher column magnification necessary for optimum performance; this, in turn, leads to an increased contribution of the gun lens aberrations which, if not obviated, minimizes the advantage of the high source brightness. As these challenges are addressed, the future of the CFE source for high-resolution probe applications appears positive. 98 L. W. Swanson and G. A. Schwind

ACKNOWLEDGMENTS

We are grateful to K. Liu and G. Magera for providing experimental data for this study and to the FEI Co. management for support. One of the authors (L.W.S.) is grateful for the early encouragement and opportunities provided by Professors R. Gomer and W.P. Dyke to study field emission phenomena.

REFERENCES

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History of the STEM at Brookhaven National Laboratory

Joseph S. Wall, Martha N. Simon, and James F. Hainfeld

Contents 1. Introduction 101 2. Instrument Design Parameters 102 3. Heavy Metal Cluster Labeling 106 4. Early User/Collaborator Projects 108 5. Recent Work 115 6. Conclusion 115 Acknowledgments 115 References 116

1. INTRODUCTION

We begin with the origins of the STEM facility at Brookhaven National Laboratory (BNL), followed by a description of the particular design of the BNL scanning transmission electron microscope (STEM). We include a short section on the early heavy metal cluster development, followed by a history of the early user/collaborators. The STEM concept was pioneered by von Ardenne at the same time as the early development of the transmission electron microscope (TEM). However, his efforts were limited by the low brightness of available sources, lack of suitable electronics, and poor recording media. Develop- ment of the field emission electron gun by Crewe et al. (1968) solved the first problem. Commercially available electronics components provided

Biology Department, Brookhaven National Laboratory, Upton, New York 11073, USA

101 102 Joseph S. Wall et al. most of the electronics needs, especially the slow-scan to TV image converters. The annular detector dark-field (DF) geometry developed by Crewe and Wall made possible imaging of unstained DNA (Crewe and Wall, 1970) and single heavy atoms (Crewe, Wall, and Langmore, 1970). Initial biological results by Wall (1971) indicated that specimen preparation and radiation damage were the main limiting factors in STEM imaging of biological specimens, but that with suitable precautions quantitative measurements could answer important questions. Even at that early stage it was possible to show that filamentous viruses were single cylinders with mass per unit length (M/L) of 1.6 kDa/A, and ‘‘split’’ structures were actually side-by-side aggregates of two particles rather than separate strands of a single virus. The early progress with the STEM at the University of Chicago attracted the attention of John Blewett and Elliott Shaw at Brookhaven, who obtained funding from the National Science Foundation, National Institutes of Health (NIH), and the Department of Energy in 1971 to develop a STEM facility along the lines of already functioning user facilities at BNL. The initial plan was to copy one of the Chicago designs and M. Isaacson was recruited to oversee the construction. When Isaacson returned to Chicago in 1973, J. Wall was recruited with the understanding that design modifications would be considered (not permitted in the original plan). By then it was clear that specimen preparation was severely limiting the quality of results. Therefore, a new design of the lens, specimen changer, and detectors was developed, emphasizing optimum conditions for the specimen.

2. INSTRUMENT DESIGN PARAMETERS

The guiding principle for the BNL design was to achieve quantitative imaging at high resolution with high reliability. Therefore, considerable attention was devoted to isolation from environmental influences using a soundproof room within a room and a 6-ton isolation block on air bags (Figure 1). Detuning and vibration damping reduced the sensitivity of the gun to vibration (50 demagnification from gun to specimen made this crucial). Lifetime of the field emission tip was a primary concern for operation as a user facility. Therefore, the operating voltage was reduced from 100 keV to 40 keV and current limiting was included, resulting in typical tip life of 5–10 years with a vacuum of 10–11 torr (ion-pumped). Optics of the STEM were optimized to provide detector acceptance angles and magnification independent of specimen position in the lens gap. A projection aperture in the gun and a condenser lens below the gun permitted rapid switching to low-magnification mode with the objective off (scan size 3 mm to 5 m with a 10-nm probe) for locating areas of History of the STEM at Brookhaven National Laboratory 103

FIGURE 1 BNL STEM located in soundproof room. From top: vibration monitor, switch- able conditioning resistor in Lucite enclosure, FE gun chamber, condenser/beam tube/ isolation valve, objective lens chamber, detectors, floating 6-ton concrete block on air springs (under outrigger frames). Extending to the right is the specimen changer with horizontal bellows drive for specimen insertion and vertical bellows drive for selecting one specimen (of six) located in the vacuum transfer cartridge (atop central block).

interest (AOIs) to high magnification with the objective on (scan size 16 m to 32 nm with a 0.3-nm probe diameter). Double deflection through the comma-free point on the axis permits offsetting AOIs 10 m from the optical axis without significant loss of resolution. Early measurements of radiation damage at room temperature versus liquid nitrogen convinced us to operate the lens cold. This enabled use of some permanently lubricated conducting plastic components in the specimen stage without compromising the 10–9 torr vacuum, thus providing smooth motions without galling. The excitation coil for the objective was encased in copper and water cooled. The remainder of the iron yoke, pole pieces, and specimen stage were thermally isolated and operated at low 104 Joseph S. Wall et al. temperature by circulating helium gas through a vacuum-insulated transfer line from an external liquid nitrogen bath. Detectors were made of europium-doped calcium fluoride and mounted on quartz light pipes connected to photomultipliers for quantum efficiency. The annular detectors were each split in half with each half on a separate light pipe to give uniform sensitivity over their surface. The detectors have fixed acceptance angles of 0–15 mrad (bright-field [BF]), 15–40 mrad (small-angle DF [SA]), and 40–250 mrad (large-angle DF [LA]). All detector signals are normalized by the sum (BF þ SA þ LA) to yield percent scattering, independent of beam current. This eliminates streaks in the image resulting from tip current variation. The external freeze dryer, vacuum transfer cartridges, specimen changer, and specimen stage were designed as a unit. To obtain quantitative images for mass measurement and mass mapping, specimens must be freeze-dried from volatile buffer. Air-drying crushes most specimens due to the surface tension of water while also forming a meniscus around objects, concentrating salts at their periphery. The ion-pumped freeze dryer (Figure 2a) accommodates one cartridge of six frozen grids (Figure 2b) during the overnight cycle. A retractable LN2 cold trap (Figure 2c) surrounds each grid on three sides and solidifies the vapors from the specimens. At the end of the computer-controlled cycle the cartridge is closed and sealed, moved to the STEM with the grids under vacuum, and inserted through an air lock into the specimen changer. A tweezers on a bellows drive picks up a grid from one cartridge slot (indexed by a perpendicular drive) and carries it into the specimen stage.

(a)

FIGURE 2 (Continued) History of the STEM at Brookhaven National Laboratory 105

(b)

(c)

FIGURE 2 (a) Ion-pumped freeze-drying system with vacuum transfer cartridge extending toward the left in the foreground. The grey panel contains the computer interface with manual override switches. The ion pump is separated from the chamber by a pneuma- tically controlled valve, as is the turbo-pumped fore line (not shown). A residual gas analyzer extends to the left in the background. A laptop computer (not shown) connects to the system via a single USB cable. (b) Specimen exchange cartridge with the specimen holding rack extended. For transfer to STEM, this rack is drawn into the outside tube and sealed via one O-ring. (c) Cold trap (retractable) that is lowered to surround the specimens on three sides during freeze-drying, after which it is then withdrawn for transfer.

The only components that have changed radically over the years have been the computer systems for control, data acquisition, and display and the off-line system for analysis. Initially we had a PDP11–10 with a 106 Joseph S. Wall et al.

PEP-400 (Princeton Electronics Products) scan converter to display the slow-scan image on a TV monitor. This was replaced by a VAX750, then a 486–66 personal computer (PC), and now a 2.8-GHz PC with digital control of scan format, position, and size, as well as acquisition of detector signals. The emphasis has always been on minimum-dose techniques, such as selecting AOIs for focusing and imaging while viewing a low-magnification (low-dose) image, then recording first-scan images. An important adjunct to the instrumentation is our off-line software for mass measurement. A key feature is repeatable masking of particles to determine the background value in clean areas between particles. The current version, PCMass, is provided to all our users and supports automated particle (or filament segment) selection and manual measurement. Numerous features are included to assess quality of structure preservation relative to predefined models and reproducibility of mass measurements. All of these features have proven their importance since the official opening of the facility in October 1977. The BNL STEM remains in the forefront of biological electron microscopes in reliability, productivity, and performance.

3. HEAVY METAL CLUSTER LABELING

Work using the STEM at Brookhaven began with a careful study of the first premise of the facility—namely, that single heavy atoms could serve as specific labels for biological specimens (Figure 3). It was determined

FIGURE 3 Uranium single atoms and clumps on a 2-nm thick carbon film imaged with BNL STEM. History of the STEM at Brookhaven National Laboratory 107 that these predictions were overly optimistic and that a dose of at least 1000 el/A˚ 2 was needed for adequate signal to noise ratio (SNR) (Wall, 1978–1979), whereas dose-response measurements for mass loss and loss of resolution on freeze-dried specimens set a useful imaging limit at 10–100 el/A˚ 2 with the specimen cooled to –140 C. Furthermore, the heavy atoms were found to move significantly from one scan to the next (Wall, Hainfeld, and Bittner, 1978). Early experiments with S. Lippard and J. Barton (then at Columbia) confirmed the low label SNR for filamentous virus fd, which had each of the 5000 coat protein subunits labeled with platinum. A promising alternative label was tetra-mercuri methane (TAMM), which had four mercury atoms within a 3-A˚ radius. This behaved well biochemically, giving 100% labeling, but decomposed in the electron beam (Lipka, Lippard, and Wall, 1979). The student, J. Lipka, came to Brookhaven to work on a variety of promising tungsten clusters. At the same time D. Safer (University of Pennsylvania) made some undecagold derivatives based on the published methods of Bartlett and Singer. This gold cluster was shown to be sufficiently stable for easy visualization in the STEM, although a dose of 100 el/A˚ 2 was required (Wall et al., 1982). The fact that the gold cluster had 21 functional groups on its surface made it interesting to work with. Biotinylating the surface groups and reacting with avidin produced spectacular ladder images that gave this effort momentum (Safer et al., 1982). Safer’s interest was in labeling actin filaments and this was much more difficult because it required a monofunctional gold cluster). At the dose used for single cluster visibility in the STEM, some disorder resulted. However, using a lower dose below undecagold visibility, cryo–electron microscopy (cryo-EM) and image-averaging studies of these same samples gave useful results (Milligan, Whittaker, and Safer, 1990). At the same time as the actin work, P. Frey (University of Wisconsin) developed his own method for preparing monofunctional undecagold and used it to label pyruvate dehydrogenase (PDH) complex. The 96 sulfhydryl (SH) groups on the ends of the flexible arms of this multi-enzyme complex were labeled and gave impressive images, which were, however, difficult to interpret (CaJacob et al., 1985; Yang et al.,1994). At this point J. Hainfeld decided that the gold and/or the tungsten clusters were important enough for the future of STEM to justify a full-time effort. The Advisory Committee supported this decision and Hain- feld obtained separate NIH funding for his work, particularly development of larger clusters and biological projects. A number of new functionalized small gold clusters were developed and used in STEM projects, including 0 glycoprotein labeling ((Lipka, Heinfeld, and Wall, 1983), small Fab -antibody-undecagold probes ((Hainfeld, 1987), tetrairidium labels (Furuya et al., 1988; Cheng et al.,1999), tungstate labels (Hainfeld et al.,1990), bacteriophage T4 baseplate labels (Watts, Hainfeld, and Coombs, 1990), transfer ribonucleic 108 Joseph S. Wall et al.

20 nm

FIGURE 4 Nanogold clusters (67 gold atoms) on a thin carbon film. Note the tendency for arrays and chains with 2.6-nm spacing. acid (tRNA) labels (Hainfeld et al.,1991), 1.4-nm nanogold (Figure 4) (Hainfeld and Furuya, 1992), phosphorylase kinase label (Wilkinson et al., 1994; Traxler et al.,2001), PDH complex labels (Yang et al.,1994), amyloid beta protein labeling (Gregori et al.,1997), fibrinogen labeling (Mosesson et al.,1998), adenosine triphosphate–gold particles (Hainfeld et al., 1999a), metallosomes (Hainfeld et al., 1999b), Ni-NTA-gold clusters (Hainfeld et al., 1999c), gold-DNA nanostructures (Xiao et al.,2002), enzyme electron transfer (Xiao et al.,2003), nanoparticle-protein arrays (Hu et al., 2007), nanoparticle size control (Brin˜as et al.,2008). Due to the usefulness of these small functionalized gold nanoparticles as general EM labels, the company Nanoprobes, Inc. (Yaphank, NY) was started by J. Hainfeld in 1990. Hainfeld also began a long-lasting interest in medical applications of heavy atom clusters (Hainfeld, 1995).

4. EARLY USER/COLLABORATOR PROJECTS

The BNL STEM facility has a long history of working with other laboratories with particular expertise in the biology/biochemistry and preparation of interesting samples. We have concentrated on maintaining the microscopy and analyses to be routinely and easily available rather than going into a field of biology. This has given us a broad range of users and collaborators. Some of the early history is recounted below. Many of these early projects have led to more recent ones, which we will try to mention. The first external biological project was a study of native and reconstituted nucleosomes by C. Woodcock (University of Massachusetts), which confirmed most of the previous EM observations (Woodcock, History of the STEM at Brookhaven National Laboratory 109

Frado, and Wall, 1979, 1980). Subsequent study of dinucleosomes permitted image averaging (Woodcock and Frank, 1984). Woodcock also measured M/L of chromatin fibers to test various nucleosome packing models (Woodcock, Frado, and Rattner, 1984). J. Langmore (University of Michigan) performed similar measurements to aid in his interpretation of X-ray patterns, and V. Ramakrishnan (then at BNL) also conducted a STEM study in conjunction with his neutron scattering work (Gerchman and Ramakrishnan, 1987). None of these studies was definitive in proving one of the competing models for the packing of nucleosomes into chromatin fibers due to the lack of uniformity of the specimens. Woodcock and his collaborators continue to be productive STEM users (Francis, Kingston, and Woodcock, 2004). The first definitive STEM biological project was on fibrinogen and set the standard for future collaborations. Trinodular TEM fibrinogen images were interpreted either as aggregates or as the active species. E. Shaw, then a protein chemist at BNL and co-founder of the STEM project, was able to persuade leading advocates of opposing views to agree on a joint STEM experiment to interpret conflicting results. M. Mosesson (then at Downstate Medical Center and a proponent of the aggregate interpretation) and R. Haschemeyer (Cornell University Medical College) each provided specimens that were freeze-dried on our carbon films. The resulting images were so clear-cut that they have become a classic example of STEM specimen preparation (Mosesson et al., 1981). Trinodular objects 430 A˚ long and 75 A˚ wide with a mass of 340 kDa were found isolated on a perfectly clean background (Figure 5). The central domain was less intense and somewhat variable in mass but the outer nodules were reproducibly 111 17 kDa, consistent with their

FIGURE 5 Fibrinogen single molecules freeze-dried on a thin carbon film. The rod-shaped object is a tobacco mosaic virus included as an internal control for mass and structural preservation. 110 Joseph S. Wall et al. assignment as D domains. Negative staining with uranyl acetate gave much greater internal detail. Interestingly, compact molecules of 340 kDa also were observed and a deletion was localized to the central domain. This study effectively answered any uncertainties about the trinodular nature of the fibrinogen molecule. Mosesson became a long-term collaborator and recruited a network of blood biochemists for follow-up studies on clotting factors (Mosesson et al., 1985, 1990a,b) and lipid vesicle-bound clotting complexes. Mosesson’s own work concentrated on the initial steps of fibrin polymerization after thrombin activation and he produced a controversial new model based on STEM mass mapping and gold labeling (Mosesson et al., 1993). Much of this has now been confirmed. Another early focus of the STEM was specific DNA–protein interactions. Initial results showed problems in maintaining the complex on the grid, so P. Hough and I. Mastrangelo (then at BNL) began an extensive investigation of the requirements for preserving significant structures for STEM imaging (Hough et al.,1982). Their first result to attract the attention of the biochemists was the demonstration that simian vacuolating virus 40 T (SV40 T) antigen bound at the adenovirus origin of replication as a double hexamer (Mastrangelo et al., 1985, 1989; Ryder et al.,1985). This is widely accepted now but was totally unexpected and had important implications for the mechanism of opening the DNA to start replication. Other studies elucidated the mechanism of action of long-range interactions (Hough et al., 1987; Mastrangelo et al.,1991). Nonspecific nucleic acid– protein interactions were also studied by Egelman and his collaborators (Stasiak et al.,1994;Turnquistet al., 1992; Yu and Egelman, 1992, 1993). Our first project with A. Steven (NIH) concerned the M/L of intermediate filaments (IF), and the demonstration that reconstituted filaments had many of the same characteristics as native (Steven et al., 1982, 1983a,b). However, controversy arose from the observation that these filaments were 15 nm wide instead of the accepted 10-nm diameter. Steven and Steinert were able to rationalize this by observing that a portion of the IF protein was very similar in all types of filament, while the remainder was quite variable. This led to the now-accepted concept that the IF structure is somewhat like myosin with the tails wound together in a tight filament, whereas the head groups protrude from the surface (Steinert, Steven, and Roop, 1985; Steven et al., 1985). The structures observed in thin sections were the central shaft but the less-dense head groups were not visible. To test this, radial mass density profiling was developed (Steven et al., 1984), which found wide application in our studies of T4 tail tubes, bacterial flagella, pili, and filamentous viruses. Work on coated vesicles extended the radial density method to spherical particles and viruses. Many of the same techniques have been applied to study of Alzheimer’s filaments (Ksiezak-Reding and Wall, 1994; Ksiezak-Reding et al., 1996), filamentous History of the STEM at Brookhaven National Laboratory 111 hemagglutinin (Makhov et al., 1994), and flagellar microtubules (Nojima, Linck, and Egelman, 1995). Consider the case of T4 tail fibers shown in Figure 6. The lower image is an average of freeze-dried whole tail fibers. The first observation is that the fibers are trimers, based on total mass, rather than dimers as stated in textbooks. A. Steven has shown that one can divide the fiber into a series of 17 nodular domains and connecting segments and correlate the mass of each nodule with the predicted folding pattern of the protein sequence. The M/L of the connecting segments is only compatible with a triple- stranded b sheet, a structure which has been predicted but never observed. Based on the STEM studies, this was proposed to be the first example. X-ray studies have now confirmed that prediction. The upper image shows an average of STEM images of tail fibers embedded in methylamine vanadate. Note the improved spatial resolution lack of phase-contrast fringes in the LA image. Alasdair Steven and his laboratory have continued as major STEM users. In the recent past, they have done some very elegant analyses of prion protein filaments (Sen et al.,2007). One of the key achievements of the STEM program was elucidation of the dynein structure (Figure 7) in collaboration with K. Johnson (then at Penn State). This motor protein for microtubules consists of three head groups connected by thin threads to a base plate and resembles a ‘‘bouquet of flowers’’ (Johnson and Wall, 1983). This success attracted a number of Johnson’s colleagues to replicate this work on their systems (Hastie et al., 1988; Marchese-Ragona and Wall, 1995; Marchese-Ragona, Wall, and Johnon, 1988; Witman et al.. 1983) which have all been shown to be variations on the same theme.

FIGURE 6 T4 viral tail fibers. The lower image is an average of freeze-dried whole tail fibers used for mass measurement, demonstrating that the fibers are trimers. A. Steven has shown that the fiber can be divided into a series of 17 nodular domains and connecting segments, correlating the mass of each nodule with the predicted folding pattern of the protein sequence. The upper image shows similar fibers embedded in methylamine vanadate (a negative stain), giving improved spatial resolution but not compatible with accurate mass measurement. 112 Joseph S. Wall et al.

FIGURE 7 Single molecules of the microtubule motor complex dynein used to determine that the mass is 1.87 MDa and to provide a model for the structure.

The ribosome projects of M. Boublik were among the most compre- hensive STEM studies. The goal was to image the reassembly process for both 30S and 50S subunits, then repeat with labeled subunits. The early retirement and untimely death of Boublik prevented us from completing the labeling work. However, several important labeling results were obtained. We also examined 16S and 23S RNA from Escherichia coli ribo- somes in distilled water and various buffers, including reconstitution buffer, demonstrating an interesting progression of structures, but none with the degree of compactness of the reconstituted ribosome. Two quantitative measures were essential elements of the entire reconstitution study. First, mass and M/L mapping demonstrated that molecules were intact and not decorated with salt (giving an objective criterion for exclusion of contaminated or defective molecules). They also demonstrated that backbone and side branches tended to be either two- or four-stranded. A secondP useful measure was the apparent radius of ¼ 2 2 gyration defined by Rg (M(x,y) (x–x0) (y-y0) ), where (x0,y0) are the coordinates of the center of mass of the selected particle and the summation is taken over all pixels within the particle image. This is not strictly comparable to the small-angle scattering Rg because it is not averaged over all possible orientations, but it does agree surprisingly well in the cases tested. Its main use is in comparing the degree of compactness of particles prepared under similar or different conditions. The aims of this study were to define intermediates in the assembly pathway and to label individual proteins and trace their progress through the entire reassembly process. The first aim was accomplished, but unfortunately the second aim has not yet been fully realized. However, several interesting labeling results were obtained with tRNA. The reassembly experiments followed protocols worked out biochemically and were highly successful. 30S (Mandiyan et al., 1991) and 50S ((Tummina et al., 1994) subunits were each subjects of major publications detailing initial folding steps, stable intermediates, major rearrangements and final assembly. The key element in both these studies was the quantitation of both mass and Rg, which (1) demonstrated that added subunits were incorporated in correct amounts, (2) allowed critical size comparison, and (3) screened out flawed experiments. History of the STEM at Brookhaven National Laboratory 113

Multi-enzyme complexes have a compact core with binding sites at the edges and faces for other enzymes and a flexible arm involved in transport of substrate along the enzyme pathway. Three projects have given significant results with the BNL STEM: (1) PDH (with P. Frey et al., University of Wisconsin, CaJacob et al., 1985; Yang et al.,1994); (2) a-ketoglutarate dehydrogenase (T. Wagenknecht, Albany; N. Francis and D. De Rosier, Brandeis) and (3) branched-chain a-keto acid dehydrogenase (M. Hackert, L. Reed et al., University of Texas). In each case the stoichiometry was established in addition to the overall arrangement of subunits. In the case of PDH, extensive gold labeling studies were performed. One of the most difficult complexes that has given useful STEM results is the basal body studies by G. Sosinsky (then at Brandeis), N. Francis and D. DeRosier (also at Brandeis) (Sosinsky et al.,1992). This structure serves as the motor to drive the flagellum, providing motive power for the bacterium. The hook, shaft, and various rings comprising this structure had been defined by cryo-EM, but the relative masses of different structures were not determined. In this case, several deletion mutants were available to determine incremental mass with and without the selected unit. These specimens are difficult to prepare entirely free of detergent and the internal TMV standard was degraded by bound detergent. Nevertheless, good differential mass measurements were obtained using the hook portion of well-preserved basal bodies as a local standard. A similar project was recently completed by Blocker and Hodgkinson (Figure 8) in which mass mapping was critical in determining the percentage of mass in the knob at the end of the needle (Hodgkinson et al.,2009).

50 nm

FIGURE 8 Needle complexes from Shigella flexneri T3SS (the cause of dysentery) freeze-dried and marked for mass measurement. 114 Joseph S. Wall et al.

The filamentous virus projects of L. Day (New York Public Health Research Laboratory) have been among the most controversial. The collaboration between Wall and Day dates back to the beginning of the STEM project. Based on biochemical, biophysical, and STEM studies, as well as extensive modeling, Day has proposed a model for the structure of Pf1, which has the DNA phosphates along the axis and the bases pointed outward (Liu and Day, 1994; Kostrikis et al., 1991). The untimely death of S. Reisberg, Day’s graduate student for much of this work, limited the number of STEM publications, but many of the concepts were based on Reisberg’s STEM data. Other types of filamentous viruses have more normal structures and almost all have been characterized with the STEM as to length, M/L, and radial density profile. Many other virus projects have been active over the years (Cheng et al., 1994). One early result is the observation that Sindbis nucleocapsid has T ¼ 4 symmetry, rather than T ¼ 3 as listed in textbooks (Paredes, Simon, and Brown, 1992). Similarly, both T7 (Steven et al., 1988) and T4 (Cerritelli et al., 1996) tail fibers were found to be trimers instead of the dimers quoted in texts. Many of the virus projects were interested in assembly intermediates and mechanisms (Briggs et al., 2004; Carlson et al.. 2008; Cerritelli and Studier, 1996a,b; Parker, Wall, and Hunter, 2001; Vogt and Simon, 1999; Wingfield et al., 1995; Yu et al.,2001). Several large invertebrate protein complexes have considerable historical significance with many interesting questions remaining. These are particularly useful as controls for specimen preparation, STEM imaging and image analysis. S. Vinogradov (Wayne State) and collaborators have studied a wide variety of giant hemoglobins (Kapp et al., 1990, Lamy et al., 1996; Martin et al., 1996; Sharma et al., 1996; Zal et al., 1996) and M. Hamilton (Fordham University) has investigated a wide variety of hemocyanin structures (Hamilton et al., 1989). Chaperone proteins and heat shock proteins (Hsps) were a lively STEM topic several years ago when A. Horwich (Yale University) began his studies on GroEL and similar proteins. The success of J. Trent (Yale, now Argonne National Laboratory) on TF55 (Trent et al., 1991) led to a series of collaborations to establish the relationship between most of the known chaperonins. The Horwich lab showed that the protein being folded was located in the central cavity of GroEL (Braig et al., 1993) and went on to obtain the three-dimensional structure of GroEL by X-ray crystallography. The Lindquist lab studied Hsp 104 (Parsell, Kowal, and Lindquist, 1994). John Flanagan’s work on ClpP (Flanagan et al., 1995) led to his solving the X-ray structure shortly thereafter. A few projects have had success with membrane proteins in the STEM. Gina Sosinsky was able to show that different types of connexins could be distinguished by mass in gap junction membranes (Sosinsky, 1995). Collier’s group was able to study pore formation with anthrax toxin (Milne et al.,1994). History of the STEM at Brookhaven National Laboratory 115

In all these projects we have tried to demonstrate a level of impartiality and dedication that has attracted projects and collaborations from all quarters. We have worked with high school teachers and leaders in many fields with equal mutual respect and motivation to find answers. In answer to the complaint that we do not have any strong in-group biological projects, we point to our track record of finding the best available collaborators in a timely fashion. Our approach eliminates any aura of competition or feeling that anyone’s ideas or results may be diluted or even co-opted. Our successes are their successes.

5. RECENT WORK

The STEM has been particularly productive recently in terms of samples requiring M/L measurements. This has included projects from David Eisenberg’s lab at UCLA to measure the M/L of some amyloid fibrils, samples from Ulrich Baxa and A. Stevens’ lab at NIH studying prions and amyloid fibrils, several plant viruses from Gerald Stubbs’ laboratory at Vanderbilt University (Kendall et al., 2008), and pili from Ed Egelman. On other projects, Nicole Francis of Harvard Medical School and Mavis McKenna of University of Florida had been earlier STEM users, as were Ed Egelman from the University of Virginia and Thomas Marlovits in Austria, who have a current joint project. Several new projects, by Ubarretxena from Mt. Sinai School of Medicine and Sakmar’s lab in Rockefeller University, are new to the STEM. Within BNL, we have projects from the Center for Functional Nanomaterials as well as those from the Biology Department.

6. CONCLUSION

The BNL STEM continues to be a unique facility offering direct mass measurement of individual biomolecules and complexes to the biological community worldwide and visualizing and developing small gold nanoparticle labels. Even though newer microscopes have been developed, the BNL STEM has remained at the forefront of EM for quantitative mass measurements and simultaneous visualization (in focus) of proteins, DNA, and heavy metal labels in the same sample.

ACKNOWLEDGMENTS

Frank Kito for design and maintenance of instrumentation. The STEM is supported by the U.S. Department of Energy, Office of Health and Environmental Research. Formerly supported by NIH Biotechnology Resources and NIBIB.

REFERENCES

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Kostrikis, L. G., Reisberg, S. A., Simon, M. N., Wall, J. S., and Day, L. A. (1991). Export of infectious particles by Escherichia coli transfected with RF DNA of Pf1, a virus of Pseudo- monas aeruginosa strain K. Molec. Microbiol. 5, 2641–2647. Ksiezak-Reding, H., and Wall, J. S. (1994). Mass and physical dimensions vary in two distinct populations of paired helical filaments in Alzheimer disease. Neurobiol. Aging 15, 11–19. Ksiezak-Reding, H., Tracz, E., Yang, L., Dickson, D. W., Simon, M., and Wall, J. S. (1996). Ultrastructural instability of paired helical filaments from corticobasal degeneration as examined by scanning transmission electron microscopy (STEM). Am. J. Pathol. 149(N2), 639–651. Lamy, J. N., Green, B. N., Toulmond, A., Wall, J. S., Weber, R. E., and Vinogradov, S. N. (1996). The giant hexagonal bilayer hemoglobins. Chem. Rev. 96(N8), 3113–3124. Lipka, J. J., Lippard, S. J., and Wall, J. S. (1979). Visualization of polymercurimethane labeled fd bacteriophage in the STEM. Science 206, 1419–1421. Lipka, J. J., Hainfeld, J. F., and Wall, J. S. (1983). Undecagold labeling of a glycoprotein: STEM visualization of an undecagold phosphine cluster labeling the carbohydrate sites of human haptoglobin-hemoglobin complex. J. Ultrastruct. Res. 84, 120–129. Liu, D. J., and Day, L. A. (1994). Pf1 virus structure: Helical coat protein and DNA with paraxial phosphates. Science 265, 671–674. Makhov, A. M., Hannah, J. H., Brennan, M. J., Trus, B. L., Kocsis, E., Conway, J. F., Wingfield, P. T., Simon, M. N., and Steven, A. C. (1994). Filamentous hemagglutinin of Bordetella pertussis: A bacterial adhesin formed as a 50–nm monomeric rigid rod based on a 19-residue repeat motif rich in beta strands and turns. J. Mol. Biol. 241, 110–124. Mandiyan, V., Tumminia, S., Wall, J. S., Hainfeld, J. F., and Boublik, M. (1991). Assembly of the Escherichia coli 30S ribosomal subunit reveals protein dependent folding of the 16S rRNA domains. Proc. Natl. Acad. Sci. USA 88, 8174–8178. Marchese-Ragona, S., Wall, J. S., and Johnson, K. (1988). Structure and mass analysis of 14S dynein obtained from Tetrahymena cilia. J. Cell Biol. 106, 127–132. Marchese-Ragona, S. P., and Wall, J. S. (1995). Scanning–transmission electron–microscopic analysis of the isolated dynein ATPase. Meth. Cell Biol. 47, 171–175. Martin, P. D., Kuchomov, A. R., Green, B. N., Oliver, R. W. A., Braswell, E. H., Wall, J. S., and Vinogradov, S. N. (1996). Mass spectrometric composition and molecular mass of Lumbricus terrestris hemoglobin: A refined model of its quaternary structure. J. Mol. Biol. 255, 154–169. Mastrangelo, I. A., Hough, P. V. C., Wilson, V. G., Wall, J. S., Hainfeld, J. F., and Tegtmeyer, P. (1985). Monomers through trimers of large tumor antigen bind in region I and monomers through tetramers bind in region II of simian virus 40 origin of replication DNA as stable structures in solution. Proc. Natl. Acad. Sci. USA 82, 3626–3630. Mastrangelo, I. A., Hough, P. V. C., Wall, J. S., Dodson, M., Dean, F. B., and Hurwitz, J. (1989). ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication. Nature 338, 658–662. Mastrangelo, I. A., Courey, A. J., Wall, J. S., Jackson, S. P., and Hough, P. V. (1991). DNA looping and Sp1 multimer links: A mechanism for transcriptional synergism and enhancement. Proc. Natl. Acad. Sci. USA 88, 5670–5674. Milligan, R. A., Whittaker, M., and Safer, D. (1990). Molecular structure of F-actin and location of surface binding sites. Nature 348, 217–221. Milne, J. C., Furlong, D., Hanna, P. C., Wall, J. S., and Collier, R. J. (1994). Anthrax protective toxin forms oligomers during intoxication of mammalian cells. J. Biol. Chem. 269, 20606–20612. Mosesson, M. W., Hainfeld, J., Haschemeyer, R. H., and Wall, J. S. (1981). Identification and mass analysis of human fibrinogen molecules and their domains by scanning transmission electron microscopy (STEM). J. Mol. Biol. 153, 695–718. History of the STEM at Brookhaven National Laboratory 119

Mosesson, M. W., Nesheim, M. E., DiOrio, J., Hainfeld, J. F., Wall, J. S., and Mann, K. G. (1985). Studies on the structure of bovine factor V by scanning transmission electron microscopy (STEM). Blood 65, 1158–1162. Mosesson, M. W., Church, W. R., DiOrio, J. P., Krishnaswamy, S., Mann, K. G., Hainfeld, J. F., and Wall, J. S. (1990a). Structural model of factors V and Va based on scanning transmission electron microscope (STEM) images and STEM mass analysis. J. Biol. Chem. 265, 8863–8868. Mosesson, M. W., Fass, D. N., Lollar, P., DiOrio, J. P., Knutson, G. J., Hainfeld, J. F., and Wall, J. S. (1990b). Structural model of porcine factors VIII and VIIIa based on scanning transmission electron microscope (STEM) images and STEM mass analysis. J. Clin. Invest. 85, 1983–1990. Mosesson, M. W., DiOrio, J. P., Siebenlist, K. R., Wall, J. S., and Hainfeld, J. F. (1993). Evidence for a second type of fibril branch point in fibrin polymer networks, the trimolecular branch junction. Blood 82, 1517–1521. Mosesson, M. W., Siebenlist, K. R., Meh, D. A., Hainfeld, J. F., and Wall, J. S. (1998). The location of the carboxy-terminal region of g chains in fibrinogen and fibrin D domains. Proc. Natl. Acad. Sci. USA 95, 10511–10516. Nojima, D., Linck, R. W., and Egelman, E. H. (1995). At least one of the protofilaments in flagellar microtubules is not composed of tubulin. Curr. Biol. 5, 158–167. Paredes, A. M., Simon, M. N., and Brown, D. T. (1992). The mass of the Sindbis virus nucleocapsid suggests it has T ¼ 4 icosahedral symmetry. Virology 187, 329–332. Parker, S. D., Wall, J. S., and Hunter, E. (2001). An analysis of Mason-Pfizer monkey virus Gag particles by scanning transmission electron microscopy. J Virol. 75, 9543–9548. Parsell, D. A., Kowal, A. S., and Lindquist, S. (1994). The S. cerevisiae Hsp 104 protein: Purification and characterization of ATP-induced structural changes. J. Biol. Chem. 269, 4480–4487. Ryder, K., Vakalopoulou, E., Mertz, R., Mastrangelo, I., Hough, P., Tegtmeyer, P., and Fanning, E. (1985). Seventeen base pairs of region I encode a novel tripartite binding signal for SV40 T antigen. Cell 42, 539–548. Safer, D., Hainfeld, J. F., Wall, J. S., and Riordan, J. (1982). Biospecific labeling with undecagold: Visualization of the biotin binding sites on avidin. Science 218, 290–291. Sen, A., Baxa, U., Simon, M. N., Wall, J. S., Sabate, R., Saupe, S. J., and Steven, A. C. (2007). Mass analysis by scanning transmission electron microscopy and electron diffraction validate predictions of stacked beta-solenoid model of HET-s prion fibrils. J. Biol. Chem. 282, 5545–5550. Sharma, P. K., Kuchumov, A. R., Chottard, G., Martin, P., Wall, J. S., and Vinogradov, S. N. (1996). The role of the dodecamer subunit in the dissociation and reassembly of the hexagonal bilayer structure of Lumbricus terrestris hemoglobin. J. Biol. Chem. 271, 8754–8762. Sosinsky, G. E., Francis, N. R., DeRosier, D. J., Wall, J. S., Simon, M. N., and Hainfeld, J. (1992). Mass determination and estimation of subunit stoichiometry of the bacterial hook- basal body flagellar complex of Salmonella typhimurium by scanning transmission electron microscopy. Proc. Natl. Acad. Sci. USA 89, 4801–4805. Sosinsky, G. (1995). Mixing of connexins in gap junction membrane channels. Proc. Natl. Acad. Sci. USA 92, 9210–9214. Stasiak, A., Tsaneva, I. R., West, S. C., Benson, C. J. B., Yu, X., and Egelman, E. H. (1994). The Eschericia coli RuvB branch migration protein forms double hexameric rings around DNA. Proc. Natl. Acad. Sci. USA 91, 7618–7622. Steinert, P. M., Steven, A. C., and Roop, D. R. (1985). The molecular biology of intermediate filaments. Cell 42, 411–419. Steven, A. C., Wall, J., Hainfeld, J., and Steinert, P. M. (1982). The structure of fibroblastic intermediate filaments: Analysis by scanning transmission electron microscopy. Proc. Natl. Acad. Sci. USA 79, 3101–3105. 120 Joseph S. Wall et al.

Steven, A. C., Hainfeld, J. F., Trus, B. L., Wall, J. S., and Steinert, P. M. (1983). Epidermal keratin filaments assembled in vitro have masses-per-unit-length that scale according to average subunit mass: Structural basis for homologous packing of subunits in intermediate filaments. J. Cell Biol. 97, 1939–1944. Steven, A. C., Hainfeld, J. F., Trus, B. L., Wall, J. S., and Steinert, P. M. (1983). The distribution of mass in heteropolymer intermediate filaments assembled in vitro: STEM analysis of vimentin/desmin and bovine epidermal keratin. J. Biol. Chem. 258, 8323–8329. Steven, A. C., Hainfeld, J. F., Trus, B. L., Steinert, P. M., and Wall, J. S. (1984). Radial distributions of density within macromolecular complexes determined from dark-field electron micrographs. Proc. Natl. Acad. Sci. USA 81, 6363–6367. Steven, A. C., Trus, B. L., Hainfeld, J. F., Wall, J. S., and Steinert, P. M. (1985). Conformity and diversity in the structures of intermediate filaments. Ann. NY Acad. Sci. 455, 371–380. Steven, A. C., Trus, B. L., Maizel, J. B., Unser, M., Parry, D. A. D., Wall, J. S., Hainfeld, J. F., and Studier, F. W. (1988). The molecular structure of a viral receptor-recognition protein: The GP17 tail-fiber of bacteriophage T7. J. Mol. Biol. 200, 351–365. Traxler, K. W., Norcum, M. T., Hainfeld, J. F., and Carlson, G. M. (2001). Direct visualization of the calmodulin subunit of phosphorylase kinase via electron microscopy following subunit exchange. J. Struct. Biol. 135, 231–238. Trent, J. D., Nimmesgern, E., Wall, J. S., Hartl, F.-U., and Horwich, A. L. (1991). A molecular chaperone from a thermophilic archaebacterium is related to a eukaryotic protein t complex polypeptide-1. Nature 354, 490–493. Tumminia, S. J., Hellmann, W., Wall, J. S., and Boublik, M. (1994). Visualization of the protein–nucleic acid interactions involved in the in vitro assembly of the Eschericia coli 50S ribosomal subunit. J. Mol. Biol. 235, 1239–1250. Turnquist, S., Simon, M., Egelman, E., and Anderson, D. (1992). Supercoiled DNA wraps around the bacteriophage Phi-29 head-tail connector. Proc. Natl. Acad. Sci. USA 89, 10479–10483. Vogt, V. M., and Simon, M. N. (1999). Mass determination of rous sarcoma virus virions by scanning transmission electron microscopy. J. Virol. 73, 7050–7055. Wall, J. S. (1971). A high resolution scanning microscope for the study of single biological molecules. University of Chicago PhD dissertation. Wall, J. S. (1978). Limits on visibility of single heavy atoms in the scanning transmission electron microscopy – an experimental study. Chem. Scripta 14, 271–278. Wall, J., Hainfeld, J., and Bittner, J. (1978). Preliminary measurements of uranium atom motion on carbon films at low temperatures. Ultramicroscopy 3, 81–86. Wall, J. S., Hainfeld, J. F., Bartlett, P. A., and Singer, S. J. (1982). Observation of an undecagold cluster compound in the scanning transmission electron microscope. Ultramicroscopy 8, 397–402. Watts, N. R. M., Hainfeld, J., and Coombs, D. H. (1990). Localization of the proteins gp7, gp8, and gplO in the bacteriophage T4 baseplate with colloidal gold F(ab)2 and undecagold Fab0 conjugates. J. Mol. Biol. 216, 315–325. Wilkinson, D. A., Marion, T. N., Tillman, D. M., Norcum, M. T., Hainfeld, J. F., Seyer, J. M., and Carlson, G. M. (1994). An epitope proximal to the carboxyl terminus of the a-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer. J. Mol. Biol. 235, 974–982. Wingfield, P. T., Stahl, S. J., Williams, R. W., and Steven, A. C. (1995). Hepatitis core antigen produced in Escherichia coli: Subunit composition, conformational analysis, and in vitro capsid assembly. Biochemistry 34, 4919–4932. Witman, G. B., Johnson, K. A., Pfister, K. K., and Wall, J. S. (1983). Fine structure and molecular weight of the outer arm dyneins of Chlamydomonas. J. Submicros. Cytol. 15, 193–197. Woodcock, C. L. F., Frado, L.-L. Y., and Wall, J. S. (1979). Direct molecular weight determination of chromatin particles by STEM. J. Cell Biol. 83, 156a. History of the STEM at Brookhaven National Laboratory 121

Woodcock, C. L. F., Frado, L.-L. Y., and Wall, J. S. (1980). Composition of native and reconstituted chromatin particles: Direct mass determination by scanning transmission electron microscopy. Proc. Natl. Acad. Sci. USA 77, 4818–4822. Woodcock, C. L. F., and Frank, J. (1984). Nucleosome mass distribution using image averaging. J. Ultrastr. Res. 89, 295–302. Woodcock, C. L. F., Frado, L.-L. Y., and Rattner, J. B. (1984). The higher order structure of chromatin: Evidence for a helical ribbon arrangement. J. Cell Biol. 99, 42–52. Xiao, S., Liu, F., Rosen, A. E., Hainfeld, J. F., Seeman, N. C., Musier-Forsyth, K., and Kiehl, R. A. (2002). Assembly of nanoparticle arrays by DNA scaffolding. J. Nanoparticle Res. 4, 313–317. Xiao, Y., Patolsky, F., Katz, E., Hainfeld, J. F., and Willner, I. (2003). ‘‘Plugging into enzymes’’: Nanowiring of redox enzymes by a gold nanoparticle. Science 299(5614), 1877–1881. Yang, Y.-S., Datta, A., Hainfeld, J. F., Furuya, F. R., Wall, J. S., and Frey, P. A. (1994). Mapping the lipoyl groups of the pyruvate dehydrogenase complex by use of gold cluster-labels and scanning transmission electron microscopy. Biochemistry 33, 9428–9437. Yu, F., Joshi, S. M., Ma, Y. M., Kingston, R. L., Simon, M. N., and Vogt, V. M. (2001). Characterization of Rous sarcoma virus Gag particles assembled in vitro. J. Virol. 75, 2753–2764. Yu, X., and Egelman, E. H. (1992). Structural data suggest that the active and inactive forms of Rec A filament are not simply interconvertible. J. Mol. Biol. 227, 334–346. Yu, X., and Egelman, E. H. (1993). The LexA repressor binds within the deep helical groove of the activated RecA filament. J. Mol. Biol. 231, 29–40. Zal, F., Lallier, F. H., Wall, J. S., Vinogradov, S. M., and Toulmond, A. (1996). The multi- hemoglobin system of the hydrothermal vent tube worm Riftia pachyptila. I. Reexamination of the number and masses of its constituents. J. Biol. Chem. 271, 8869–8888. Chapter 4

Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes

Hiromi Inada, Hiroshi Kakibayashi, Shigeto Isakozawa, Takahito Hashimoto, Toshie Yaguchi, and Kuniyasu Nakamura

Hitachi High-Technologies Corp., Tokyo, Japan

123 124 Hiromi Inada et al.

1. INTRODUCTION

2. THE DAWN OF HITACHI ELECTRON MICROSCOPES (BY HIROMI INADA)

FE tip

V 1 1st anode V 0

2nd anode

FIGURE 1 Schematic diagram of FE gun with Butler electrode lens.

TABLE 1 Characteristics of Emitters

Thermionic emission Field emission W<310> (Tungsten filament)

Ion-pump (2) ϫ −8 Aperture 5 10 torr

Condenser lens (stigmator) Objective lens (deflector) Ion-pump (3) Specimen stage

Detector 1 ϫ 10−6 torr (P.M. tube)

FIGURE 3 Tissue section of biological specimen showing double-layered structure of cell membrane. Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 131

FIGURE 4 Photograph taken at meeting with Professor Crewe of Chicago University and Hitachi colleagues during his consultancy (1972). 132 Hiromi Inada et al.

500 Å

16 HSM-2 14 Thermionic electron SEMs 12

HSM-2A 10

6 HFS-2 (5 nm) SEM resolution (nm) 4 Field emission SEMs S-700 S-800 2 S-900 S-5000 S-5200 S-5500 0 1960 1970 1980 1990 2000 2010 Year

Emitter destroyed E

I by vacuum arc

Current decreasing Current increasing due to adsorption due to ion bombardment Emission current

IMIN

Time Flashing Cyclical lifetime

− 4 ϫ 10 8 Pa − 4.5 ϫ 10 7 Pa − 1 ϫ 10 6 Pa − 4.5 ϫ 10 6 Pa 10 (%) ∞ /I ∞ D I 5

0 − − − − − 10 13 10 12 10 11 10 10 10 9 P ϫ I∞(Pa.A)

V Ј 1 V1

REC. REC.

Emitter Emitter I II

Anode

1cm

FIGURE 10 Experimental apparatus of Gun B. Two FE tips were placed face to face and had the same anode distance of 10 mm. Reproduced with permission of Todokoro, Saito, and Yamamoto, 1982.

Emitter I 1.1

1.0 5 mA m 10 mA 50 mA 100 mA 250 A 0.9 1 min

3 ϫ 10−7 Pa 1.1 Emitter II 1.0

5 mA5mA 5 mA5mA5mA 0.9

FIGURE 13 Overview of 50-kV FE-TEM (Tonomura, 1972).

Insulator housing

Field emission tip

Electrodes

Insulator

FIGURE 14 Cross-sectional image of FE gun with multistage acceleration tube (Tonomura, 1984).

C-F discri

SEM. image SEM. det.

0 EDX. det. I P1 Def. coil P2

EELS. control EELS. det

STEM. det Slit Ratemeter

TABLE 2 Speciﬁcations of the prototype FE-STEM

Item Specifications

Field emission gun CRT for display

Probe forming lens

X ray detector Specimen

Electron detector(A)

Electron energy Slit loss spectrometer

Electron detector(B) (B)

FIGURE 17 General view of FE-STEM.

3.4 Å

FIGURE 18 Lattice fringe of carbon graphite (bright-field image).

3nm

FIGURE 19 A 0.2-nm lattice fringe of single-crystal Au (bright-field image). 144 Hiromi Inada et al.

0.20 )

0 0.15 (I/I

A thorium atom 0.10

0.05 Carbon film (15 C atoms) Scattered electrons intensities

0 0 50 100 150 (mrad.) Scattered angle

FIGURE 20 Intensity angular distribution of scattered electrons (calculated by Nomura). Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 145

4.0

3.0 Probe size

2.0 5Å Contrast 10Å 1.0

0 02040 60 80 100

Atomic number

FIGURE 21 Atom observation contrast dependence on probe size (calculation).

100 − I0=1 ϫ 10 10A 50 100 kV Wgun ( Å )

10 F W LaB6 LaB 5 E FE gun 6 gun Electorn probe diameter 1 106 107 108 109 (A/cm2.sr.) Brightness

(A) (B1) (B2)

9mrad 9mrad 9mrad

18mrad 60 9mrad mrad 9mrad 9 mrad

18mrad

FIGURE 23 Schematic diagrams of STEM detecting conditions. Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 147

(A) (B1) (B2)

10 nm

FIGURE 24 Dark-field images of carbon film (2-nm thick) captured under respective detection conditions of Figure 23.

5nm

FIGURE 25 Dark-field image of carbon film approximately 2-nm thick.

5nm

FIGURE 27 Developed EELS equipped with STEM.

Estimating that the probe diameter was 1 nm and that the diameter of the boron nitride was 20 nm, Nomura concluded that 7 10 21 g of boron and 9 10 21 gofnitrogenweredetected.

(d) (a)

ϫ1000 B,K

(b) ϫ100

C,K Relative intensity

N,K ϫ10 200Å

(c)

0 300 600 Energy loss (eV)

FIGURE 29 FE-TEM developed by Hitachi CRL. 152 Hiromi Inada et al.

FIGURE 30 200-kV FE-TEM (HF-2000).

FE-gun

Condenser lens SE detector

Scan coil EDX detector*

Objective lens Specimen Scattered electron Display Projector lens

Retractabl SEM DF-STEM DF-STEM detector Retractabl Scan generator

Image sensor* Signal processor (CCD camera) Transmission BF-STEM electron Control unit detector EDX analyzer

FIGURE 32 Configuration of Gate Viewer FE-STEM (HD-2000) (with optional EDX and charge-coupled device (CCD) camera).

= 4l 1 4 a ¼ (4.2) Opt Cs

Measured probe

0.42 nm

FIGURE 33 Smallest measured probe.

8 0.17 nm

FWHM

Intensity (a.u.) 4

0 −0.6 −0.4 −0.2 0.0 0.2 0.4 0.6 Distance (nm)

FIGURE 34 Line profile of Figure 33. 158 Hiromi Inada et al.

HD-2000 200 kV x 5000 k TE 6.00 nm

FIGURE 35 A BF-STEM image of thin single-crystal Au film taken with HD-2000.

Virtual source

C1 lens C1 cross over C1 cross over

C2 lens

OBJ.AP. C2 cross over

OBJ lens

Specimen Emissive mode High resolution mode

(a) (b) SiN a-Si

Poly-Si

100 nm SiO2

(semi-spherical grained capacitor TEG) Poly-Si

SiO2

FIGURE 37 Images of Si device. (a) SEM, (b) BF-STEM, and (c) DF-STEM.

FIGURE 38 Photograph of Gate Viewer FE-STEM (HD-2000).

(a) (b)

Incident beam

Specimen

Objective lens Transmission <= B.F.P. electron (diffraction pattern)

Simplified TEM Actual TEM

Objective lens Reduced Objective lens convergence Specimen post-field angle Transmission electron CBED pattern BF-STEM Simplified STEM Actual STEM CCD camera detector STEM nano diff. mode

1.0E + 01

1.0E + 00

Diffraction Spheerical aberr. Probe size (nm) - 1.0E 01 e- source image RMS

1.0E - 02 0.1 1 10 100 Convergence angle (mrad)

FIGURE 40 Convergence angle and probe size.

(a) (b)

Standard STEM (emissive) Nano diffraction mode

Si crystal grain ONO layer

Insulator

Capacitor

Substrate

HD 2000 200 kv ϫ 60.Dk ZC 500 nm DF-STEM image

Insulator Substrate (amorphous SiO2) (Si single crystal)

FIGURE 42 DF-STEM image of Dynamic Random Access Memory (DRAM) and diffraction patterns for nano-diffraction mode.

(a)

5nm

(b)

1 2

60 −4 50

−3 30 40

20 −2

Position (nm) 10 −1

0 0 (start)

−10 1 23 Position (nm)

FIGURE 44 Measured STEM image drift during 61-minute observation. Numbers in graph denote elapsed time in minutes.

1nm Eu

(a) (b) (c)

0.5 nm

La, Sr Mn

(d) (e) (f)

5.2/9.2 1.2/13.0 3.5/10.2

(a) (b)

50 nm 50 nm

8. ABERRATION-CORRECTED CFE-STEM (BY KUNIYASU NAKAMURA)

Microscope column with whole cover Cold-FEG

Condenser aperture Condenser lens Operating console

Spherical aberration Ion pump corrector

Objective lens Specimen stage

FIGURE 48 Appearance (left) and several elements inside whole cover (right) of aberration-corrected HD-2700 CFEG-STEM. 172 Hiromi Inada et al.

p cð Þ¼ Á 2 wðoÞ Á ðoÞ r FT exp i l A (4.8)

AðoÞ¼1 for o a; AðoÞ¼0 for o > a; (4.9) where FT represents Fourier transform operation. 0 For incorporating chromatic aberration, extended defocus C1 is introduced:

0 DE C ¼ C þ C Á ; (4.10) 1 1 C E Hitachi’s Development of Cold-Field Emission Scanning Transmission Electron Microscopes 173

0.35

0.3 ΔEo = 0.3 eV ΔEo = 0.8 eV 0.25

0.2

0.15 probe size (nm) 59

d 0.1

0.05

0 0 102030405060 Convergence angle (mrad)

FIGURE 49 Calculated d59 probe size as a function of convergence angle.

(a)

(c)

(b)

(d) 444

335

004

0.136 nm

FIGURE 50 DF-STEM images of Si (110) single crystals for (a) uncorrected and (b) corrected states; images (c) and (d) are power spectra corresponding to images (a) and (b), respectively.

6e - 3

5e - 3

E : 80 keV 4e - 3 ΔE0 : 0.3 eV

C3 : 10 m m 3e - 3 C5 : 2 mm

A5 : 2 mm

Intensity (a.u.) 2e - 3

1e - 3

0e + 0 −0.5 −0.4 −0.3 −0.2 −0.1 0.0 0.1 0.2 0.3 0.4 0.5 Distance (nm)

FIGURE 51 Calculated 2D-probe profile at 80 kV and its line profile. 176 Hiromi Inada et al.

333

0.136 nm

(a)

0.2 0.1 0 −0.1 Energy (eV)

0 1020304050607080 90 100 110 120 130 Time (s) (b) 4 9.0 mA 2.0 ϫ 10 m 9.0 A 4.0 mA 4.0 mA 2.0 mA 1.0 mA 2.0 mA

ϫ 4 m 1.2 10 1.0 A 0.4 eV

4 ϫ 103

0 −1.0 −0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Energy (eV)

(b) ϫ105 HAADF-STEM 120

(a) 0.08 nm 80 Intensity (arb. unit) Spectrum imaging 40 0 0.2 0.4 0.6 0.8 1.0 (nm) (c) ) − U-O4,5 mapping 1200

2nm Survey image 0.13 nm 800

Electron counts(e 400 0 0.2 0.4 0.6 0.8 1.0 (nm)

C-k 10 400 Pt−M4,5 8 300 Pd−M4,5 −

e 6 200 Carbon 4 support 100 2 0 0 20 nm 300 400 500 600 2000 2100 2200 2300 2400 eV eV 20370 e− 3780 e− 64400 e− (d) (e) (f) (g)

5nm

HAADF-STEM Pd mapping −2560 Pt mapping −6390 C mapping −5198

0.5 nm 1nm

(b) (d) (e) Spectrum image

30 Ca

ϫ 3 20 2.19 2.19 nm 10

Spatial drift ϫ −

e O 10 Co

0 300 400 500 600 700 800 900 1nm eV

Ca O Co 2nm ADF-STEM survey image

9. CONCLUSION AND FUTURE PROSPECTIVE (BY HIROSHI KAKIBAYASHI)

(a) (b)

2nm

Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1

P. W. Hawkes

Contents 1. Introduction 188 2. The AEI STEM-1 188 3. The Siemens ST100F 191 3.1. The New ELMISKOP ST100F Scanning Transmission Electron Microscope 195 Introduction 195 Construction of the ELMISKOP ST100F 195 Advantages of the Scanning Transmission Electron Microscope 198 Imaging with Low Radiation Damage 198 Conclusion 201 3.2. Image Forming Systems 203 General Remarks 203 The Imaging Process 204 Image Forming Properties of Magnetic Lenses 205 Strong Lenses 208 Lens Systems for CTEM and STEM: Similarities and Differences 210 Requirements for Analytical Microscopy 215 Acknowledgments 217 References 217

CEMES-CNRS, Toulouse, France

187 188 P. W. Hawkes

1. INTRODUCTION

The images published by Crewe and colleagues showing that scanning transmission electron microscopy (STEM) was a real rival to the conventional transmission electron microscope (CTEM) and to the recently developed scanning electron microscope (SEM) caused a flurry of excite- ment among the constructors of electron microscopes and the potential users of this new instrument. Only three years had elapsed since the first commercial SEM, the Cambridge Instrument Company’s Stereoscan, had been launched and one might have expected the same company to develop a STEM. In practice, however, it was two well-established builders of transmission electron microscopes who embarked on the production of commercial models of the STEM—AEI and Siemens—and a newcomer to the electron microscope field, Vacuum Generators. Despite considerable efforts, no one from Siemens or AEI could be found to write the history of their STEM instruments. The present short account is based on the few publications devoted to these microscopes and some unpublished material that has been made available to me. The earliest paper I have found on the AEI STEM appeared in 1973, while the Siemens ST100F was launched at the 1975 meeting of the German Electron Microscopy Society (DGE), held in Berlin.

2. THE AEI STEM-1

The earliest indication of interest in developing a STEM at AEI that I have encountered is a two-page note by Alderson (1971) in the AEI house journal. The prototype STEM was first described at conferences in Eng- land and the United States by Banbury and Bance (1973a,b), at which date it was sufficiently advanced to provide early images. Its later history can be followed in the papers by Banbury (1974) at the International Congress on Electron Microscopy at Canberra and by Banbury et al. (1975) and Ray et al. (1976) at the Electron Microscope Society of America and the Electron Microscopy and Analysis Group meetings, respectively. The 1973 papers tell us that The AEI gun has a triode structure which differs significantly from the well- known design of Butler (1966) and Crewe et al. (1968). The geometry chosen produces a divergent beam from a virtual source whose position remains constant over a wide range of operating voltages, thus simplifying changes of working voltage. The cathode and first anode assembly forms a pre- aligned structure mounted on a single substantial insulator, eliminating the need for precise mechanical tip alignment in ultra high vacuum.... The vacuum system is in three sections using ion and sublimation pumps throughout, to maintain a clean vacuum system in which the specimen Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 189

contamination rate is much lower than for a conventional microscope. The actual rate is largely dependent on the care with which the specimen/specimen holder and the internal surfaces of the specimen chamber are handled. Typically the specimen chamber will reach a vacuum below 10–7 torr in under two hours from atmospheric pressure with the unbaked system in clean condition; it is bakeable up to 1000 C for further reduction of the working pressure. The gun is isolated by a double differential system, and a vacuum liner tube extending up to the lower pole of the objective lens keeps all coils isolated from the high vacuum. The operating pressure in the gun is normally at or below 10–10 torr and the gun has been designed for operation up to 100 kV. The prototype has a top-entry specimen stage based on the well-tried EM802 stage (Lucas, 1970) with some minor modifications for operation in the region of 10–7 torr. The instrument is fitted with an airlock through which the specimen can be changed in two to three minutes.... The modular electronic design is all solid state with digital scan and compact EHT supplies. The system has provision for convenient extension of the video processing facilities and for remote control of the electron probe. The authors go on to describe the operating modes envisaged and, in their summary, they tell us that ‘‘The initial resolution was of the order of 10 A˚ , which is being improved in parallel with the development of the instrument. The design of the AEI field emission gun has resulted in stable and reliable operation up to and beyond 80 kV.’’ A figure shows a thin section of conventionally stained brain tissue, photographed directly from a 312-line monitor, at 80 kV; the exposure time was 5 s and two magnifications are displayed, 80,000 and 200,000. Two years later, the prototype had been installed in the MRC Labora- tory of Molecular Biology in Cambridge. Ray et al. (1976) tell us that It has a triode cold field emission gun operating over the range 20 kV to 80 kV which uses a <111> oriented single crystal tungsten tip. The tip has given 1000 hours of trouble-free operation in a vacuum of 5 10–9 Pa. A total emission current of up to 10 mA can be drawn from the tip and this produces a brightness of approximately 1013 Am–2sr–1 in a 0.3 nm probe with a probe current stability of better than 1% rms over a period of several minutes. The source size produced by the gun is estimated to lie between 3 nm and 5 nm in diameter. The source is demagnified by two magnetic lenses, a condenser and an objective, to give a gaussian spot size 0.l nm in diameter in high resolution operation. Two lenses give a certain degree of freedom in setting up the final probe size and probe current. Specimens are mounted on conventional 3 mm grids which are accommodated by a top- entry stage. The specimen chamber is pumped by an ion/sublimator pump combination and the pressure is maintained at 10–6 Pa. An airlock permits specimens to be changed in about 5 minutes.The electronics incorporates a digital scan generator which allows computer control of the picture point period, raster resolution and mode of operation. The number of picture points can be varied from 256 256 to 4096 4096. 190 P. W. Hawkes

FIGURE 1 Friedrich Thon with the Siemens ST100F.

Routine resolution of 0.3 nm can be obtained as measured using lattice fringe images or the background phase contrast detail in the image of a carbon film. These images are recorded using a relatively small collector aperture (5 millirads) and a wide objective aperture: Figure 1,1 shows a lattice fringe image from an area of graphitized carbon, the fringe spacing corresponding to 0.34 nm. This was recorded at 80 kV with an instrumental magnification of 8 million and a recording time of 16 s. Figure 2,2 shows (111) lattice fringes in a single crystal silicon specimen with a 0.3l nm spacing, recorded under similar conditions. The facility for displaying images directly at very high magnifications combined with the possibilities of contrast enhancement through signal processing make STEM a potentially very powerful instrument for studying lattice fringe images and their variation with instrumental parameters. Contamination is not a problem with this type of specimen and an image such as Figure 1 can be displayed continuously on the screen for 30 minutes or more. The microscope is equipped with a double focusing magnetic sector electron energy analyser, with a resolution of approximately 1.4 eV at 80 kV. The analyser can be used to give an energy spectrum from a stationary probe in switched ranges from 0–10 eV to 0–1000 eV with a continuous offset of 0–1000 eV. Alternatively the probe can be scanned in the usual way whilst the energy analyser is set to some particular energy band-pass. In this manner a filtered image can be obtained. A particular advantage of STEM

1 Not reproduced here. 2 Not reproduced here. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 191

FIGURE 2 Dieter Willasch and the Siemens ST100F.

is the ease with which such high-resolution energy-loss images can be obtained since the analyser transmits a time resolved image rather than a spatially resolved image. Once it was installed in the Laboratory of Molecular Biology, the STEM-1 failed to live up to its promise. The spot was indeed small, but the efficiency of the detector was poor and the vacuum system defective. After a year of frustration, the instrument, which had been purchased for about £70,000, was sold to the European Molecular Biology Laboratory (EMBL) in Heidel- berg for £10,000 in the hope that some parts could be reused but this was apparently not the case. A cryoSTEM was built at the EMBL and performed much better ( Jones et al., 1985; Jones and Haider, 1989).

3. THE SIEMENS ST100F

Information about this instrument is far more abundant and includes detailed descriptions in German and English (Willasch, 1975, 1976a,b; Gauger et al., 1975; Kempin et al., 1975; Krisch et al., 1975, 1976a,b, 1977; Hubert et al., 1978). The articles by Blaschke (1972) and Thieringer (1972) are perhaps forerunners of the STEM development. The first description that I have seen is the paper by Willasch delivered at the meeting of the DGE in 1975, in Berlin, which includes nine sets of micrographs obtained with the instrument: 192 P. W. Hawkes

1. A comparison of STEM and CTEM bright-field images of an adenovirus preparation 2. A bright-field image of muscle tissue 3. STEM and CTEM bright-field images of a 1-mm thick liver section 4. A bright-field image of an oxygen-doped chromium foil, 200 nm thick 5. Bright-field images of the 0.34-nm lattice fringes of graphitized carbon with two different scan fields 6. Bright-field and dark-field images of mitochondria 7. Bright-field images of gametocytes, 1 mm thick 8. Bright-field and dark-field images of the alloy MP35N þ Ti, as recorded and after differentiation 9. An example of selected-area diffraction, showing the bright-field image of a brass specimen and the diffraction pattern from a region about 10 nm across. Several other papers in the same volume of BEDO describe individual features of the instrument in more detail: Krisch et al. (1975) describe the signal-processing unit, Gauger et al. (1975) present a 100-kV field-emission system, and Kempin et al. (1975) give information about the objective lens. Similar information is provided in the article in the Siemens Review by Krisch et al. (1976a; in German, 1976b). The most recent account is to be found in a typescript by Schliepe (1978), which includes many technical details and extensive discussion; this is the text of a paper delivered at a Colloquium u¨ ber Elektronenschreiber und Rasterelektronenmikroskopie: Physikalische Grundlagen, Gera¨tetechnik und Anwendungen, held in the Optisches Institut of the Technische Universita¨t, Berlin, and organized by B. Lischke and H. Niedrig. The papers were not, however, published other than for internal circulation. Rather than paraphrase all this material, I reproduce below the contents of two Siemens Analytical Application Notes prepared by D. Willasch: Note 204 on ‘‘The new ELMISKOP ST100F scanning transmission electron microscope’’ and Note 212 on ‘‘Image forming systems.’’ The late Friedrich Thon, Dieter Willasch, and Reinhard Schliepe were largely responsible for the design of this microscope. Figures 1 and 2 show Thon and Willasch with the instrument. Figure 3 shows the image of an adenovirus, about 50 nm in diameter. Figure 4 gives an overall view of the control panel and the column. Other members of the development team were the late Karl-Heinz Mu¨ ller and Burkhard Krisch. I have found no reliable published information about the number of ST100F microscopes built and sold. I am therefore especially grateful to Dr. Cilly Weichan, formerly of Siemens, who has provided many of the following details. The order in which the microscopes are listed has no particular significance. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 193

FIGURE 3 Image of an adenovirus, obtained with the Siemens STEM.

FIGURE 4 Overall view of the ELMISKOP ST100F. The column and control panel of the ST100F. 194 P. W. Hawkes

1. A demonstration model was installed in Karlsruhe and at least one publication refers to it explicitly (Ruel et al., 1978, who studied softwood specimens). It was subsequently returned to Berlin for final checking before delivery to a Siemens customer. The late Dr. Joachim Stabenow at BASF in Ludwigshafen-am-Rhein showed interest in it, but BASF did not purchase an ST100F. 2. An ST100F was ordered by Don Williams, director of the Medical Research Council Laboratory in Belville, adjacent to the Tygerberg Hospital, Cape Town. It worked very well but there were specimen preparation problems. With a few exceptions contamination was very fast and this problem slowed us down. The installation of a quartz window into the specimen chamber through which the specimen could be irradiated with UV from an HBO 200 burner (for varying periods) before the beam was turned on helped somewhat. But the problem was never really solved. More interesting was the work we did interfacing the STEM directly to an HP 2000 system using a 16-bit IO card from HP. We overcame the problem of interruption from the RTE IV operating system by the use of a »flywheel » driver that bypassed the OS. In this way very fast image collection was possible. In 1982, however, Don Williams left South Africa and his successor, Wynand Murray, failed to obtain any results with the microscope and it was scrapped, even though in good working order, as was one of the latest Siemens transmission electron microscopes, a CT150. 3. A third Siemens STEM was destined for a research laboratory in the University of Sa˜o Paolo in Brazil. Funding came from Hospitalia, jointly supported by Siemens and the University. Both partners contributed to the construction and equipment of a new laboratory. But when the time came to install the microcope, it was found that the air conditioning was not capable of maintaining the necessary room temperature (18–19 C): The power supply was insufficient for the demands of the system. The microscope was therefore never installed. 4. The remaining instruments remained in Germany. One was ordered by the late Karl Zierold for the Max-Planck-Institut fu¨r Molekulare Physio- logie in Dortmund, where is was installed toward the end of 1978. This instrument performed perfectly and was in regular use in Zierold’s laboratory until 1999, when the Institute moved from the Rheinland- damm to new premises and the ST100F was left behind. The first field- emission cathode functioned for about one year; its replacement continued to operate efficiently throughout the lifetime of the microscope. Zierold was a specialist of cryo–electron microscopy and many special accessories were constructed in the workshop of the Institute: a cryo- transfer system, specimen holders, and a cooling system in high vacuum, among others. This very satisfactory performance was largely due to the expertise of Mr. Gerd Trogemann, the Siemens service engineer. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 195

5. Another instrument was ordered by the late Walter Hoppe, director of the Max-Planck-Institut fu¨ r Biochemie in Munich. He planned to use it for ptychography (Hoppe, 1982) but it seems that no results were obtained with it though the technician responsible for it claimed that it worked satisfactorily. Toward the end of the 1980s, it was transferred to Tu¨ bingen, where Hannes Lichte used it to pursue STEM holography, which he had launched in 1988 using a modified Siemens Elmis- kop 1A. Two Ph. D. dissertations resulted (Leuthner, 1992, see Leuthner et al., 1989; Sum, 1995, see Leuthner et al., 1990). 6. A final ST100F was ordered by Dr. Blank for the Institut fu¨r Transurane [Institute for Transuranium Elements] in Leopoldshafen; it seems to have failed to reach its guaranteed performance and been refused by the Institute.

3.1. The New ELMISKOP ST100F Scanning Transmission Electron Microscope by D. Willasch

Introduction Scanning transmission electron microscopy, the origins of which go back to work done by M. von Ardenne (1938) in the Thirties, has met with amazingly increasing interest since about 1969 as a new technique in electron microscopy. Scanning transmission microscopy closes the gap between conventional transmission electron microscopy and surface scanning microscopy by applying the scanning principle to imaging of objects which can be penetrated by the electron beam. The starting point for constantly increasing activities in this field was formed by the pioneering work done by Crewe and his coworkers at the University of Chicago (Crewe and Wall, 1970). They built the first, high-resolution scanning transmission microscope. Now there is no more doubt that the scanning transmission electron microscope (STEM) will assume a position of equal importance with the conventional transmission microscope (CEM or CTEM) and the surface scanning microscope (SEM), although its broad application has not been possible yet so far, especially in the area of high resolution. However, the main reason for this is that such microscopes have been available to only a few users until now.

Construction of the ELMISKOP ST100F For these reasons we have developed a high-performance scanning transmission microscope which has been designed for a great variety of different fields of application and combines ease of operation with wide 196 P. W. Hawkes technical possibilities: the ELMISKOPÒ ST100F (Fig. 4) (Krisch et al., 1976). The microscope has been designed as a compact unit, i.e. the entire electronic instrumentation is incorporated in the apparatus. No further supply units are required. The internal arrangement is: microscope stand with microscope column and control panel. The microscope column consists of a 100 kV field emission gun with a cathode which is specially processed to achieve particularly long operating times even at the relatively uncomplicated UHV of 10 9 mbar. The brightness of the source at 100 kV is better than 109 A/cm2 ster, and is thus higher by a factor of 1000 than in a thermionic system. In addition to oxygen-treated (100)-oriented tips, other tips as well may be used. An electrostatic single-lens condenser is incorporated in the electron gun. It allows for optimum beam adjustment to various operating conditions; magnetic condenser lenses can thus be dispensed with. Below the electron gun there is a differentially pumped unit with automatic valve, equipped with two differential apertures and evacuated by ion getter pumps. This unit separates the electron gun vacuum from the oil-free specimen stage vacuum which is better than 10 7 mbar. The objective lens leaves adequate room for accommodating a eucentric goniometer as well as additional analytical equipment while offering minimum aberration coefficients which permit a resolution of 0.2 nm. The detector unit containing one detector each for bright-field and dark-field imaging adjoins the objective lens at the bottom. An energy spectrometer can be connected to it below. The column cross-section and the ray path for imaging are illustrated in Fig. 5. The cross-over formed by the einzel-lens system is focused onto the specimen by the objective lens in the case of high and medium magnifications, and by a mini-lens in the case of low magnifications. Two dynamic deflection systems scan the probe over the specimen. Two more dynamic deflection systems behind the specimen adjust the beam to the detectors and the energy analyzer, thereby permitting diffraction techniques too. Two static deflection systems are used for the alignment. The arrangement of the controls and CRT’s as well as the recording system can be seen in Fig. 4. The settings of all essential operating functions are displayed by a display panel, most of them in digital form. The most important electronic components are: Vacuum control: The entire vacuum system is monitored automatically; if vacuum malfunctions occur, the appropriate protective measures are taken automatically. Scanning system: The magnification can be set from 50: 1 to 10,000,000:1. A great variety of scanning modes can be selected, including TV frequency and digital scan. Focusing and control system for the objective lens: The sensitivity of the focusing control is adjusted automatically to the respective Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 197

Field emission gun Field emission gun with einzel-lens

SF6

Insulator Static Illumination deflection Differentially aperture systems pumped unit with valve Dynamic deflection Mini lens systems Objective Objective aperture lens Specimen

Detector aperture Detector unit Dark-field Energy detector spectrometer Bright-field detector

FIGURE 5 Microscope column and ray path of the ELMISKOP ST100F. magnification. The defocusing value is indicated digitally. The stage coordinates and the goniometer tilt and rotation angles are adjusted by means of a motor with feedback of the coordinates or tilting angles, resp. High-voltage and field emission system: The acceleration voltage can be set in five fixed steps from 20 kV to 100 kV and continuously from 10 kV to 30 kV. Various illumination modes and the beam current can be set by means of the field emission control. Signal detection and processing system: Three separate measuring channels for bright-field, dark-field, and reference signals are set automatically to optimum values. Combination of the signals by addition, subtraction or division as well as further processing of the signals is possible. CRT’s: The image can be observed on two CRT’s with diagonals of 31 cm each. If an image storage unit is used, further advantages as well as optimum control of low exposure work will result. Recording system: The resolution of the recording scope is better than 2000 lines. A motor-driven 6 6 roll-film camera is used for recording. 35-mm or Polaroid cameras can also be connected. The controls for aligning, trimming and additional functions which are not required for microscope operation are arranged above the control panel and are normally hidden by a flap. 198 P. W. Hawkes

Advantages of the Scanning Transmission Electron Microscope Images which can be interpreted directly Bright-field imaging with large circular detector (objective aperture detector aperture) or dark-field imaging with annular detector furnishes contrast transfer conditions which can be interpreted directly (Crewe, 1974). Phase contrast effects, which occur in the images of conventional microscopes or the STEM with small detector aperture in the form of granulation, are avoided in this way. Figure 6 demonstrates the reduced phase contrast influence when Adeno viruses are imaged, as compared to the CEM image of the same specimen. A bright-field image of a thin section of muscle tissue is shown in Fig. 7. When focusing such periodic structures, uncertainties in selecting the focus do not arise. Ambiguities during interpretation are excluded by the optical transfer properties of the microscope. Imaging with Low Radiation Damage In dark-field imaging with large annular detector – the optimum mode of operation of a scanning transmission microscope – almost all elastically scattered electrons can be collected without loss of resolution. Damage to sensitive objects can thus be kept distinctly lower in the scanning microscope than in a conventional microscope (Crewe et al., 1974). Moreover images with minimum exposure can be achieved by shifting the scanning field to a previously non-irradiated specimen area without any noticeable change in focusing. This is quite easy because only that specimen area is irradiated by electrons which appears on the observation CRT.

(a) (b)

FIGURE 6 Comparison of STEM bright-field image (left) and CEM bright-field image (right), using an Adeno virus preparation. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 199

FIGURE 7 Bright-field image of muscle tissue.

Better imaging conditions in the case of thick specimens The inelastic scattering which is unavoidable in thick specimens does not lead in the STEM to any deterioration in resolution due to chromatic aberration, because no imaging lenses are used behind the object. For this reason, it is possible – especially in the case of amorphous specimens of low atomic number – to image even relatively thick layers with a resolution which can be achieved in a conventional microscope at much higher acceleration voltages only (Cowley, 1974; Groves, 1975). An example is presented in Fig. 8 on a section of about 1 mm thickness, as compared to the CEM. Signal processing and electronic contrast enhancement Due to the fact that the information is serially displayed there is a quantitative electronic signal available which can be processed electronically in a defined manner. This allows for processing and combining various signals in a manner adjusted optimally to the respective application. It is also possible to feed the image information directly into a computer. The contrast possibilities are illustrated in Fig. 9 on an oxygen-doped chromium layer. Optimum analysis possibilities The field emission gun offers ideal conditions for generating extremely high currents in extremely small probes. Quantitative analysis of minute specimen details thus is possible at short recording times. Diffraction examinations in micro-areas of less than 10 nm diameter as 200 P. W. Hawkes

FIGURE 8 Comparison of STEM bright-field image (left) and CEM bright-field image (right), using a liver section sample (thickness about 1 mm).

(a) (b)

FIGURE 9 Bright-field image of an oxygen-doped chromium layer; thickness about 200 nm. well as topography and material contrast imaging by means of secondary and backscattered electron detectors are possible. The electrons can be selected according to energy by means of an electron energy loss spectrometer. The signals of the elastically scattered and inelastically scattered electrons can thus be separated and can be linked to one another in a defined manner. Ease of operation Constant image brightness and focusing, regardless of the magnification, are the direct result of the scanning principle. Changes in magnification do not result in image rotations or image shifts (zoom effect). Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 201

FIGURE 10 Bright-field images of the 0.34 nm lattice planes of carbon black.

FIGURE 11 Bright-field image (left) and dark-field image (right) of mitochondria.

A few applications on various specimens are illustrated by Fig. 10 to 14.

Conclusion The new ELMISKOP ST100F high-resolution scanning transmission electron microscope was developed with the goal of providing users with an apparatus combining the advantages of STEM technique (analytical microscopy) and of great ease of operation. The major optical components of the microscope are: Field emission gun with a maximum acceleration voltage of 100 kV. Stable emission and a long life time of the cathode are achieved even at a vacuum of 10 9 mbar by using an oxygen-processed tip and a three- stage accelerator. An electrostatic einzel-lens located in front of the accelerator ensures optimum adjustment to all acceleration voltages and modes of operation. 202 P. W. Hawkes

(a) (b)

FIGURE 12 Bright-field image of gametocytes in different states; section thickness about 1 mm.

(a) (b)

FIGURE 13 Images of MP 35 N þ Ti alloy: (a) bright-field imaging; (b) as (a), but differentiated; (c) dark-field image in the light of a reflection; (d) as (c), but differentiated. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 203

(a) (b)

FIGURE 14 Micro area diffraction by means of the ELMISKOP ST100F. (a) bright-field image of an etched brass specimen; (b) diffraction pattern of an area of (a) having a diameter of about 10 nm.

¼ ¼ Objective lens combining low aberrations (Cs Cc 1.3 mm) with a large gap, thereby providing adequate space for specimen manipula- tors (e.g., a fully eucentric goniometer) and accessories (EDAX, SEM). Energy spectrometer for investigations involving inelastic electrons and energy-loss spectroscopy. A number of possibilities and advantages in using the ELMISKOP ST100F on various specimens are shown.

3.2. Image Forming Systems* by D. Willasch

General Remarks The intention of this paper is to give a short review of the most important image forming systems and their properties for the conventional transmission electron microscope (CTEM) and the scanning transmission electron microscope (STEM). For the better understanding of those, who are not concerned with imaging theory, lens design, and electron optics, it will be necessary to repeat some principles and definitions. It is not intended to deduce and evaluate solutions for electron optical problems in a rigorous way as they can be found in many textbooks. They are used

* Abstract of a key-note paper given at the workshop on ‘‘Analytical Electron Microscopy’’ at Cornell University, Ithaka, August 1976. 204 P. W. Hawkes in this context only to discuss the properties of certain systems, and to give an insight into the difficulties of designing electron microscope imaging systems. The main interest will be directed towards the electron optical and design problems of advanced microscopes, especially in analytical microscopy.

The Imaging Process Generally speaking, the purpose of an image forming system is to transform an object information into an image, which then can be interpreted by an observer. One of the fundamental problems of electron microscopy results from the fact that the specimens are three-dimensional and that the object information commonly sought in electron microscopy (i.e. the arrangement of atoms and molecules making up the specimen) has to be described by a complex function with three variables. The complete information is carried in amplitude and phase of the electron wave field which has passed through the object. But the image can be observed only as a two-dimensional intensity distribution, represented by a real function with two variables. That is why in general several assumptions and approximations are necessary to interpret an image and to understand the imaging process. In order to describe the imaging, it is useful to project the function containing the object information into a plane shortly behind the object, so c a complex function 0(x0, y0) is obtained. x0, y0 are the coordinates in this projection plane. With very thin objects it is identical with the object plane. The image forming system transforms this function into the wave c function i(xi, yi) in the image plane and the recording system squares it ¼jc j2 so that the image intensity distribution is I i(xi, yi) xi, yi are the ¼ coordinates in the image plane. In an ideal imaging system, xi Mx0, ¼ yi My0, with M as the system magnification. In order to get a faithful image of an object property, the image forming process should be linear, i.e. the output signal has to be linearly related to the input signal, in the case of coherent illumination the image c intensity I(xi, yi) to the wave function 0(x0, y0). In integral form this means ðð

Iðxi; yiÞ¼ Gðxi; yi; x0; y0ÞC0ðx0; y0Þdx0dy0 (5.1) where the function G describes the imaging qualities of the image forming system. It is clear that by far not all imaging modes and systems will obey this equation. Also certain approximations in respect to the object func- c tion 0 have to be made to obtain linear imaging. It is known, for instance, that high resolution coherent bright field imaging of a weak phase or amplitude object is linear, but not the coherent dark field image. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 205

However, if an incoherent imaging mode is used as in high resolution bright field and dark field STEM, equation (5.1) is fulfilled, since then c jc j2 instead of 0 0 is the input signal. Another requirement on a good imaging system, especially the objective lens, is that its aberrations shall have the same effect on all image points, i.e. that the aberration discs depend only on the relative distance to the corresponding Gaussian image point (the image point obtained with an aberration free system). If this is fulfilled, the image is called isoplana- tic and (5.1) can be written as ðð

Iðxi; yiÞ¼ Gðxi x0; yi y0ÞC0ðx0; y0Þdx0dy0; (5.2) i.e. as a convolution integral. Fourier transforming this (denoted by F) we get:

F½I¼F½G F½C0; (5.3) where the Fourier spectrum of the object function is directly related to the spectrum of the image intensity. F[G] is called the contrast transfer function and G the pulse response function, i.e. G is the response of the system to a very steep pulse, for instance a delta function. The concept of contrast transfer functions is very important for describing the properties of optical systems, as resolution and contrast, and is nowadays widely used. Transfer functions can be determined both by theory and experiment.

Image Forming Properties of Magnetic Lenses In modern electron microscopes magnetic lenses are in nearly all cases used as image forming elements. Despite their different use and design the basic configuration is always the same (Fig. 15): A current that runs in iron-shrouded coils generates a magnetic field distribution in the gap of

FIGURE 15 Schematic cross section of a magnetic lens. 206 P. W. Hawkes the shroud, that can be specially formed by pole pieces. The field is rotationally symmetrical about the optical axis z, and the field distribution B(z) along this axis approximates very often a bell-shaped curve. The excitationpffiffiffiffiffiffiffi of such lenses is conveniently described by the parameter NI= U, where N I are the ampere turns and U* the relativistically corrected accelerating voltage of the electron beam. In order to determine the imaging properties of a lens one has to solve, for a certain lens field, the Lorentz equation (second order differential equation) of electron motion. The ray path is determined when e.g. the ¼ starting point in the object plane z z0 and the slope are given. If one has ¼ ¼ two independent solutions, e.g. s(z) and t(z)withs(z0) 1, s’(z0) 0, and ¼ ¼ t(z0) 0, t’(z0) 1, all other solutions can be found by superposition. From these trajectories the image plane and magnification of the lens can easily be attained, as is illustrated in Fig. 16. The focal length of a weak lens, where the focal length is large compared to the extent of the lens field in z-direction, is given by 1ð 1 e 2 ¼ B ðzÞdz (5.4) f0 8mU 1 with e ¼ electron charge and m ¼ electron mass. The image rotation which occurs in magnetic lenses is given by rffiffiffiffiffiffiffiffiffiffiffiffi 1ð e F ¼ BðzÞdz; (5.5) 0 8mU 1 i.e. the rotation can be reversed by reversing the excitation of the lens. Focal length, focal position and principal planes are found, when trajec- ’ ’ tories with s(1) ¼ 1, s (1) ¼ 0 and sþ(þ1) ¼ 1, sþ (þ1) ¼ 0 are chosen as shown in Fig. 17.

Object plane lmage plane

t(z) 1 45Њ g z

s(z) M

zo zi

FIGURE 16 Ray trajectories in a magnetic lens to determine image plane and magnification. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 207

H− H+ s− (z) s+ (z)

1 1 F+ F− z

FIGURE 17 Ray trajectories in a magnetic lens to determine focal length f0, focal points Fþ and F and principal planes H and Hþ.

The aberrations that occur in electron lenses can be classified as geometrical aberrations, chromatic aberrations, and diffraction error. Geometrical and chromatic aberrations are determined from the ray trajectories by calculating the deviation of the aberrated ray trajectory from an ideal one without aberrations. In the third order approximation of this calculation there are 8 geometrical aberrations: spherical aberration, isotropic and anisotropic astigmatism, field curvature, isotropic and anisotropic distortion, isotropic and anisotropic coma. If there is not good rotational symmetry of the lens field, we have in addition to these also axial astigmatism; if there is defocussing, we have to consider this, too. As to chromatic aberration one distinguishes between axial chromatic aberration, i.e. the shift of the image plane due to the chromatic error and the chromatic aberrations of magnification and image rotation, which occur on off-axis points. It would lead too far to discuss these aberrations in detail, but we can say which aberrations have significant influence on the image in different modes of operation. Since all geometrical aberrations except spherical aberration vanish on the axis, they can be neglected in high resolution microscopy where only a small area around the optical axis is imaged (case of isoplanasy). The same is true for the chromatic aberrations: only axial chromatic aberration remains at high magnification. So one can conclude that for assessing the resolution of an electron lens system only spherical aberration, axial chromatic aberration, diffraction error and defocussing have to be considered. If correction of axial astigmatism is not sufficient this has to be considered, too. The situation is different at medium or low magnifications, when off-axis regions are imaged. Then means for correcting some of the other aberrations have to be found, as we will see later. The theoretical resolution limit of a lens system is obtained by calculating the aberration discs of the different aberrations and adding them properly. The resolution limit in electron microscopy, imposed by spherical aberration and diffraction error under optimum focus 208 P. W. Hawkes

1/4 l3/4 ¼ l ¼ conditions is c Cs (Cs coefficient of spherical aberration, electron wavelength). The factor c lies between 0.4 and 0.6 and depends on the degree of beam coherence. In modern electron microscopes the resolution limit due to axial chromatic aberration lies in the same order of a DW ¼ magnitude. It is given by Cc W , with Cc coefficient of axial chromatic aberration, a ¼ objective aperture and DW/W ¼ lens current and high voltage instabilities including the energy spread of the gun.

Strong Lenses The two main aberrations that limit the resolution of electron microscopes besides the diffraction error are spherical and axial chromatic aberration. Since it is very difficult to correct them, one has to find proper design and operating conditions to minimize their influence. This can be done by choosing high lens excitations, i.e. short focal length, since the coefficients of spherical and chromatic aberrations decrease with increasing excitation in the same order as the focal length (Fig. 18). Then the object plane lies within the lens field, and the lens has to be considered a strong lens. The magnetic field between the pole pieces is proportional to the lens excitation divided by the gap width and should stay in the order of 10 kG, since beyond this value saturation effects in the iron occur. That means that with a large gap one needs high excitation values to get small aberration coefficients. But not only the gap width, also the dimensions of the pole piece bores influence the field distribution. That is why the way the specimen is introduced influences the design of a lens strongly. With a side entry specimen stage one keeps the bores quite small, so that the gap width is the determining factor. With a top entry stage the upper bore has to be larger, which then is the determining factor while the gap is small.

f0.z0 cd 5

(mm) f1 f0 f0 cd cF z 1 0 5 10152025 NJ VU

FIGURE 18 Dependence of focal length and lens aberrations on lens excitation. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 209

1 10 9 2 3

8 4 7 6 5

1 Specimen stage 8 Bottom objective pole piece section 2 Specimen (pole piece) 3 Aperture slide 9 Cooling rod 4 Thermistors 10 Top objective pole piece section 5 Objective coil (pole plate) 6 Cooling water chambers 11 Specimen cartridge in the working 7 Objective stigmator position

FIGURE 19 Schematic cross section of the objective lens of the ELMISKOP 102.

To illustrate the design of a high resolution lens, Fig. 19 shows the cross section of a top entry stage lens, as used in the ELMISKOPÒ 102. The specimen is introduced through the 9 mm diameter upper bore into the gap. The stage mounts on the upper pole piece, the specimen in the cartridge is coupled with the stage by cone fitting. High mechanical stability can be achieved by providing good coupling between the upper and lower pole piece and between the upper pole piece and the stage. The coils are beneath the pole piece, so that the space above the lens is kept free for additional equipment. One of the advantages of such a lens is the rotationally symmetrical design, providing very good mechanical and thermal stability. It is demonstrated by the fact that with this lens a line resolution of 0.7 A˚ was attained. Since focal length, spherical, and chromatic aberration decrease with increasing excitation it is interesting to find out what happens at very high excitations. Figure 20 shows the ray paths within a lens field at three different excitation values for parallel illumination. Path I is the case used in conventional objective lenses: the optical axis is crossed only once; the object is located in the first half of the field. Path II represents the telecentric mode, which is used in the condensor-objective lens according to Riecke and Ruska. Now the object is in the center of the field, the first half of the field acts as a condensor, the second half as objective lens, thus reducing focal length and aberrations. With this kind of lens, aberration coefficients below 1 mm can be obtained at the expense of a very small gap (/3 mm). A disadvantage of the lens for routine use in CTEM is the fact that only small areas of the specimen can be illuminated which is very 210 P. W. Hawkes

B (z) B max 1

III II I

Z BA

−1

FIGURE 20pffiffiffiffiffiffiRay trajectories for parallel illuminationpffiffiffiffiffiffi for different lens excitations: (I) Nl= pUffiffiffiffiffiffi¼ 14, conventional mode; (II) Nl= U ¼ 19, telecentric mode; (III) Nl= U ¼ 28, second zone mode. (A) Object position for CTEM; (B) object position for STEM. inconvenient at low and mediumpffiffiffiffiffiffi magnifications. Path III illustrates the case of an excitation of Nl= U ¼ 28. Now the ray path crosses the optical axis twice, and if one arranges the object in the second half of the lens field for CTEM, focal length and aberration coefficients of the objective part are further reduced, while nearly two thirds of the lens field act as condensor system for parallel illumination (second zone mode). Thus the disadvantages of the telecentric mode are avoided. The most important point, however, is that this lens can combine low aberrations with a large gap and therefore it is very well suited for analytical attachments and STEM.

Lens Systems for CTEM and STEM: Similarities and Differences Now we come to design principles and properties of lens systems, i.e. combinations of electron lenses that are widely used in advanced electron microscopes of the CTEM and STEM type. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 211

Electron gun

First condensor lens

Second condensor lens

Airlock and beam deflection system

Specimen Objective lens

First intermediate lens Second intermediate lens

Intermediate viewing screen

Projector lens Shutter

Viewing chamber

Final viewing screen

Plate camera

TV flange

FIGURE 21 Schematic cross section of the ELMISKOP 102 column.

In CTEM as well as in STEM the resolution is determined mainly by the objective lens, in a STEM also called the final probe-forming lens. In CTEM one uses in addition to the objective lens several imaging lenses (Fig. 21) to further magnify the image, change magnification in a large range, compensate certain aberrations and switch from the imaging mode to diffraction. In a relatively simple lens system the objective lens is succeeded by an intermediate lens and a projector lens. The intermediate lens images the image plane of the objective into the object plane of the 212 P. W. Hawkes projector. This again is imaged onto the screen of the microscope. With this combination it is possible to apply Boersch’s ray path for diffraction, i.e. by changing the excitation of the intermediate lens one can alternatively image the back focal plane of the objective lens, where the first diffraction pattern occurs, onto the final screen. By introducing an aperture into the plane of the first intermediate image, selected area diffraction is obtained. If another intermediate lens is used (double intermediate lens), as shown in Fig. 21, one gets a high degree of flexibility, thus being able to keep the objective lens focus constant in a wide range of magnifications, to gain in the low and the high magnification range and to compensate for aberrations and image rotation. Magnetic lenses show image rotation, which depends according to equation (5) on the lens excitation. So at magnification changes the final image tends to rotate. Since the direction of rotation reverses with reversed excitation, it is possible to compensate this effect, in some degree, by proper combination of excitation changes in several of the lenses. More important then compensating this effect is the compensation of some geometrical and chromatic aberrations that cannot be neglected at low and medium magnifications, e.g. isotropic distortion, chromatic aberration of magnification and rotation. Isotropic distortion is known also as pin cushion and barrel distortion. Chromatic aberrations of magnification and rotation must be considered when examining thicker objects with energy losses at lower magnification. By proper combination of imaging lenses, distortion and other aberrations can partly be compensated because of two facts: 1. The coefficients of geometrical aberrations are excitation dependent and change sign at certain excitation values. This is demonstrated in Fig. 22. 2. By reversing the lens polarity some aberration coefficients change sign. When knowing this, one can optimize a lens system in respect to the desired compensations by computer. The basic arrangement of a high resolution STEM imaging system, as first introduced by Crewe, consists, besides of the objective lens, of one or two additional condensor lenses. They have the purpose of demagnifying the tip. As long as the demagnification factor is large enough (i.e. that the Gaussian image of the tip is smaller than the aberration disc) the resolution in STEM with high brightness guns is the same as in CTEM. Since in STEM change of magnification is done by deflection systems, the condensor system can be run at a relatively constant excitation. But it is possible to do without a magnetic condensor, when an electrostatic condensor system in the field emission gun itself is used. This way it is easy to adjust to different illuminating modes and the distance between tip and specimen gets rather small. Another feature of STEM is quite Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 213

Isotropic coma Chrom. aberr. of rotation 1 Chrom. aberr. of magnif.

Field curvature

Isotropic distortion Anisotropic astigmatism

Isotropic astigmatism NI/√U* 06 7 8 9 10111213 14

FIGURE 22 Aberration coefficients of an intermediate lens as a function of lens excitation. important: Chromatic aberrations, as far as they originate from energy losses in the specimen, do not exist, because no imaging lenses are used behind the specimen. Besides of this, as was shown by Crewe, all geometrical aberrations, except spherical aberration, can be compensated for by proper alignment and feeding of the deflection systems. This means, that in STEM it is not necessary to use lens systems for this purpose as in CTEM, but electronic means, which is a great advantage. That is why the requirements on the condensor system are relatively low in STEM, except for the last probe forming lens (objective lens), which must fulfill the same requirements with respect to resolution as in CTEM. Because of this in STEM strong magnetic lenses are used as objective lens. Since its purpose is to form a very small probe with a diameter in the order of the resolution limit of the microscope (2 to 5 A˚ ) and not to illuminate a larger field of view it seems obvious to use the telecentric mode of operation (preceding section). The disadvantages are, however, that to obtain low aberrations quite a small gap must be used, and the separation between scattered and unscattered beam, which is necessary for an efficient detector design, is unsatisfactory. Therefore one can either use an excitation near the telecentric mode, lower excitation or very high 214 P. W. Hawkes excitation (second zone case), which seems to be the optimum, since then a large gap can be combined with low aberrations. Another important design principle should be mentioned: The most sensitive region in respect to mechanical vibrations and stray fields is in a CTEM between objective lens and first intermediate lens, since here the first magnification takes place. Therefore it is necessary to couple these elements very rigidly. In a STEM the same holds between tip and objective lens. Figure 23 shows an example for the design of an advanced STEM imaging system; it is the column of the ELMISKOP ST100F. The objective lens represents a high excitation lens with large gap for analytical attachments and goniometer devices. The coefficients of spherical and chromatic aberration are 1.3 mm both, allowing a resolution of 0.2 nm. The field emission gun incorporates an electrostatic einzel-lens condensor. For SEM imaging and obtaining a wide field of view at low magnifications, a mini-lens is integrated into the upper part of the objective lens. Since the objective lens is run in the second zone mode and the stage allows movement in z-direction, the scattering angles can be matched very well to the detectors. For adjusting to optimum illuminating conditions for the energy analyzer another mini-lens in the lower part of the objective lens could be incorported.

F.E.gun Field emission cathode with ‘einzel’ lens system SF 6

Insulator Illumination Static deflection aperture systems Differentially pumped aperture with valve Dynamic deflection systems Mini lens

Objective aperture Objective lens Specimen

Detector aperture

Detector area Dark-field detector Energy analyzer Bright-field detector

FIGURE 23 Schematic cross section of the column and ray path of the ELMISKOP ST 100F. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 215

Requirements for Analytical Microscopy Although the first and, until recently, main interest in electron microscopy was to create electron images and interpret them, there is a steadily increasing trend to get more information out of the instrument. This is due to the fact that an electron beam impinging on a specimen is not only scattered, but generates several secondary events that carry a lot of information about the specimen. Figure 24 demonstrates this by showing some of the signals that can be obtained from a specimen. Besides studying transmission electron images, there have been many successful efforts to use especially the surface image and X-ray and energy loss analysis in addition. In a STEM, a combination of these different techniques is relatively easy to attain, since the information is collected seriesly. That is why scanning attachments to CTEMs have been designed in recent years. When combining analytical techniques with an electron microscope one always has to compromise between the requirements for an optimum analysis system and the imaging system. This also holds when different imaging modes, e.g. STEM and CTEM, are to be combined. Generally, one needs for analytical microscopy a wide lens gap or a large upper pole piece bore, to position the X-ray detector (e.g. energy-dispersive system) as close as possible to the specimen to get a high yield. In addition, it is desirable, especially in material science investigations, to have specimen

XMA Signal of backscattered electrons

BS Signal of hnR secondary electrons SE SEA hnL Specimen current signal SEA Dark-field SEA signal

Signal of Signal of SEA energy luminescence loss

Bright-field SEA signal

FIGURE 24 Possibilities for obtaining signals from a specimen. 216 P. W. Hawkes manipulation facilities, which also require space around the specimen. On the other hand, by opening the lens gap or the upper bore, there will be a loss of resolution, which can only be tolerated to a certain extend. A way to solve this problem is to use a high excitation lens in the second zone mode, as described in section ‘‘Strong Lenses’’. With this type of lens one can obtain with a gap width of 8 mm aberration coefficients of 1.3 mm and with a gap width of 12 mm coefficients around 2 mm. Such a lens has another important feature: If the specimen is not located in the second half of the lens field as for CTEM, but in the first half, this part then acts as probe forming lens (Fig. 20) with low aberration coefficients for STEM. Now we will consider the case, where CTEM, STEM, and surface imaging are to be used alternatively with the same instruments as in CTEM with scanning attachments. To combine CTEM and STEM, one usually uses the complete illumination system of the CTEM including the two condensor lenses. Besides working in the telecentric mode there are two possible ways for the change from CTEM to STEM mode: 1. One uses an objective lens, that is capable of high excitations and keeps the position of the object constant while the excitation of the lens is changed between two values. In one excitation state a probe is focussed into the object plane (STEM), in the other the object plane is imaged into the intermediate lens (CTEM). Since one of these states uses lower, the other higher excitation there is always a difference in aberration coefficients and possible resolution between them. Due to the large difference in excitation there is also the danger of loosing alignment when changing the excitation. 2. Change of excitation can be avoided, when the specimen is shifted to the back focal plane for STEM, then the aberrations and alignment are the same in both cases. But, of course, for this a good mechanical lift stage is necessary. In order to obtain surface images, either secondary or backscattered electrons can be used. The backscattered electron detector is a solid state detector that can be arranged within or above the upper bore of the objective lens. To use secondary electrons is more difficult. Since they are slow, and the specimen is located within a strong magnetic field, they follow the lines of force of the field. Thus it is necessary to use an objective lens with relatively large upper bore to extract them from the lens field. Therefore, the SE-detector can only be arranged properly in a lens with asymmetric shroud as demonstrated in Fig. 25. For a lens with symmetric iron shroud it is best to switch off the objective lens for SEM imaging and arrange the detector in the gap, but then one must take care that the demagnification of the source is still large enough to get good SEM resolution. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 217

10 kV

300 V

SE-detector

FIGURE 25 Detection of secondary electrons in a high resolution objective lens.

ACKNOWLEDGMENTS

I am most grateful to the many people who have provided the information on which this chapter is based. For the AEI STEM, Mrs. John Banbury and several present or past members of the MRC Laboratory of Molecular Biology in Cambridge: Richard Henderson, Nigel Unwin, Aaron Klug, and Annette Faux. For the Siemens ST100F, Frau Alexandra Kintner at the Siemens archives, Dieter Willasch and Reinhard Schliepe, who were directly involved in the development of the instrument, and Frau Dr. Cilly Weichan, formerly of Siemens; Don Williams and also Robin Cross, Alan Hall, Dave Johnson, and Bryan Sewell (the Cape Town STEM); Frau Sabine Dongard (the Dortmund instrument); Wolfgang Baumeister and Hannes Lichte (the Munich–Tu¨ bingen model); Manfred Essig (the BASF microscope in Ludwigsha- fen); and Antonio Sesso (the Brazil microscope). The instrument destined for the Institute for Transuranium Elements remains a mystery. The micrographs were clearer in the original documents, they have lost some sharpness in reproduction.

REFERENCES

Alderson, R. H. (1971). High resolution scanning transmission electron microscopy. AEI Electron Microscope News 2, 2. Ardenne, M. von (1938). Das Elektronen-Rastermikroskop. Praktische Ausfu¨ hrung. Z. techn. Physik 19, 404 Banbury, J. R. (1974). Operation of an experimental high voltage field emission STEM. Proc. 8th Int. Cong. Electron Microscopy, Canberra 1, 44–45. Banbury, J. R., and Bance, U. R. (1973a). A prototype field emission scanning transmission electron microscope. In ‘‘Proc. Scanning Electron Microscopy, Newcastle,’’ (W. C. Nixon ed.), pp. 164–168. Institute of Physics, London. Banbury, J. R., and Bance, U. R. (1973b). A prototype field-emission scanning transmission electron microscope. Proc. EMSA 31, 296–297. Banbury, J. R., Drummond, I. W., and Ray, I. L. F. (1975). Performance and applications of a high resolution field emission STEM. Proc. EMSA 33, 112–113. Blaschke, R. (1972). Einige anwendungstechnische Gesichtspunkte fu¨r Raster-Elektronenmik- roskopie im Durchstrahlungsbereich. Beitra¨ge zur elektronenmikroskopischen Direktabbildung von Oberfla¨che 5, 965–976. 218 P. W. Hawkes

Butler, J. W. (1966). Digital computer techniques in electron microscopy. Proc. 6th Int. Cong. Electron Microscopy, Kyoto 1, 191–192. Cowley, J. M. (1974). Scanning transmission electron microscopy of thick and crystalline specimens. Proc. 8th Int. Cong. Electron Microscopy, Canberra I, 18–19. Crewe, A. V. (1974). High resolution scanning microscopy. Proc. 8th Int. Cong. Electron Microscopy, Canberra I, 20–21. Crewe, A. V., and Wall, J. (1970). A scanning microscope with 5A˚ resolution. J. Molec. Biol. 48, 375–393. Crewe, A. V., Eggenberger, D. N., Wall, J., and Welter, L. M. (1968). Electron gun using a field emission source. Rev. Sci. Instrum. 39, 576–583. Crewe, A. V., Langmore, J. P., Lamvik, M. K., and Pullman, J. (1974). Scanning electron microscopy of unstained and selectively stained biological specimens. Proc. 8th Int. Cong. Electron Microscopy, Canberra II, 162–163. Gauger, W., Gottschlich, H.-J., Krisch, B., Veneklasen, L. H., and Zschimmer, M. (1975). 100kV Feldemissionsstrahler. Beitra¨ge zur elektronenmikroskopischen Direktabbildung von Oberfla¨che 8, 331–338. Groves, T. (1975). Thick specimens in the CEM and STEM. Resolution and image formation. Ultramicroscopy 1, 15–31. Hoppe, W. (1982). Trace structure analysis, ptychography, phase tomography. Ultramicro- scopy 10, 187–198. Hubert, G., Willasch, D., and Krisch, B. (1978). Results on obtaining strutural and analytical information with high lateral resolution in a STEM. Proc. 9th Int. Cong. Electron Microsc. Toronto 1, 22–23. Jones, A. V., and Haider, M. (1989). Modular detector system for scanning transmission electron microscope. Scanning Microsc. Part 3, 33–42. Jones, A. V., Homo, J.-C., Unitt, B. M., and Webster, N. (1985). The CryoSTEM: a STEM with superconducting objective lens. J. Microsc. Spectrosc. Electron. 10, 361–370. Kempin, H.-J., Mu¨ ller, K. H., Rauch, M., von Scha¨fer, N., and Vollborn, K. (1975). Ein Hochauflo¨sungsobjektiv mit Goniometer. Beitra¨ge zur elektronenmikroskopischen Direktab- bildung von Oberfla¨che 8, 339–346. Krisch, B., Mu¨ ller, K.-H., Rindfleisch, V., and Schliepe, R. (1975). Signalelektronik fu¨ r hochauflo¨sende Durchstrahlungs-Rastermikroskopie. Beitra¨ge zur elektronenmikroskopischen Direktabbildung von Oberfla¨che 8, 325–330. Krisch, B., Mu¨ ller, K.-H., Schliepe, R., and Willasch, D. (1976a). The ELMISKOP ST100F, a high-performance transmission scanning elecron microscope. Siemens Rev. 43, 390–393. Krisch, B., Mu¨ller, K.-H., Schliepe, R., and Willasch, D. (1976b). ELMISKOP ST100F, ein Durchstrahlungs-Rasterelektronenmikroskop ho¨chster Leistung. Siemens Z. 50, 47–50. Krisch, B., Mu¨ ller, K. H., Rauch, M., von Schliepe, R., and Thieringer, H. M. (1977). Analytical microscopy using a high-resolution STEM. Scanning Electron Microsc 423–430. Leuthner, T. (1992). Off-axis STEM-Holographie. Dissertation, Tu¨bingen. Leuthner, T., Lichte, H., and Herrmann, K.-H. (1989). STEM-holography using the electron biprism. Phys. Stat. Sol. (A) 116, 113–121. Leuthner, T., Sum, J., Lichte, H., and Herrmann, K.-H. (1990). STEM-holography. Proc. 12th Int. Cong. Electron Microscopy, Seattle 1, 224–225. Lucas, J. H. (1970). A 60 double tilt stage for the AEI EM820. Proc. 7th Int. Cong. Electron Microscopy, Grenoble 1, 159–160. Ray, I. L. F., Drummond, I. W., and Banbury, J. R. (1976). A high resolution field emission scanning transmission electron microscope with energy analysis. In ‘‘Developments in Electron Microscopy and Analysis, Proceedings of EMAG1975,’’ (J. A. Venables ed.), pp. 11–14. Academic Press, London & New York. Ruel, K., Barnoud, F., and Goring, D. A. I. (1978). Lamellation in the S2 layer of softwood tracheids as demonstrated by scanning transmission electron microscopy. Wood Sci. Technol. 12, 287–291. Two Commercial STEMs: The Siemens ST100F and the AEI STEM-1 219

Schliepe, R. (1978). Das ELMISKOP ST 100 F. Aufbau, Funktionsweise und Anwendungen eines hochauflo¨senden Durchstrahlungs-Rasterelektronenmikroskops (STEM). Type- script, Siemens Archives. Sum, J. (1995). Off-Axis STEM-Holographie. Erweiterte Untersuchung der Grundlagen und Einsatz eines Computers zur Mikroskopsteuerung und Signalauswertung. Dissertation, Tu¨ bingen. Thieringer, H. M. (1972). Transmission scanning electron microscopy and energy analysis with the Siemens ELMISKOP 101. Proc. EMSA 30, 450–451. Willasch, D. (1975). Das neue Transmissions-Rasterelektronenmikroskop ELMISKOP ST100F. Beitra¨ge zur elektronenmikroskopischen Direktabbildung von Oberfla¨che 8, 757–770. Willasch, D. (1976a). High resolution scanning transmission electron microscopy; aspects and trends of a new method. J. Microsc. Spectrosc. Electron. 1, 505–506. Willasch, D. (1976b). Features and applications of a high resolution scanning transmission electron microscope. Proceedings 15th Annual Conference of EMSSA, pp. 1–2. Chapter 6

A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope

† Ian R. M. Wardell* and Peter E. Bovey

Contents 1. The Early Days 222 2. Design Considerations for a Commercial STEM 224 2.1. The Objective Lens 225 2.2. The Specimen Stage 225 2.3. The Electron Gun 226 2.4. Location of Analytical Facilities 226 2.5. The Resultant Assembly 227 3. The First STEMs and HB5 Development 227 3.1. The Optical Column 232 3.2. Stage Development 238 3.3. Early Airlock and Manipulator 240 3.4. The Dark-Field Detector 241 3.5. Secondary Electron Detector 245 3.6. Diffraction Facilities 246 3.7. The Energy Analyzer Mk1 249 3.8. Electronics 251 4. Improved Resolution 254 4.1. The MIT HB5 254 4.2. The University of Illinois HB5 255 5. Other Developments 256 5.1. Beam-Blanking Plates 256 5.2. Detectors and the Virtual Objective Aperture 257 5.3. Stage Motor Drives 261

* Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom { Lindfield, West Sussex, United Kingdom

221 222 Ian R. M. Wardell and Peter E. Bovey

5.4. New Airlock 262 5.5. Energy Analyzer Mk2 264 5.6. Gun Lens 266 5.7. High-Excitation Objective Lens 266 6. Stages and Cartridges 268 6.1. Basic Cartridges 268 6.2. Beryllium Cartridges 269 6.3. Cold Stage 269 6.4. Cryo-Transfer System 270 7. The HB501 271 7.1. General Development 271 7.2. HB501UX and High-Resolution Imaging 273 8. Special and Variant Instruments 273 8.1. University of Glasgow’s HB5 275 8.2. The HB501A 276 9. The HB601 278 10. Postscript 280 Acknowledgments 282 References 283

1. THE EARLY DAYS

Vacuum Generators (VG) was founded in 1962 by Bernard Eastwell and the late Vic Treasure as an ultra-high vacuum (UHV) technology company. The primary objective was to meet the growing worldwide need of academic and industrial research and development laboratories for UHV systems and associated components. The company’s growth strategy, however, was not simply to satisfy this generic market demand but to develop instrumentation that could address a wide variety of research projects requiring a UHV environment. The first example of this strategy was the development of a low-energy electron diffraction (LEED) system, such as that used by Reid and Mykura (1969), for the study of surface structures in a clean environment. Shortly thereafter retarding field LEED Auger was introduced (Bishop, Rivie`re, and Coad, 1971), followed by the development of a hemi-cylindrical Auger electron spectrometer (Bishop, Coad, and Rivie`re, 1972/3). The latter device was the result of a cooperative project between VG and Hugh Bishop and John Rivie`re at the United Kingdom Atomic Energy Laboratories, Harwell. VG’s X-ray photo-electron spectrometer (XPS) system (Brundle et al., 1974) was introduced in the early 1970s; the work by Brundle and Roberts (1972) using this equipment helped establish electron spectroscopy for chemical analysis (ESCA) as an important surface analysis technique. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 223

All these instruments required UHV conditions for their operation and it was against this background of UHV technology–based developments that the imagination of VG’s founders was captured by the widely pub- licized work of Albert Crewe (Crewe et al., 1968, 1969, 1971; Crewe and Wall, 1970) on his high-resolution cold field-emission source scanning transmission electron microscope (STEM) in Chicago. Eastwell and Trea- sure were familiar with field emission electron sources as a result of their time at the Mullard Research Laboratories, Salfords, UK, where they had worked before establishing VG. Instrumentation based on the field emission source therefore presented another opportunity for the company to exploit its core UHV technology. A collaborative project was set up with a group at the National Physi- cal Laboratory (NPL), Teddington, that included Peter Stuart, Martin Seah, Milt Lilburne, and Albert Franks. The objective was to develop jointly a field emission source STEM with 10-A˚ resolution, to be known as the HB10. The nomenclature ‘‘HB’’ was derived from the high brightness of the field emission source. VG would supply the UHV and other engineering expertise and NPL the electron optical design. In June 1970, VG recruited one of the authors (PEB) to liaise with NPL on the project and, in particular, to work with them to develop a stable field emission source for a commercial instrument (Lilburne, Herd, and Stuart, 1970). This resulted in the decision to select <310>-oriented single-crystal tungsten as the emitter of choice. An early result of this collaboration was VG’s first field emission instrument, the 30-kV HB200 scanning electron microscope (SEM). This used a modified version of the field emission electron gun design described by Butler (1966) and Crewe (Crewe et al., 1968, 1969, 1971; Crewe and Wall, 1970). The instrument had a horizontal optical column that generated a 250- A˚ probe. It was totally UHV with the electron gun and specimen chambers differentially pumped by separate ion pumps and sublimation pumps. A version, the HBA200, was designed to accept VG’s hemi-cylindrical electron energy analyzer enabling Auger analysis to be performed at better than 100-nm resolution (Griffiths, Jones, and Wardell, 1973). In late 1971 and during 1972, one of the authors (IRMW), John Colling, John Morphew, and the late Jeff Lucas, all of whom had previously worked with the Electron Microscope Division of AEI Scientific Appara- tus, substantially reinforced the microscope effort at VG. This group had diverse engineering and design experience of microscope electronics, specimen stages, electron lenses, electron energy analyzers, and X-ray microanalysis. There was also some familiarity with cold field-emission. All the expertise was now in place to design a high-resolution, high- voltage field emission STEM. The design of some of the building blocks, mainly electronic features, had started in late 1971, and early 1972 saw the microscope specification and general design principle take shape. 224 Ian R. M. Wardell and Peter E. Bovey

One consequence of the arrival of the former-AEI group was the decision to change the direction of the collaboration with the NPL. VG would supply the NPL with the basic vacuum system that it had specified and this would enable it to continue to develop the HB10. For its part, VG would develop independently a STEM with a resolution of 5 A˚ , the HB5, which was believed to be better aligned with potential market demands (see Section 3). On April 26, 1972, VG’s microscope division was incorporated as a subsidiary of Vacuum Generators. It was first incorporated as VG Micro- scan Limited until it was realized that the ‘‘Microscan’’ name had been previously registered by Cambridge Instruments (now Leica/Cam- bridge) for one of their SEM instruments. On March 21, 1974, the company name was changed to VG Microscopes (VGM) Limited.

2. DESIGN CONSIDERATIONS FOR A COMMERCIAL STEM

It was important that certain key features of the first VG field emission STEM were correctly implemented. It would have been an expensive mistake if the basic design were wrong. The prime consideration was the necessary means of achieving the target resolution, 5 A˚ , which imposed a stringent requirement on electrical and mechanical stabilities and magnetic interfer- ences. These aspects needed to be adequately specified and controlled for the performance required but not excessively so that unnecessary cost was incurred. Crewe and Wall (1970) had shown that 5-A˚ resolution was possible and that it was possible to achieve an image of adequate quality in a reasonable time. A commercial STEM, however, had to be capable of achieving this performance in laboratory environments that were not necessarily ideal. There were other considerations beyond the need to produce good, high-resolution images. STEM clearly had the potential to be a powerful analytical tool. The plan was that the initial VGM instrument would be capable of producing diffraction patterns and that later developments would permit electron energy-loss and X-ray analysis. A prime mechanical feature to be addressed was whether the optical column should be horizontal, as in the case of VG’s first field emission instrument, the HB200, or vertical, as in the case of all transmission electron microscopes (TEMs). The prospect of a horizontal column was rapidly dismissed and the debate then centered on whether the gun should be at the bottom or the top of the optical column. It was thought that potential customers might find it difficult to accept an ‘‘upside-down’’ transmission microscope with the gun at the bottom, but there were other issues to be considered before it was possible to decide which orientation was preferred. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 225

2.1. The Objective Lens Cowley (1969) had discussed the reciprocal relationship between a probe- forming STEM and the imaging system of a TEM. The resolution and image contrast obtained with a STEM should be substantially the same as that obtained with a TEM for a given objective lens geometry, provided the TEM illuminating beam and the STEM probe-forming beam approached the lens from opposite directions and the aperture conditions were made to correspond. Ideally, the objective aperture that defined the incoming angle of the STEM probe electrons needed to be the same size and in the same position with respect to the objective lens as the objective aperture of the TEM. Similarly, the defined collection angle in the STEM needed to be the same as the defined illumination angle in the TEM. It was also necessary to ensure that the Gaussian diameter of the STEM probe was small compared to the aberration limitation of the lens. (This was equivalent to saying that the resolution of the recording media in a TEM when referred to the sample needed to be small compared to the aberration limitation of the lens.) This could be achieved by ensuring that adequate demagnification of the field emission source was used. In effect, this meant that the dimensions and excitation of a STEM objective lens needed to be very similar to those of a TEM.

2.2. The Specimen Stage Another very important consideration was the type of specimen stage that should be used. The options were side entry or top entry. A side-entry stage, possibly combined with a symmetrical objective lens, was viewed as a possibility but at the time was considered to be the more difficult to implement, more difficult to stabilize, and more restrictive when adding the analytical facilities, which were anticipated but as yet undefined. However, side entry would permit the use of an electron gun firing electrons either upward or in the more conventional downward direction. A top-entry stage, on the other hand, was particularly suited to an asymmetrical set of pole pieces with the larger bore uppermost. The specimen cartridge would be located in this larger upper bore. This cartridge and pole piece geometry was well suited to a TEM with the incoming electron beam approaching from above. With the specimen plane in such a TEM positioned near the top of the lens gap, the electron beam would be weakly focused before striking the specimen and strongly focused in the imaging field after leaving the specimen. To achieve the same resolution with this lens in a STEM application an electron probe- forming beam would need to approach the sample from the opposite direction. This was simply a manifestation and consequence of the reciprocity principle. So, in the STEM case, the use of a top-entry stage and 226 Ian R. M. Wardell and Peter E. Bovey cartridge meant that the electron gun would need to be firing electrons upward through the small bore of the objective lens. Although some manufacturers had marketed TEMs and SEMs with electron guns firing electrons upward, this went against most conventional practice of the day. One concern was the possibility of dirt particles, particularly from the sample region, falling onto apertures or into the field emitter region and causing charging problems.

2.3. The Electron Gun The field emission electron gun needed to be operated in a good vacuum. Based on the observations of Crewe (Crewe et al., 1968, 1969, 1971; Crewe and Wall, 1970) and the experience gained by the VGM designers, a pressure better than 10 8 Pa was considered a necessary vacuum for the gun chamber. In equipment that VG was accustomed to manufacturing, this pressure was routinely achieved with an ion pump and a sublimation pump. However, the prospect of a gun chamber on top of an electron optical column with an ion pump, a sublimation pump, and an attendant baking facility was considered a potential source of mechanical instability. Attaching a reduced-sized pumping system with an ion pump magnet close to the electron gun was an unattractive development route, which might impair gun performance in several ways. Smaller pumps might result in reduced pumping speed while greater proximity might increase the probability of sputtered material contaminating the gun. It could also become difficult to shield the field emitter region from the ion pump’s strong magnetic field, and possible saturation of mu-metal screening in the vicinity of the gun could weaken shielding against ambient fields. A gun chamber with pumps securely anchored to a strong framework and beneath an electron optical column was a preferred route to achieving good mechanical stability, good vacuum, and minimal impairment of gun performance by the pumping system.

2.4. Location of Analytical Facilities It was central to VGM’s thinking that the STEM had great potential for incorporating a variety of analytical techniques. Non-dispersive X-ray, electron energy-loss, and Auger analysis were some of the considered options. In this respect, a STEM was more akin to an SEM than a TEM but here the analogy ended because SEM design was dictated by the particular needs of reflection specimens. As a result, most SEMs had previously been designed with the specimen/analysis chamber beneath the electron optical column. The analyzed specimen surface faced upward and most analytical facilities were above the specimen. TEMs were usually designed with image-viewing facilities under the electron optical column in chambers attached to the top surface of a bench. Many varied add-ons, A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 227 such as energy analyzers and image-recording devices, had been successfully attached below the TEM bench but designs were usually restricted by the available space. The nature of any analytical additions to the future STEM column was not well defined initially. Clearly it would be much easier if the gun were located at the bottom of the electron optical column and the specimen chamber was at the top, where the physical restriction to adding analytical devices and post-specimen optical systems would be minimal.

2.5. The Resultant Assembly The above issues were the primary concerns of the VGM team in deciding the general geometry of the STEM design. The arguments heavily favored locating the electron gun at the bottom of the optical column with a top- entry specimen stage and an asymmetrical objective lens design that greatly resembled TEM objective lenses of the late 1960s. The benefits heavily outweighed the negative aspects of dirt falling on apertures and the emitter and of possible market preference for the inverse geometry. Thus the decision was made to put the gun at the bottom and this general structure was used in all VGM STEMs. Subsequent experience proved that any concerns about the choice of orientation were largely unfounded and the fact that the specimen chamber was on top of the electron optical column permitted many design variants that might otherwise have proved very difficult to construct.

3. THE FIRST STEMS AND HB5 DEVELOPMENT

Early in 1972, VG received its first order for a STEM. This was to be a very simple imaging microscope with none of the many options available later. Ron Burge, professor of physics at Queen Elizabeth College (QEC) in Kensington, London, placed this order for the sum of £20,000, and the first HB5 was installed in March 1974. Later, when King’s College and QEC amalgamated, this instrument was moved to King’s College in The Strand, London, where it was used by Ron Burge and colleagues for electron beam lithography. VG’s second HB5 was delivered in November 1974 to Drs—later Professors—Mick Brown and Archie Howie in the Metal Physics Depart- ment at the Cavendish Laboratory in Cambridge. Peter Hirsch headed the department at the time, shortly before his appointment as professor at the Department of Metallurgy at Oxford University. The packing note accompanying this delivery is illustrated in Figure 1; although it shows that the instrument was invoiced to the Old Cavendish Laboratory in Free School Lane, the installation in fact took place in the New Cavendish Laboratory on Madingley Road, Cambridge. 228 Ian R. M. Wardell and Peter E. Bovey

FIGURE 1 Packing note sent with the second HB5. (Photograph courtesy of Cavendish Laboratory.)

Like the QEC HB5, which was moved into storage when Ron Burge moved to Cambridge, the Cavendish Laboratory’s HB5 is not functioning today. However, the electron optical column of the Cavendish instrument, modified and adapted by various workers since its installation, has been preserved in the museum at the Cavendish Laboratory. Expertly sectioned by David Clarke in the Cavendish Laboratory’s workshop, it now reveals much of the HB5’s inner detail. Photographs of this exhibit and some of its component parts taken during preparation have been invaluable in producing some of the illustrations in this chapter. Figures 2 and 3 are photographs of the QEC and the Cavendish instruments, respectively, taken shortly after they were installed in 1974. The design of these first two instruments established many of the building blocks that were featured in VGM’s STEMs in the following 24 years. The QEC order created a focus for VGM’s design team and momentum for the project. The Cavendish order allowed VGM to extend the range of options. At the outset it was clear that the instrument would need to compete not only with other commercial STEMs but also with established TEMs of the day. To meet this challenge a spatial resolution of 5 A˚ or better was considered to be an absolute requirement. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 229

FIGURE 2 Queen Elizabeth College HB5 at installation, March 1974.

FIGURE 3 Cavendish Laboratory HB5 at installation, November 1974. (Photograph courtesy of Cavendish Laboratory.) 230 Ian R. M. Wardell and Peter E. Bovey

The performance specification agreed with QEC was 5 A˚ at an accelerating voltage of 80 kV, and the ability of the microscope to observe 3.44-A˚ fringes from graphitized carbon was also anticipated. This was an ideal situation from VGM’s point of view; there was now a customer for the prototype STEM and the order would go some way in paying for initial development costs. It also may have set a precedent for the future since a policy of continuous market-led development was to become VGM’s modus operandi. It took about two years to design and build the first HB5, which produced initial images at VGM’s East Grinstead factory premises in late 1973 before being dispatched to QEC (Wardell, Morphew, and Bovey, 1973). Those engaged in testing the instrument remember the relief, and perhaps the surprise, when 3.44-A˚ graphite fringes actually appeared, even though Crewe and Wall (1970) had demonstrated they could be viewed at 25 kV in their STEM instrument. Figure 4 shows one of these first lattice images, which was much improved upon with time. Although the first instruments were specified at 80 kV, with an overvoltage capability of 100 kV, the basic design anticipated eventual gun operation at 100 kV with an overvoltage capability of 120 kV. The overvoltage was necessary to burn off field emitting points from the high- voltage extraction electrode. The process, sometimes referred to as ‘‘spot knocking‘‘, was necessary to avoid unwanted discharges during normal gun operation that could destroy the tungsten field-emitting tip.

FIGURE 4 3.44-A˚ graphite lattice fringes from QEC’s HB5. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 231

A basic structure of the STEM evolved where the optical column was supported on a solid box-shaped bench, which consisted of five massive rectangular inch-thick slabs of steel plate bolted together. Two separate pumping systems were developed; one was dedicated to the gun and the other to the rest of the system that included the electron optical column, the sample region, and the future analytical facilities. It was reasoned that the sample vacuum could on occasion be poor due to the nature of the sample, sample processing, or simply the process of sample entry into the system. On the other hand, the cold field-emitter in the gun needed to be maintained in a good vacuum at all times if stable electron emission was to be achieved. The gun pumping system, consisting of an ion pump and a sublimation pump, together with the gun, were located within the box-shaped bench. The pressure achieved in the gun system was routinely better than 10 8 Pa. An aperture of 0.6-mm diameter located on the optical axis of the microscope (Figure 5) divided the gun vacuum from the sample vacuum and was capable of supporting a pressure differential of three to four orders of magnitude between the two vacuum systems. Located adjacent to this aperture was a valve that allowed the two vacuum systems to be isolated from each other. It was decided not to use an ion pump and sublimation pump for the sample environment. The pumping system used was one developed earlier by VG for their ESCA instruments. It was a diffusion pump system with a specially constructed liquid nitrogen cold trap, which was capable

FIGURE 5 View of sectioned anode showing 600-m differential pumping aperture and isolation valve housing. (Photograph courtesy of Cavendish Laboratory.) 232 Ian R. M. Wardell and Peter E. Bovey

of handling large gas loads as well as producing a 10 8-Pa base pressure. The sample environment in the STEM was intended to be operated at a pressure of 10 6 Pa or better. To achieve these levels of vacuum it was, of course, necessary periodi- cally to bake the entire microscope to outgas the internal surfaces. The gun pumping system was permanently housed in an oven. An oven section surrounding the gun itself was removed after baking, allowing the high-voltage gun cable to be connected. A system of heaters and demountable bake-out oven panels was designed to surround the microscope column and vacuum system above the bench. The gun region was all metal sealed and could be baked at 200 C. The region above the bench, although mainly metal sealed, had some Viton seals and was baked to a lesser temperature, usually 150 C. This general arrangement was to survive for the lifetime of VGM’s 100-kV STEMs. The occasional exception occurred when a customer would specify that an ion pump should replace the diffusion pump to provide an oil-free pumping system. It should be noted that the need to bake the entire microscope and operate at high or ultra-high vacuum at all times imposed considerable constraints on the general engineering design of the system and the choice of materials used in its construction. Bearing surfaces of mechanisms in vacuum, for example, had to survive the temperature cycling and continue to operate smoothly in UHV at all times. Both materials and lubrication problems had to be overcome. In this respect it was, perhaps, fortunate that VGM was able to approach these issues in an environment without the constraints of a mindset born of conventional TEM construction. Although essential for successful operation of the field emitter, the need to achieve UHV conditions before operating the electron gun meant that instrument development was a slow process. Any problem in the gun region of the instrument necessitated an overnight bake-out of the system that resulted in a cycle time of around 24 hours. In the early days, therefore, nighttime visits to the factory to check that all was well with the baking process were a regular precautionary activity for the VGM team.

3.1. The Optical Column 3.1.1. The Gun In Figures 2 and 3 the gun is hidden below the bench top under the optical column. The design of the gun differed from the Butler (1966) design, which had been used by Crewe (Crewe et al., 1968, 1969, 1971; Crewe and Wall, 1970) and the similar design incorporated in VGM’s HB200 (Griffiths, Jones, and Wardell, 1973). It was, however, rather similar in appearance to a conventional triode thermionic gun. The field emission tip was located about 1 mm behind a 1-mm diameter hole in a A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 233

FIGURE 6 Sectioned HB5 extraction electrode showing pumping slots and a replaceable field emission tip assembly. The <310>-oriented single-crystal tungsten tip, located just below a small hole in the center of the extraction electrode, is welded to the arc filament. The coiled filament is used for electrode outgassing. (Photograph courtesy of Cavendish Laboratory.) high-voltage extraction electrode mounted (Figure 6) on the end of a long alumina ceramic, about 270 mm in length (Figure 7). Unlike a triode gun, this electrode was held at a stabilized positive voltage, typically in the 2-kV to 4-kV range, with respect to the emitting tip to effect emission. The tip was held at the accelerating voltage and electrons were accelerated to and through a grounded anode, which was spaced about 10 mm from the high-voltage extraction electrode. This is shown in the schematic diagram, Figure 8, which is reproduced from an early VGM brochure. Because of the appreciable source demagnification needed to achieve a 5-A˚ probe it was calculated that the aberrations attributable to this triode design would not be significant compared with those of the objective lens. The extraction electrode was made from a spinning of thin stainless steel sheet, which gave it little mass and made it relatively easy to heat and outgas. There were two filaments on the replaceable field emission tip assembly, as shown in Figure 6. One of these, a helical, thoriated, iridium filament, was used to generate thermionic electrons with which to bom- bard the extraction electrode during an outgassing procedure at the end of a system bake cycle. The tungsten field-emitting tip was spot-welded to the other, arc-shaped filament. During microscope operation there was a gradual fall in emission current due to gas absorption on the tungsten tip, which needed, therefore, periodic desorption to restore the emission current to its initial value. This was achieved by occasionally, and briefly, heating the arc-shaped filament using preset controls. 234 Ian R. M. Wardell and Peter E. Bovey

FIGURE 7 HB5 electron gun. The field-emission extraction electrode is on the 100-kV alumina insulator, which is bellows-sealed. Adjustment keys for lateral alignment are visible. (Photograph courtesy of Cavendish Laboratory.)

3.1.2. Basic Imaging and Diffraction Modes The probe-forming column of the original VGM STEMs, which included the QEC and Cavendish HB5s, was based on one condenser lens and one objective lens. Later, however, a second condenser lens was introduced; Figure 9a is a ray diagram illustrating the basic imaging mode of the two- condenser lens STEM. The diagram shows the first condenser lens (C1) and second condenser lens (C2) set to focus on the selected area diffraction (SAD) aperture and the objective lens set to focus on the specimen. Changing the relative excitations of C1 and C2 will change both the demagnification of the source at the specimen and the current striking the specimen for a given size of objective aperture. Bright-field and dark- field images are formed by signals collected through the collector aperture and on the annular detector, respectively. The scan coils in imaging mode are excited such that the beam is tilt-scanned about, and therefore stationary on, a point in the back focal plane of the objective lens where the objective aperture is located. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 235

Exit slit and detector

Electron spectrometer

Detector aperture and dark field detector Microdiffraction scan coil assembly Specimen airlock Specimen Objective aperture Stigmator Objective lens

Diffraction aperture

Scan and alignment coil assembly Condenser lens Beam blanking plates Gun stigmator Differential Column isolation valve aperture Anode Field-emission tip and extraction electrode 100 kV insulator

FIGURE 8 Schematic diagram of an HB5 column from an early brochure.

Figure 9b shows the SAD mode where the objective lens has been set to focus the SAD aperture into the specimen and C2 set to focus the beam into the back focal plane of the objective lens. The current striking the specimen is again dependent on the relative excitations of C1 and C2 and, in this case, the size of the SAD aperture. In diffraction mode the scan coils are tilt-scanned about the SAD aperture and consequently, because of the objective lens setting, the specimen. As the beam is tilt-scanned about the specimen, the image of the diffraction pattern is formed from the signal collected through the collector aperture. It will be apparent from these diagrams that the early instruments with one condenser lens had relatively limited control of source demagnification and current striking the specimen. 236 Ian R. M. Wardell and Peter E. Bovey

(a) (b)

Collector aperture Collector aperture

Dark field detector

Specimen Specimen Objective lens Objective lens Objective aperture Back focal plane SAD aperture SAD aperture

Scan coils Scan coils

C2 C2

C1 C1

Gun Gun

Unscanned incident and Unscanned incident and bright field beam main beams Example of incident Example of beam tilt-scanned scanned beam about selected area Unscanned dark field Unscanned diffracted beams

FIGURE 9 Ray diagrams for (a) normal imaging and (b) selected area diffraction.

3.1.3. The First Optical Column Design The detailed design of the optical column varied considerably during the 1970s. As stated previously, the first instruments consisted of just one condenser lens and an objective lens. Between the two lenses were located scan and alignment coils and, in the bore of the objective lens, was an electrostatic stigmator. These are illustrated in the schematic diagram (Figure 8), which shows the general layout of early microscopes. The diagram also illustrates some features such as an energy analyzer, dark- field detector, microdiffraction coil assembly, and a condenser lens stigmator, none of which were developed by VGM until after the delivery of the instrument to QEC. All lenses and coil assemblies between the gun and the objective lens/objective stigmator unit were outside the vacuum envelope, which, in this region, comprised a thin stainless steel tube around the optical column axis. Column alignment in both the QEC and Cavendish instruments was performed with a mechanical condenser lens shift in conjunction with the shift and tilt alignment coils between the two lenses. Magnetic screening of the probe-forming system was provided by lining the inside of the column support tube with two concentric tubes of mu-metal that are clearly visible in Figure 10. Because the source size of the field emitter is of the order 50 A˚ the gun also needed to be well screened from ambient A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 237

FIGURE 10 View of sectioned HB5 shows the 100-kV insulator, extraction electrode, anode, isolation valve in an open position, gun stigmator coils, and condenser lens. The twin mu-metal shields inside the column support tube and surrounding the condenser lens and the belled-out shield around the upper end of the insulator are also visible. (Photograph courtesy of Cavendish Laboratory.) magnetic fields. To achieve this the walls of early gun chambers were made from mu-metal. Later gun chambers were made from stainless steel and lined internally with mu-metal. The gun chamber screening was linked to the upper screens with an overlapping, bell-ended mu-metal screen (visible in Figure 10), located inside the top of the gun chamber. This provided continuous screening from the gun to the objective lens. An asymmetrical objective lens geometry, which had an upper bore of 12 mm, lower bore of 3 mm, and gap of 4 mm, was chosen along with the top-entry specimen cartridge and stage. This lens was designed with a removable pole piece assembly so that alternative designs could be fitted. The assembly was approximately 45 mm long by 45 mm diameter. It consisted of two soft iron pole pieces permanently shrunk into a phosphor bronze spacer such that the assembly could be final-machined with good concentricity and hence low astigmatism. ‘‘Shrunk’’ was VGM’s engineering term for the process of allowing an outer diameter of a soft iron pole piece cooled by liquid nitrogen to expand into a recessed diameter in the phosphor bronze spacer heated to about 100 C. The final, machined assembly was gold plated, which protected it against corrosion. 238 Ian R. M. Wardell and Peter E. Bovey

1/4 l3/4 ˚ The resolution of this lens, 0.43 Cs , was 3.15 A at 80 kV and it was expected that the target specification would be easily achieved. The resolution at the intended 100-kV operation of future instruments was 2.93 A˚ . Three aperture-alignment mechanisms, each of which could carry up to three apertures, can be identified in Figure 2. Located just below the top of the microscope is the collector aperture mechanism; the objective aperture mechanism is seen lower, and lower still is the selected area aperture mechanism. At delivery, the QEC instrument had only a bright-field detector. The relevant photomultiplier assembly can be seen in Figure 2 located at the top of the instrument where later STEMs would accommodate energy analyzers. The photomultiplier was mounted on a glass window, close behind a scintillator.

3.1.4. Column Development The limitations of a single condenser lens (see Section 3.1.2) were not addressed until several instruments had been delivered. The second lens offered greater control of probe demagnification and provided a fixed crossover in the plane of the SAD aperture that acted as the source for the objective lens. Introducing a mechanical tilt as well as a shift to each condenser lens provided much-needed additional degrees of freedom for alignment. This was achieved by equipping the lenses with pole pieces that could be moved laterally with respect to each other. A condenser stigmator and more shift and tilt coils were introduced between the gun and the first condenser lens (C1), and corresponding front panel controls were added to the electronics console. The demagnification of the field emission source to form a sub–5-A˚ probe needed to be 30. In conventional terms this was low and meant that the objective lens and electron gun had to be mechanically linked in an extremely rigid manner. This was achieved in the initial design by means of a set of tie bars (visible in Figures 2 and 3), linking flanges at the top of the gun chamber and the top of the objective lens. An early design change altered the way the column support tube interfaced with the gun flange and the objective lens. This both eliminated the need for the tie bars and improved the microscope’s appearance.

3.2. Stage Development The stage facilities of the QEC instrument were very basic. The specimen cartridge accommodated standard 3-mm grids but no tilting facility was available. The major stage development occurred later. The tapered cartridge was loaded into a simple stage base that slid across the surface of the objective lens. The specimen grid was appropriately located in the A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 239 objective lens field, a few centimeters below the top surface of the objective lens. The stage was pushed in x and y across the surface of the lens by micrometer drives operating through 10:1 levers. Because the intention was to motorize these drives at a later date, the micrometers were not positioned very conveniently for manual operation but located (as seen in Figure 3) toward the rear of the specimen chamber just above the objective lens. It was commented at the time that the instrument really was suitable only for microscopists with unusual physical characteristics because it was not easy to view an image on the display monitors at the same time as adjusting the position of the specimen! However, an electronic image shift applied by means of the scan and alignment coil assembly was available, which made fine image movements easy to achieve. A more complete stage with a 60 double-tilt cartridge had been ordered by the Cavendish Laboratory with their HB5 but the development of stage mechanics proved to be a long, tedious process and the final version of their stage was eventually delivered as a retrofit after the main installation was complete. The stage shift drives, like those at QEC, were still manual, although the Cavendish added motors themselves (Figure 11) soon after installation. The tilt drives, which are absent in Figure 3 but visible in Figure 11, were also manual but because of their location on the front of the specimen chamber they were not difficult to use. Figure 12 is a photograph of the HB5 stage sitting on the surface of the objective lens and shows some of its complicated system of levers, springs, and push rods. This stage design remained substantially

FIGURE 11 HB5 specimen chamber. Clockwise from top right: dark-field detector (Cavendish design), stage shift motor drive (Cavendish design), airlock, manual tilt drives, cartridge manipulator, microdiffraction coil assembly, and collector aperture mechanism. (Photograph courtesy of Cavendish Laboratory.) 240 Ian R. M. Wardell and Peter E. Bovey

FIGURE 12 Stage base on objective lens. The stage is driven in x and y through the 10:1 levers (left and rear). A tilt drive mechanism is visible on the tilt block (right). A rod pushes the ball on the right-hand spring-loaded lever. Independent of stage x and y position this actions, by means of a horizontal plate, a second ball-ended lever on the stage base causing a horizontal push rod to drive the tilt mechanism in the cartridge (not present). (Photograph courtesy of Cavendish Laboratory.) unchanged and was used, with a few exceptions, for all of VG Micro- scope’s 100-kV STEMs. Stage-related development did not stop here, however. Many different specimen cartridges (Section 6) were to follow.

3.3. Early Airlock and Manipulator Loading a specimen cartridge into the QEC HB5 was very much a manual process. The airlock can be seen in Figure 2 on the right-hand side of the specimen chamber pointing toward the vacuum control rack of electronics. A door on top of the airlock gave access to a socket, which carried the cartridge into and out of the specimen chamber. At a pressure of about 10 1 Pa the airlock was manually actuated, which opened a door and moved the socket into the specimen chamber. Here a hand-operated bellows-sealed manipulator (visible on the front of the specimen chambers in Figures 2 and 3) was used to transfer the cartridge between the airlock socket and the stage socket. Figure 13 illustrates the ball-grabbing inner end of the manipulator poised over a ball-topped cartridge in the stage and, partially hidden behind the airlock door, the socket in the airlock. Windows into the specimen chamber were provided to allow observation of the process but, as can possibly be appreciated from Figure 2, it was a distinct advantage to be left-handed! It was not uncommon for the occasional, unfortunate operator to inadvertently release the cartridge from the manipulator when it was A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 241

FIGURE 13 Cavendish museum exhibit. HB5 specimen chamber showing, from bottom to top, the electrostatic stigmator in the objective lens bore, stage base with cartridge inserted, cartridge manipulator above, and shift drive to the left. At the rear is the airlock door with cartridge socket behind and to the right are the microdiffraction coils. (Photograph courtesy of Cavendish Laboratory.) neither in the stage nor in its location in the airlock socket. This resulted in the need to break vacuum to effect a cartridge recovery with probably an overnight bake of the system to restore good vacuum. The above method of sample entry was also used on the Cavendish HB5, where both the airlock and the manipulator had been significantly modified for easier use. A large (50-mm diameter) phosphor bronze nut located at its outermost extremity actuated the QEC airlock (see Figure 2). This nut was replaced by a lever (see Figure 3) on the Cavendish airlock. The external part of the manipulator (visible in Figure 3) was also changed on the Cavendish HB5. For reason unknown to the authors it was changed yet again, as can be seen in Figure 11 to the left of the tilt control box, before the instrument was finally decommissioned. An automatic airlock was high on the list of priorities and was developed soon afterward.

3.4. The Dark-Field Detector VGM had not developed a dark-field detector (DFD) when the HB5 was delivered to QEC. However, much work was done on detectors by Ron Burge at QEC, and he and his colleagues (Browne, Lackovic, and Burge, 242 Ian R. M. Wardell and Peter E. Bovey

1975) designed and built an annular DFD for their HB5. Meanwhile VGM designed a relatively simple annular DFD, which was delivered with the Cavendish HB5. This consisted of a photomultiplier viewing, through a glass window, the reverse side of an annular disk of scintillator, which was angled at 45. This design of DFD was used for many years and is illustrated, minus a photomultiplier, in Figure 14; this schematic diagram

Electron spectrometer and bright field detector

Diffraction screen Collector aperture

Annular dark field detector Airlock fitted with specimen outgassing facility

Microdiffraction and beam tilt coil assembly

Objective stigmator Objective aperture Diffraction aperture High excitation objective lens

Scan and alignment coil assembly

Condenser stigmator and gun Double condenser lens system alignment coil assembly

Gun isolation valve High brightness field emission gun

FIGURE 14 Schematic diagram of the HB5 optical column (dated 1981) and used in an HB501 brochure, 1986. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 243 was generated in the early 1980s (Wardell, 1981) and is reproduced from a 1986 VGM brochure. Maximum effort was taken to ensure minimum light loss between the scintillator and the photomultiplier, which were separated by the radius of the specimen chamber (150 mm) and optically connected by an internally silvered glass tube. The hole through the center of the annular disk permitted the passage of the bright-field beam to the collector aperture, energy-loss analyzer, and the bright-field detector. The original DFD was static, but with the introduction of the Energy Analyser Mk2 (Section 5.5), which had a much-increased collection angle, it was necessary to make it retractable. The schematic diagram of Figure 15, which is reproduced from a 1989 VGM brochure, shows the DFD mounted on a retractable bellows movement. Single-electron counting with the DFD was possible by paying careful attention to the choice of scintillator and by selecting a low-noise

Electron spectrometer and bright field detector.

Collector aperture. Diffraction screen (retractable).

Annular dark field detector (retractable). Microdiffraction and beam tilt coil Specimen assembly. airlock. Specimen stage and holder. X-ray detector. Objective aperture. Objective lens. Objective stigmator. Selected area diffraction aperture. Scan and alignment coil assembly.

Condenser lens system. Condenser stigmator and gun alignment coil assembly. Gun isolation valve. Virtual objective aperture.

FEG2100 gun lens.

High brightness field emission electron source.

FIGURE 15 Schematic diagram of the optical column from an HB501 brochure, 1989. 244 Ian R. M. Wardell and Peter E. Bovey photomultiplier. This was a result of a development with Andreas Engel, now professor at the University of Basel, who took delivery of one of the first HB5s in Europe. This meant that the HB5 had the ability to generate a dark-field image with close to 100% efficiency, compared with the TEM’s 1% to 10% efficiency. The DFD, particularly when used in conjunction with beam blanking (see Section 5.1), was to be of value for biological specimens where limiting specimen exposure to the electron beam was important. Engel, Dubochet, and Kellenburger (1976) showed (Figure 16) that a DFD image of tobacco mosaic virus from the HB5 was better quality than a similar TEM bright-field image that had been recorded with five times the dose. They also showed that a dark-field image in a TEM required 150 times the 50 nm

Dark field STEM (HB5) Dark field CTEM Bright field CTEM Dose: 12 electrons/sqÅ Dose: 1800 electrons/sqÅ (reversed contrast) Dose: 60 electrons/sqÅ

FIGURE 16 Comparison between a dark-field STEM image of TMV and dark-field and bright-field TEM images. The STEM image has a lower electron dose but shows better quality than the TEM images. (From Engel, Dubochet, and Kellenburger, 1976; courtesy of Academic Press.) A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 245

STEM

500 Å

CTEM

FIGURE 17 Dark-field micrographs of a DNA strand with similar signal to noise. Good detail is shown in the STEM image with a dose of 22 electrons/A˚ compared with the TEM image and 600 electrons/A˚. (From Engel, Dubochet, and Kellenburger, 1976; courtesy of Academic Press.)

dose required for a DFD STEM image to achieve the same signal to noise ratio. They were also able to obtain, due to the reduced dose, improved dark-field images of DNA strands in the STEM compared with those obtained from a TEM (Figure 17).

3.5. Secondary Electron Detector A specimen cartridge to allow reflection microscopy was an early HB5 development, which required a secondary electron detector to be added to the specimen chamber. This was a simple Everhart–Thornley detector (Everhart and Thornley, 1960) with a biased mesh cage to attract or repel, depending on bias polarity (Section 3.8.1), the secondary electrons. Behind the cage a scintillator, which was held at þ10 kV, was attached to the end of a straight light pipe. The light pipe butted up against a zero-length window to which a photomultiplier was attached. 246 Ian R. M. Wardell and Peter E. Bovey

3.6. Diffraction Facilities Diffraction facilities on the HB5 fell into three categories: selected area diffraction (SAD) (described in Section 3.1.2), microdiffraction, and a means of viewing diffraction patterns directly on a phosphor screen. SAD was available on the first HB5. Microdiffraction and the means to directly observe diffraction patterns were introduced later.

3.6.1. Microdiffraction Coils Grigson (Grigson, 1961; Denbigh and Grigson, 1965) developed microdiffraction during the 1960s at the Engineering Laboratory in Cambridge. The technique was performed in the HB5 using a set of double deflection coils positioned just over the specimen cartridge. The electron probe was focused onto the specimen, producing an (angular) diffraction pattern in space immediately after the specimen. Scanning this pattern across the collector aperture and collecting the transmitted signal with the bright- field detector resulted in the diffraction pattern being displayed on the cathode ray tubes (CRTs). The scanning needs to be double deflection (as shown in Figure 18a), rocking the deflected beam about a fixed point in

(a) (b) Collector aperture Collector aperture

Post-specimen (Grigson) Post-specimen DC coils scan coils

Specimen Specimen Objective lens Objective lens Objective aperture Objective aperture

C2 C2

C1 C1

Gun Gun

Unscanned main beam Bright field Example of scanned main beam Selected weak beam Unscanned diffracted beam

FIGURE 18 Ray diagrams illustrating (a) the formation of diffraction patterns with the microdiffraction scan coils and (b) weak beam imaging with the DC coils. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 247 the specimen plane. In this way, any beam deflected through the collector aperture appears to be originating from a stationary point on the specimen. With an energy-loss analyzer (see Section 3.7) fitted after the collector aperture, diffraction patterns with a selected energy loss are then possible. Two sets of coil windings were incorporated in the microdiffraction unit. One set was for scanning to produce diffraction patterns and a second set for DC operation to enable weak beam imaging by deflecting a selected diffracted beam back onto the microscope axis (Figure 18b). These scan coils were necessarily relatively high powered and their windings could not easily be located directly in the vacuum. They were, therefore, encased at atmosphere in a stainless steel can with a thin-walled, stainless steel liner tube running through their center. Because they were located just above the specimen cartridge, they needed to be retractable to permit cartridge removal. The microdiffraction coils are illustrated schematically in Figure 14, and a sectioned view is shown in Figure 13. Figure 19a shows a bright-field image of an MoS2 specimen, which has been used to generate typical SAD patterns. The image in Figure 19c is an SAD pattern with the energy analyzer set to pass all electrons from zero to

(a) (c)

3 mm (b) (d)

FIGURE 19 (a) Micrograph of an MoS2 specimen and (b) a weak beam image from the same area. (c) Selected area diffraction pattern from MoS2 using energy-loss electrons in the range 0 eV to 1000 eV. (d) A pattern from the same area with energy-loss electrons in the range 0 ev to 3 eV. 248 Ian R. M. Wardell and Peter E. Bovey

1000-eV energy loss. The image in Figure 19d is the same except the energy analyzer has been set to pass electrons in a limited energy-loss range from zero to 3 eV. Figure 19b shows a weak beam image from the same area as in Figure 19a where a selected diffracted beam has been steered back on axis by the DC microdiffraction coils and through the energy analyzer in place of the zero-order beam. Figure 20a shows a bright-field image of graphite flakes, and Figure 20b illustrates a typical microdiffraction pattern, which was obtained from one of these flakes less than 100 A˚ in size.

3.6.2. The Diffraction Pattern Observation Screen Another diffraction facility, the diffraction pattern observation screen (DPOS), was introduced about the same time as the microdiffraction. The DPOS is located in the specimen chamber (shown schematically in Figure 14). A diffraction pattern is formed on a phosphor-coated glass slide, which is located on the microscope axis facing the oncoming electron beam. The pattern is viewed horizontally through a window from outside the vacuum system and a 45 mirror positioned just behind the glass slide. Small central holes in the slide and the mirror allowed the axial electron beam to pass through to the collector aperture, energy analyzer, and bright-field detector. This enabled a DPOS diffraction pattern to be viewed simultaneously with a bright-field image but not a dark- field image because the DPOS necessarily obscured the DFD scintillator.

(a) (b)

20 nm

FIGURE 20 (a) Bright-field micrograph of graphitized carbon. (b) Microdiffraction pattern from a flake of graphitized carbon, which is less than 100 A˚ in size. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 249

The DPOS was mounted on a bellows-sealed mechanism that allowed it to be retracted when a DFD image was required. Figure 14 illustrates the relative position of the DPOS with respect to the DFD and the microdiffraction coil assembly. A diffraction angle at the specimen up to about 5 could be observed with the original objective lens pole pieces. This angle increased to around 20 following the introduction of the high-excitation objective lens pole pieces, which are described in Section 5.7. The pattern in HB5s originally was viewed with a binocular microscope to which a 35-mm camera could be attached. Subsequently the pattern was viewed with a charge-coupled device (CCD) camera through a hinged mirror, which, for recording purposes, could be moved so that the image was viewed directly by the 35-mm camera (see Figure 27).

3.7. The Energy Analyzer Mk1 Figure 14 provides a schematic illustration of the energy analyzer Mk1, and Figure 3 shows the analyzer with mu-metal covers fitted to the top of the specimen chamber on the Cavendish HB5. This was a simple 90 sector magnet, which refocused a point on the specimen onto an adjustable-width slit, located beyond the magnet where an energy-loss spectrum was formed. A scintillator—photomultiplier combination was located behind the slit. To maintain a stationary focus on the slit a small amount of descanning was incorporated to compensate for the scanning of the probe on the sample. With a wide-open slit a normal bright-field image was formed. With a narrow slit an image with a selected energy loss could be formed by appropriately adjusting the magnet current. Alternatively, a spectrum could be displayed on the microscope CRTs by scanning the magnet current. Eventually it became routine to display energy-loss spectra on non-dispersive X-ray systems when they were interfaced with the HB5. There was some hysteresis associated with scanning the magnet current. Wilson et al. (1977) at the Cavendish resolved this by adding an alternative set of air-cored coils after the magnet to scan the spectrum; this solution was subsequently adopted by VGM. With a slit of approximately 1 m the initial energy resolution of this analyzer was better than 1.5 eV at 80 kV on the Cavendish instrument. After reducing interference from many electrical and magnetic sources a resolution of 0.75 eV at 100 kV eventually was guaranteed on later instruments. The original method of making the micron-wide slits was to select under an optical microscope two cleaved pieces of silicon wafer and cement them in place on the micrometer-driven slit mechanism. This resulted in edges that were straight to within a fraction of a micron over 250 Ian R. M. Wardell and Peter E. Bovey several millimeters and could be totally closed to 100-kV electrons. Subsequently a method of producing slits with machined edges was adopted after realizing that Alan Craven had successfully had a set made and installed on the Cavendish HB5. This solution brought to an end the laborious task of cleaving, selecting, and mounting fragments of silicon wafer; this was a result for which the VGM team was very grateful. Focusing the analyzer was a slightly tedious mechanical process of moving the whole analyzer assembly with respect to the column axis, moving the slit with respect to the magnet, and trimming the input field shape with a set of adjustable pole pieces. These can be seen in Figure 14, as can the bifurcated vacuum tube through the center of the magnet. This gave the option, which was seldom employed, of using a second scintillator—photomultiplier combination on the top flange and switching rapidly between an energy-loss mode with the magnet energized to an unfiltered, bright-field mode with the magnet not energized. Figure 21 illustrates spectra that were initially possible with this analyzer with a 5-A˚ probe diameter on the specimen.

Plasmon loss peaks

0 100 200 300 400 500 600 700 Energy loss (eV)

FIGURE 21 Energy-loss spectrum using the energy analyzer Mk1 showing the presence of titanium and carbon in an alloy precipitate. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 251

3.8. Electronics 3.8.1. The Microscope Control Console The microscope electronics, designed in the early 1970s, was modular and non-digital. A control console from 1986 is shown in Figure 22. Compari- son with Figure 2 shows that the design remained substantially unchanged throughout this period. A key feature of the design was a common reference system that ensured that alignment currents, lens excitations, and scan currents automatically tracked with accelerating voltage. Stabilized power supplies and the gun high-voltage generator were located in the base of the sloping control console, and the instrument control electronics were mounted above desk level in standard 19-inch crates. Over time, additional modules were designed and another tier of crates was added on top of the console to house overflow. On a general note, it is to be borne in mind

FIGURE 22 An HB5 control console from 1986 showing few differences from the original delivered to QEC in 1974. 252 Ian R. M. Wardell and Peter E. Bovey that, although the VG STEMs were transmission microscopes, their control and imaging systems were closely related to those of an SEM. Two viewing CRT displays were located in the central bay with the gun controls above and the lens and stigmator controls below. Image magnification, image rotation, and diffraction (camera length) controls were located on the lower right. Alignments and image electrical shifts were on the lower left with the right-hand side of this panel being occupied by energy analyzer controls. In Figure 22 these analyzer controls, which relate to the Mk1 analyzer, are quite simple. With the advent of the Mk2 version (see Section 5.5), additional controls were introduced for analyzer focus and aberration correction. The bay to the right of the central display CRTs was reserved for detector and image processing controls. When there was insufficient space in this bay, modules were placed in the extension bay directly above (see Figure 22). Immediately to the right of the central CRTs was a video control module, which provided a metered indication of the signal level to the two display monitors. The initial version of the module (visible in Figure 2) allowed separate switched control of signal bandwidth and signal inversion in the two channels. After a short time the Mark 1 module was replaced by a new module (visible in Figure 22)withautomaticbandwidthcontrol dependent on line scan speed. This module also provided gamma control and a range of controls to enhance image detail and contrast. Control modules for the bright-field and dark-field detectors allowed adjustment of photomultiplier gain and signal black level. A similar module, for control of a secondary electron detector, provided an additional 10-kV scintillator accelerating voltage and an adjustable (100 V to þ300 V) bias supply. An X-ray input module routed pulse and ratemeter outputs from energy-dispersive X-ray systems to the display CRTs. A low-current electrometer was included with the range of detector options. The electrometer control module was housed with all the other detector control modules. Its head amplifier, which was suitable for measuring beam currents in the 10 5-A to 10 10-A range, was mounted close to the optical column and near the point where the current was being measured. This was usually the current being collected by an electrically isolated aperture blade where the aperture mechanism had been provided with a suitable feed-through outlet for the current. A head amplifier is shown in Figure 23 mounted on the surface of the microscope bench next to a virtual objective aperture (VOA) mechanism (see Section 5.2.3), which has been fitted with a feed-through. The electrometer output was usually used to normalize imaging signals against fluctuations in electron beam current. To achieve this the output was permanently connected to the C channel of the microscope’s sum-difference-ratio (SDR) video-processing unit. Additionally the output could be channelled to an energy-dispersive X-ray system for the same purpose. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 253

FIGURE 23 Glasgow University’s HB5 showing three post-specimen lenses and the motor drive for the z-drive of the specimen stage to the left of the tilt motor drives. The bottom left shows the virtual objective aperture mechanism with a cable leading to the electrometer. The new airlock is visible on the right.

The SDR unit (visible in Figure 22) was fitted to most microscopes and was introduced to facilitate signal processing in a manner similar to that described by Crewe, Wall, and Langmore (1970). The unit produced an output signal, V, which was a function of three input signals described by the following formula: V ¼ðAu Bv CwÞ=ð Ax By CzÞ where A, B, and C are input signals, and u, v, w, x, y, and z are controllable multiplying factors. A and B signals could typically be the bright-field and dark-field detector signals and C, as mentioned previously, was the normalizing signal. The output signal, V, could be routed to the CRT displays by means of gain and black-level controls. The SDR unit required a careful setup procedure and was probably rarely used for anything other than simple normalization against beam current fluctuations. 254 Ian R. M. Wardell and Peter E. Bovey

To the left of the central display CRTs was the time base module and a CRT for photographic recording of images, which was slaved to one or other of the two viewing CRTs depending on user choice. The time base generated a range of line and frame speeds, which were selected by front panel switches. TV rate could be selected or canceled at the push of a single button. As with SEMs at the time, scan formats comprised full-area scan, reduced-area scan, area y-modulation, line scan, spot (no beam scanning), and spectrum. This last function enabled an energy-loss spectrum, for example, to be displayed on the CRTs. An adjustable locate spot indicated the beam position in spot, spectrum, and line scan modes. Recording images presented some problems at that time. Photograph- ing a live image off a CRT was the only viable option in the absence of any readily available means of image storage, which would be commonplace today. The chosen method, relatively conventional at the time, was to use a Polaroid camera to photograph the image on a blue phosphor CRT. The blue, short-persistence phosphor produced a slightly sharper focus than the green phosphors of the viewing CRTs, which had the advantage of longer persistence and being more friendly to the eye. Recording a good-quality image required careful adjustment of the screen brightness, a process requiring operator judgment and skill that usually improved with experience. Provided signal levels were carefully controlled using the meters on the video level module, good images were regularly achieved.

3.8.2. Vacuum Controls The manufacture of and techniques for handling UHV vacuum systems had become a routine activity at VG by the time the microscope development was taking place, and much of the STEM vacuum system was based on this technology. Standard VG vacuum hardware and electronics, which had been developed and marketed for general UHV applications, were used wherever possible. The post office rack (seen on the right in Figure 2) houses all the microscope vacuum controls. At the top are standard VG controllers for two ion gauges and the gun ion pump. In the center are some specially adapted controls for the diffusion pump, the rotary pumps, and the bake-out. At the bottom is a standard VG sublimation pump controller.

4. IMPROVED RESOLUTION

4.1. The MIT HB5 By 1976 VGM had delivered the first HB5s to laboratories in Europe and was actively trying to sell its STEM in the United States. It was common knowledge in the mid-1970s that Siemens was engaged in building a A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 255 commercial STEM known as the Elmiskop ST100F. Their approach to the design, not surprisingly, resulted in an instrument that looked similar to one of their TEMs and was known to have a specification competitive with that of the HB5. With their long experience in the TEM field, Siemens was viewed as serious competition for VGM. The critical time came when potential orders in the United States materialized, the first being from Professor J. B. Vander Sande at MIT, Boston. This specified 100-kV accelerating voltage and the capability of demonstrating 2.04-A˚ gold lattice fringes, which, at the point of bidding for the order, was an unproven performance for the HB5. However VGM considered that its instrument was capable of meeting the specification and so submitted a bid guaran- teeing that performance. VGM then learned that their bid had been unsuccessful and that MIT was in final negotiations with Siemens. How- ever, several weeks after this major disappointment the prospect of the MIT order suddenly resurfaced and, after a brief negotiation held mostly over the telephone, the contract was finally placed with VGM. It subsequently transpired that MIT and Siemens had not been able to agree on contract terms and negotiations had been halted. Despite this situation Siemens was still considered by VGM as a serious threat in the marketplace. It was therefore with great surprise and considerable relief that VGM learned in the following months that Siemens had withdrawn the ST100F from its product line. The HB5 therefore remained the only cold field-emission STEM on the market. The acceptance trials of the MIT instrument took place at VGM’s East Grinstead factory in the late spring of 1977, where, with the assistance of John Vander Sande, the first 2.04-A˚ lattice fringes were obtained by the VGM team (Figure 24a). It is fair to say that all present were greatly excited, but also greatly relieved, by the result. The microscope was delivered to MIT a few weeks later.

4.2. The University of Illinois HB5 Obtaining 1.44-A˚ gold lattice fringes was the next performance breakthrough with the HB5. These were achieved with the help of Profes- sor Hamish Fraser from the University of Illinois, who was to take delivery of an HB5 soon after the MIT system had been delivered. This early 1.44-A˚ lattice fringe image is reproduced in Figure 24c. The dark-field 2.04-A˚ fringes in Figure 24b were produced at the same time using (020) and (022) reflections from the gold lattice. Although this seemed like a major step at the time, there was never a problem demonstrating the 1.44- A˚ fringes with subsequent instruments. 256 Ian R. M. Wardell and Peter E. Bovey

(a)

(b) (c)

FIGURE 24 The first gold lattice fringes. (a) 2.04-A˚ fringes obtained on the MIT HB5. (b) and (c) Dark-field 2.04-A˚ fringes using (020) and (022) reflections and 1.44-A˚ fringes, respectively, obtained on the University of Illinois HB5.

5. OTHER DEVELOPMENTS

5.1. Beam-Blanking Plates A set of beam-blanking plates located just above the differential pumping aperture (see Figure 8) allowed the operator to minimize specimen exposure to the beam between the moment of identifying an area of interest and recording an image of the area with the camera. This was easily achieved by identifying the area of interest at low magnification, positioning the area at the magnification center of the image, blanking the beam to set recording conditions, and then simultaneously starting to record as the image was switched to a predetermined high magnification. It is noteworthy that, because of their location, the blanking plates needed A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 257 only a very small voltage to cause a significant movement of the effective source position. To avoid any image interference it was a necessary feature of the design that the voltages applied to the plates by the beam- blanking electronics were very rapidly locked solidly to zero volts when the beam was returned to the unblanked state.

5.2. Detectors and the Virtual Objective Aperture 5.2.1. Non-Dispersive X-Ray Detector Another step in the continuous development program at VGM was the addition of a non-dispersive X-ray detector to the HB5 (Figure 25). Four access ports had been incorporated into the original design of the objective lens looking horizontally to where the specimen was positioned in the pole piece gap. One of these ports was already used to mount the objective aperture mechanism, and a second port at right angles to the aperture port was used to mount an X-ray detector. With the aid of slightly

FIGURE 25 HB501 fitted with Link Systems non-dispersive X-ray detector on extreme left. 258 Ian R. M. Wardell and Peter E. Bovey

Detector crystal Specimen 20° • • • • 0.181 sr

Electron beam

FIGURE 26 Typical X-ray detector geometry in HB501. modified objective lens pole pieces the X-ray detector crystal could be brought very close to the microscope axis (Figure 26). This permitted a solid angle of collection of 0.18 steradian at a take-off angle of 20. Standard X-ray detectors were usually connected to a microscope by a bellows and a small gate valve attached to the microscope port (see Figure 25). When the volume contained by the bellows had been rough pumped to about 10 1 Pa, the gate valve was opened and a linear drive then moved the detector crystal through the valve to its operating position close to the microscope axis. This meant that a detector could be added or removed without breaking the microscope vacuum.

5.2.2. Windowless X-Ray Detector Windowless X-ray detectors were connected to the microscope by a bellows and two gate valves (Figure 27). The extra valve, which was permanently attached to the detector by the bellows, ensured that the detector nose, which was maintained at liquid nitrogen temperature, remained evacuated and did not become frosted. In this case, the inter- space between the two valves had to be rough pumped before both gate valves were opened and the detector crystal moved forward to its operating position. A windowless X-ray detector was not much more difficult to fit to an HB5 than a standard windowed detector. The windowless version extended the energy range of detectable X-rays down to include atomic number 5, boron (Figure 28), and 4, beryllium. As a result of the high vacuum in the microscope column, windowless detectors could be used for long periods before a warm-up cycle was needed to desorb gas from the detector crystal. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 259

FIGURE 27 HB501 fitted with Link Systems windowless non-dispersive X-ray detector. Above the tilt drives, front right, are the charge-coupled device and 35-mm cameras for viewing the diffraction pattern observation screen.

5.2.3. Virtual Objective Aperture The combination of highly efficient X-ray detection with a sub-nanometer probe was not sufficient for good microanalysis without further changes to the HB5. It was important that the probe-forming system did not generate stray X-rays or scattered electrons, which could irradiate remote parts of the specimen and its holder and produce a spurious X-ray background. This particular requirement formed an important aspect of the development of the optical column design. One method of detecting the presence of unwanted stray radiation was to measure the ‘‘hole count’’ using a disk specimen of an element such as molybdenum, which has a low- and a high-energy X-ray line in the typical 0 to 20-keV energy range of most non-dispersive X-ray detectors. The disk was prepared with a small hole etched close to its center. If the electron probe is positioned in the hole, in principle, there should be no signal; that is, the hole count should be zero. Using the standard objective aperture just a few millimeters below the specimen to define the convergence angle, and hence the size of the probe, resulted in a very large hole count. This was caused by many electrons scattered by the aperture 260 Ian R. M. Wardell and Peter E. Bovey

Counts (ϫ102) 20 Noise Mo 18

16 Mn,Fe

14 V,Cr 12 B 10

6 V Mo 4

0 012345 Range (keV) Mo2(B,C) in steel

FIGURE 28 X-ray spectrum from a precipitate in a forging steel containing molybdenum, recorded using a windowless detector showing a boron peak. (Courtesy of J. R. Michael, Bethlehem Steel Co., USA.) finding a path to strike the specimen or by X-ray fluorescence of the specimen created by the X-ray flux from electrons stopped by the aperture. The problem was overcome by introducing a VOA (Craven and Buggy, 1981), which was an idea suggested by Alan Craven. The VOA was held in an aperture mechanism located as close as possible to the gun (see Figure 15). The condenser lenses were operated such that the VOA was conjugate to the back focal plane of the objective lens where it created an effective objective aperture. However, radiation and electrons scattered from the VOA had much farther to travel before striking the specimen and its immediate surround, consequently giving rise to a much- reduced background. The probe current was defined by the size of the VOA. The effective objective aperture size and convergence angle at the specimen were defined by the magnification between the VOA and the back focal plane of the objective lens. With two condenser lenses this could be varied over a wide range by adjusting their relative excitations. It was important to set this angle to the correct value to optimize the probe shape and size; if the angle were too large, spherical aberration would broaden the probe size, and if the angle were too small, diffraction would broaden the probe size. Using the VOA produced a good hole count. Inserting an SAD aperture improved the hole count by further attenuating A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 261

X-ray: 0–20 keV X-ray: 0–20 keV Live : 100 s Preset: 100 s Remaining: 0 s Live : 100 s Preset: 100 s Remaining: 0 s Real: 137 s 27% Dead Real: 111 s 10% Dead

< – .2 10.040 keV 20.3 > <–.2 10.040 keV 20.3 > FS = 8k ch 512= 27 cts FS = 31 ch 512= 0 cts MEM1: 50μm VOA 20nm into Mo Foil MEM1 Hole Count 50μm VOA 60nm into Hole

FIGURE 29 HB501 hole count measurement using a molybdenum foil specimen. Spectra are recorded with the probe 20 nm into the foil (left) and 60 nm into a hole (right), showing greater than 200 reduction in the height of the Mo K peak. the radiation and number of unwanted electrons reaching the specimen. Although this restricted the field view on the specimen, it was of no consequence at high image magnification. Further improvements could be achieved by inserting an objective aperture as long as its diameter was greater than that defined by the VOA. Figure 29 illustrates a typical hole count measurement that could be obtained by using the VOA and following the other precautions mentioned. The VOA aperture mechanism was subsequently fitted with an electrically isolated aperture blade that allowed the beam current that it inter- cepted to be measured (see Section 3.8.1). This was used as a normalizing signal with the microscope’s analytical X-ray and electron energy-loss spectroscopy (EELS) modes and overcame to a considerable extent any field emission instabilities. With this normalizing feature, it became possible to perform quantitative X-ray microanalysis with much-improved accuracy.

5.3. Stage Motor Drives The original manual stage drives were never intended to be a permanent feature. The motor drives to the specimen stage were always a priority development and were introduced soon after the QEC and Cavendish Instruments had been delivered. Four stepper motors were used—one for each shift movement and one for each tilt movement—and the controls were set in a recess in the console desk top below the two viewing CRTs (see Figure 22). There were lever keys for forward and reverse movement of each motor with meters to indicate position. Motor speeds were selectable or 262 Ian R. M. Wardell and Peter E. Bovey single-stepped. The shift motors could be tracked with magnification to give constant speed on the CRT displays. Importantly, there was a switch that set the stage to a central position with the tilt drives fully disengaged from the specimen cartridge ready for a specimen change. A red lamp indicated when this process was complete and that it was safe to remove a cartridge without attempting to remove the stage base as well. The two tilt-drive motor units can be seen on the front of the microscope in Figures 23, 25, and 27, and one of the shift motor drive units is visible to the right of the new airlock in Figure 30.

5.4. New Airlock The original manual transfer of cartridges between airlock and stage was used in only a few of the early microscopes after which a much-improved airlock (see Figures 14 and 23) was introduced. This was a precision

FIGURE 30 HB501 column showing a stage shift motor drive in the foreground to the right of the new airlock. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 263 mechanism that required a detailed sequence of operations to be performed to achieve a specimen change. It was not a truly automatic airlock, but it had the advantage that it did not require any particular operator skills. To change a specimen cartridge the microdiffraction coils needed to be temporarily retracted and, as described in Section 5.3, the stage needed to be put into the change position. The procedure to remove a sample from the stage, after ensuring the airlock was rough pumped, was simple. A locking catch on the airlock was released and the handwheel manually rotated clockwise. This opened the airlock door into the specimen chamber and advanced an arm into the center of the chamber (Figure 31). The handwheel was further rotated, lowering the arm down on top of the specimen cartridge, until a stop position was reached. At this point the arm latched onto the cartridge by means of a ballpoint pen–type mechanism. Reversing the direction of wind returned the arm, with the cartridge, into the airlock and closed the airlock door to the chamber. The airlock was then vented, the specimen changed, and the cartridge reat- tached to the loading arm. The procedure for inserting the cartridge into the stage was a repeat of that for its removal. The time required to change a specimen and be back in operation with an image was about 5 minutes.

FIGURE 31 Top view of an HB501 specimen cartridge being loaded into the stage base on the floor of the specimen chamber. The square-topped cartridge is hanging from the airlock loading arm beneath the open airlock door. The top left area shows a 10:1 lever block driven by a push rod that provides one of the stage shifts. At the bottom right is the mechanism that provides cartridges with a tilt motion that is independent of stage position. 264 Ian R. M. Wardell and Peter E. Bovey

An occasionally useful feature was a small port located below the position where the cartridge was suspended on the loading arm in the airlock (see Figure 14). A radiant outgassing facility (Wardell, 1981) could be fitted to this port to heat a specimen. Alternatively, a gas jet could be fitted for simple in situ chemical reactions. The airlock design remained substantially unchanged for about 20 years; the same design was fitted to the last HB601 (see Section 10) manufactured by VGM in 1996.

5.5. Energy Analyzer Mk2 The Energy Analyzer Mk1 did not have properly terminated magnetic fields at its entrance and exit and no attempt had been made to minimize aberrations. The analyzer had only a limited angular acceptance of about 0.6 mrad for a 3-eV energy resolution at 80-kV accelerating voltage (Batson and Craven, 1979). Several attempts were made to improve the performance of magnetic sector analyzers. Crewe, Isaacson, and Johnson (1971) had incorporated a 100 analyzer on a STEM in the University of Chicago. This had pole pieces with curved entrance and exit faces to correct for median plane aberrations and their measurements indicated a 25 times improvement in angular collection in the median plane over a straight-edged analyzer. Further calculations on aberration correction were done by Parker, Utlaug, and Isaacson (1978) and improved analyzers were built by a number of laboratories and substituted for the original VG analyzer. Colliex at Orsay in France had added an analyzer built by Gatan Inc. to an HB5 and reported (Jeanguillaume, Krivanek, and Colliex, 1981; Colliex and Trebbia, 1982) analyzer input semi-angles in the region of 5 mrad for 1-eV resolution at 100 kV. Meanwhile, at Cornell University, Isaacson and Scheinfein (1983) had designed and built an aberration-corrected analyzer for their HB5. This was an 80 analyzer, which had entrance and exit faces that were curved and terminated with mirror plates. Aberration correction was achieved by two quadrupoles and an octupole, which were located at the entrance to the analyzer. Another feature of the design was that the tilt of the focal plane was zero to within 0.5. The resulting performance was a great improvement on the original VGM analyzer. It demonstrated a resolution of better than 0.5 eV at 100-kV accelerating voltage with an entrance semi-angle of 7.8 mrad, which corresponded to an increased solid angle of acceptance in excess of two orders of magnitude. With Mike Isaacson’s help VGM adopted this design for the HB5 and later HB501 (see Section 7). Figure 32 is an outline drawing of the ELS501, as it was designated, illustrating the essential features of this analyzer. De-scan coils were added at the exit of the analyzer. The photomultiplier was mounted horizontally behind the slit and a yttrium-aluminum-garnet (YAG) scintillator; YAG had become the A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 265

Scintillator Slit mechanism Main magnet and coils Descan coils

Corrector coils

Slit drive Aperture assembly

FIGURE 32 Outline drawing of the Energy Analyzer Mk2. preferred scintillator material at that time. Because the input acceptance angle of this analyzer was so much greater than that of its predecessor an additional entrance aperture mechanism that accommodated larger apertures than those previously used was required. Its location is indicated in Figure 32. The ELS501, like its predecessor, had been designed to collect data serially. There were, however, clear benefits in terms of collection efficiency if the ELS501 could be fitted with a parallel detector. By 1989 VGM had addressed the issue and had designed a parallel detection version of the ELS501 designated ELS6000P. This was achieved by adding extra quadrupole lenses to the output side of the ELS501 analyzer to increase the dispersion of the spectra and by introducing a cooled CCD array (von Harrach and Colling, 1989) to effect the parallel detection. This array was optically coupled to YAG scintillators that were positioned slightly offset from the axis of the analyzer. An on-axis YAG scintillator behind a narrow slit was coupled to a photomultiplier and used for serial EELS and energy-selected STEM imaging. A deflection quadrupole was used to switch the spectra between the serial and parallel detection systems. The system, which was controlled from a personal computer, was capable of fast parallel acquisition, time-resolved spectral acquisition, and serial acquisition. However, the parallel EELS (PEELS) system presented VGM with a major software problem that they were unable to resolve. The competitive 266 Ian R. M. Wardell and Peter E. Bovey software that Gatan offered with their parallel EELS analyzer, designated 666-UHV PEELS, was more advanced than that which VGM could offer at the time with the ELS6000P, particularly for the processing of EELS spectra. The timing was unfortunate because VGM had a heavy commit- ment at that time to digitizing the microscope electronics (see Section 9), which left insufficient effort available to address the problem of the PEELS software. The result was that very few ELS6000Ps were ever made. A number of Gatan’s 666-UHV PEELS were retrofitted to HB501s. Initially there were a few vacuum problems, particularly during bake- outs, but these were resolved and from 1992 most VGM microscopes were equipped with the Gatan PEELS analyzer instead of the VGM ELS501.

5.6. Gun Lens In addition to its STEM instruments, VGM also developed a 60-kV UHV SEM designated HB50, which shared features with the HB5 including the field emission source. Some of these instruments were fitted with a cylindrical mirror analyzer (CMA) for Auger analysis and designated HB50A. J. A. Venables, professor of physics at the University of Sussex, UK, had received the first HB50A with which he conducted detailed spatial resolution measurements (Venables and Janssen, 1980a,b). Subse- quently he suggested that source aberrations of the VGM field emission source could be reduced by introducing a magnetic lens field in the acceleration region of the electron gun (Venables and Archer, 1980). His calculations suggested that an order of magnitude increase into a 20-A˚ probe at 60-kV accelerating voltage was possible with a ‘‘gun lens’’ and some alterations to the objective lens geometry. In conjunction with Venables, VGM designed and manufactured such a gun lens and it was retrofitted to the HB50A at Sussex University. A version of this lens, known as the FEG2100, was designed as an option for the HB5. Illustrated diagrammatically in Figure 15, it subsequently became a standard feature of VGM STEMs. Measurements on an HB501 with the FEG2100 showed an increase in current of 3 into probe diameters of 1 nm to 2 nm when operated at 100 kV (Figure 33). This was a useful improvement for analytical applications of the microscope that typically used a probe size in this range.

5.7. High-Excitation Objective Lens As indicated in Section 5.5, the angular acceptance from the specimen of the Energy Analyzer Mk1, which was not aberration corrected, was limited to 0.6 mrad for 3-eV resolution at 80 kV. Before the introduction of the aberration-corrected Energy Analyzer Mk2 there was a need for A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 267

FEG2100 gun with magnetic lens

1 Standard gun Probe current (nA) 0.1

0.01 0.1 1 10 Probe diameter (nm)

FIGURE 33 Graph showing the relative performance of the field emission gun with (FEG2100) and without (Standard) a magnetic gun lens. increased angular acceptance, which was achieved by introducing a replacement high-excitation objective-lens pole piece. The probe-forming focal length and aberrations of this pole piece were similar to those of the original pole piece. However, a strong focusing of the beam leaving the specimen produced a substantial beam compression of about 4 (Figure 34). This translated into a 16 gain in solid angle accepted by the analyzer from the specimen. The dimensions of the new lens (original in parentheses) were an 11.5-mm (4-mm) gap, a 5-mm (3-mm) lower bore, and a 15-mm (12-mm) upper bore. This pole piece was first fitted to the HB5 delivered to Glasgow University (Section 8.1) and became a standard pole piece option thereafter. Another consequence of the high-excitation lens was a 4 reduction of the camera length of the DPOS and microdiffraction facility. A corresponding increaseto8 alsobecameavailablewiththemicrodiffractionDCtiltcoilsfor weak beam imaging. Unlike Glasgow’s HB5, the majority of high-excitation objective lenses were not accompanied by z-motion stages and in consequence were not operated at a totally constant excitation. This created a specimen drift problem that necessitated a redesign (illustrated diagrammatically by the 268 Ian R. M. Wardell and Peter E. Bovey

Incident beam

FIGURE 34 Schematic ray diagram of high-excitation lens. The compression ratio / typically is equal to 4. difference between Figures 14 and 15) of the objective lens coil cooling to extract more efficiently the additional heat generated.

6. STAGES AND CARTRIDGES

The top-entry stage base, which was manufactured primarily from phosphor bronze, was developed for the first HB5 and survived as the basic design for all of the 100-kV HB-series microscopes. The original design allowed the specimen to move 1.5 mm in x and y. With the introduction of motor drives it became necessary to reduce this movement to 1.0 mm, and when the high-resolution pole piece (Section 7.2) was introduced, it was necessary to limit the movement even further to 0.7 mm.

6.1. Basic Cartridges A variety of cartridges, which were also manufactured primarily from phosphor bronze, were developed for this stage as the need arose. The late Jeff Lucas, who had previously designed stages for AEI TEMs, was responsible for the original stage base and cartridge designs and then the A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 269 late George Brown continued much of the design work and manufacture locally in his precision engineering workshop. The first cartridge to be made was a simple, non-tilting version that was supplied to QEC. This was followed by the 60 double-axis tilting cartridge for the Cavendish instrument. After receiving requests from early customers a tilting cartridge for SEM work that positioned the specimen above the objective lens was introduced. As pole piece dimensions were reduced to provide better image resolution performance, cartridges with 30 and 10 double tilt were developed. A single-axis 0 to 45 tilt cartridge was also developed to satisfy the need for a robust tilting holder when working with magnetic specimens.

6.2. Beryllium Cartridges When microscopes were fitted with X-ray detectors, cartridges with beryllium noses were required to reduce the background X-ray signal. Various cartridges of this type were initially designed and produced by George Brown. Later when it became necessary to find other manufacturers for these items, difficulties were encountered in locating workshops with the ability to handle beryllium. The problem led VGM to establish an in-house facility that was capable of coping with this hazardous material and carrying out the machining and assembly of all VGM’s stages and cartridges. The eventual range of cartridges with beryllium noses included a non-tilt, a fixed 20 tilt, and a 10 double-tilt version.

6.3. Cold Stage As with most developments at VGM, the cold-stage development was customer driven. A socket (see Figure 35), which could be cooled by liquid nitrogen, was mounted in a modified stage base and locked in position. The outer taper of the socket matched the stage taper, and an inner taper accepted special cartridges that could be inserted through the airlock in the usual way. The lower end of a cartridge came into spring contact with the cooled lower end of the socket and could be cooled to 150 C. Figure 35 also shows two of the cartridges that were designed for the stage. To the left is a simple non-tilt cartridge and to the right is a 30 double-tilt beryllium-nosed cartridge. Both the cartridges and the socket had high thermal impedance between their upper and lower ends. Visible with the cooling pipes in Figure 35 is a pair of wires leading to a small heater that is attached to the cold part of the socket. This provided a means of rapidly bringing the cooled cartridge back to room temperature so that it could be removed for a specimen change. Installation of the cold stage necessitated a vent of the specimen chamber to atmosphere and the use of standard cartridges was not possible when the cold stage was installed. 270 Ian R. M. Wardell and Peter E. Bovey

FIGURE 35 Cold stage components. In the center is the stage insert, which is liquid nitrogen cooled. This shows the two cooling pipes connected to the thermally isolated socket, into which specimen cartridges are loaded, and two wires connected to an integral heater. Two specimen cartridges are shown: on the left is a non-tilt cartridge, and on the right is a 30 double-tilt cartridge with beryllium nose.

6.4. Cryo-Transfer System Three cryo-transfer systems were built to special order. The purpose of the systems was to transfer a specimen from a cold source outside the microscope to a cold cartridge in the microscope’s analytical position while keeping the specimen cold and free from condensation. The cold source outside the microscope could be, for example, a cryomicrotome where the specimen would be prepared and mounted on a small capsule at liquid nitrogen temperature. If the source was remote, the specimen on its capsule could be transported to the microscope under liquid nitrogen. A special tool was developed to grab the capsule from its source or cold transfer container and transport it into the microscope’s cryo-transfer mechanism, which was held at liquid nitrogen temperature. The tool provided a flow of dry nitrogen over the specimen to prevent condensation, as did the cryo-transfer mechanism when it was at atmosphere. It took about 15 seconds to transfer the capsule into the mechanism. A special cold cartridge was developed that was, in effect, a combined cold socket and non-tilt cartridge (see Section 6.3) that fit into a redesigned stage base. The bottom end of the cartridge was designed to accept the capsule, which was square-shaped with just enough thickness to accommodate a specimen. The capsule held the specimen in the microscope’s analytical location at an angle of 20 toward an X-ray detector. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 271

FIGURE 36 HB501Cryo. The mechanism below the airlock on the right accepts a liquid nitrogen–precooled specimen from other equipment and transfers it, still cold, into the analysis position in the microscope’s cold stage.

The cryo-transfer system was, of course, an airlock and once rough pumped was able to transfer and ‘‘post’’ the capsule with its specimen into the cold cartridge. One of the microscope’s redundant tilt drives operated a small spring- loaded clamp that held the capsule in place. The clamp was released to allow the capsule to be put into or taken out of the cartridge by the cryo-transfer mechanism. In Figure 36 the drive system for the cryo-transfer mechanism with a liquid nitrogen Dewar assembly can be seen on the right-hand side of the optical column. Figure 37 shows images from a cryomicrotomed specimen (Andrews and Leapman, 1989). One of these images shows a very small amount of condensation on the specimen that occurred during preparation and transfer.

7. THE HB501

7.1. General Development Evolution of the HB5 was continuous over the years; Sections 3 and 5 describe a number of instrumental developments that took place in the 1970s and 1980s. In 1981 the decision was made to introduce the HB501 and incorporate many of these developments into the basic instrument. This enabled VGM to offer a ‘‘standard’’ high-performance analytical microscope to the increasing number of industrial research laboratories that were 272 Ian R. M. Wardell and Peter E. Bovey

(a)

(b)

FIGURE 37 (a) A cryomicrotomed frozen hydrated sample of 10% albumen in simulated sea water with added synaptosomes imaged at 170 C. Despite extended observation (4 hours) there was little sign of etching. Knife marks typical of cryomicrotomed samples are visible. The few widely dispersed white particles are frost formed during transfer. (b) The same specimen imaged at 170 C after warming to 60 C to drive off water. Much more internal detail, including synaptosomes, is visible and because the specimen was 90% water there has been some shrinkage and tearing. (From Andrews and Leapman, 1989; courtesy of San Francisco Press.) purchasing the instrument for routine work. The more specialized developments were then made available as options. Figures 14 and 15 (which date from 1981 and 1989, respectively) show that development was ongoing with the energy analyzer Mk2, the gun lens, and redesigned aperture mechanisms being incorporated into the standard instrument in the period. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 273

7.2. HB501UX and High-Resolution Imaging In the late 1980s interest grew in the annular dark-field imaging potential of the HB501 and this led to the introduction of a new version of the microscope, designated HB501UX. An objective-lens pole piece geometry was designed for high-resolution imaging applications to exploit this growing interest. The bore and gap dimensions of this pole piece were smaller than for previous designs with correspondingly smaller focal ¼ ¼ length (2.3 mm) and aberrations (Cs 1.3 mm and Cc 1.6 mm). The 1/4l3/4 resolution (0.43 Cs ) for this lens was 0.22 nm at 100 kV. The instrument was also equipped with a modified DFD, which was suitable for high-angle dark-field imaging. The expected resolution in this mode was 65% of that in a bright-field image and the image contrast was strongly atomic number (Z) dependent, approaching a Z2 dependency at large scattering angles. The theory of image formation and use of this detector are discussed by Pennycook et al. (Pennycook, 1989; Pennycook and Boatner, 1988; Pennycook et al., 1989; Pennycook and Jesson, 1990) and Xu et al. (1990). Figure 38 illustrates schematically the different classes of electrons scattered by a specimen. The central region very close to the axis gives rise to coherent, bright-field phase-contrast, the intermediate region primarily to diffraction contrast, and the outer region to Z-contrast. A modified DFD was equipped with a means of inserting and aligning one of a set of three masks, which permitted annular detection with a choice of inner angle ( 1). An improvement also was made to the coupling between the scintillator and photomultiplier in the new design. Examples of Z-contrast images are shown in Figure 39 (Pennycook, 1992). Columns of atoms, viewed end-on, can be seen with varying contrast corresponding to Ba (Z ¼ 56), Y (Z ¼ 39), and Cu (Z ¼ 29) with the heaviest atoms generating the greatest intensity. Standard analytical features such as energy-loss analysis and X-ray analysis were not compromised by the addition of the Z-contrast facility. The specimen stage was standard top entry and accommodated a 10 double-tilt beryllium-nosed cartridge, which was specially designed to fit into the small bore of the high-resolution objective pole piece.

8. SPECIAL AND VARIANT INSTRUMENTS

Some of the special instruments developed by VGM over the years are worthy of specific mention. Elsewhere in this volume a heavily modified HB501, the microscope for imaging diffraction and analysis of surfaces (MIDAS) (Venables, Cowley, and von Harrach, 1987), which was constructed for the late Professor J. M. Cowley at Arizona State University, is described by Sebastian von Harrach. Two further instruments are described here. 274 Ian R. M. Wardell and Peter E. Bovey

Axial Coherent, bright field phase contrast image, detector 1/4 λ3/4 with resolution = 0.66 Cs

Intermediate angle elastic and inelastic scattering dominated Incoherent, high angle by diffraction contrast dark field image with resolution 1/4 λ3/4 = 0.43 Cs

Annular detector

b >∼ 1 50 mrad High angle elastic scattering b ≈ dominated by 2 150 mrad Z-contrast

Thin specimen

Incident electron beam, 0.22 nm diameter

FIGURE 38 This diagram summarizes the detector geometry for Z-contrast imaging in the HB501UX. The high-angle annular dark-field detector collects electrons that are elastically scattered with intensity proportional to Z2. The result is an incoherent image for which the theoretical resolution is 35% better than the conventional bright-field phase- contrast image. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 275

Cu Y Ba

FIGURE 39 The two possible projections shown schematically for a substitutional defect in a YBa2Cu3O7-x high Tc superconductor are clearly differentiated in these Z-contrast images of atom stacks of Ba(Z ¼ 56), Y(Z ¼ 39), and Cu(Z ¼ 29). (Photographs courtesy of S. J. Pennycook, ORNL, USA.)

8.1. University of Glasgow’s HB5 A heavily modified HB5 was developed for Professor R. P. Ferrier and his associates, Alan Craven and John Chapman (both now professors) at the University of Glasgow, UK, and is shown in Figure 23. Important aspects of this particular instrument were a set of three projector lenses, called the 276 Ian R. M. Wardell and Peter E. Bovey post-specimen lens (PSL) system, a high-excitation objective lens (see Section 5.7), and a 0.7-mm z-motion of the stage. In addition to the high- excitation pole pieces, this was the first instrument to have two condenser lenses and was also equipped with a VOA. The considerations leading up to the building of this instrument and the anticipated performance can be found in the publication by Craven and Buggy (1981), and it is not intended to do more than summarize their reasoning here. The high-excitation objective lens was incorporated to provide post- specimen focusing of the beam. The condenser lenses were operated with variable source demagnification but a fixed objective lens source position at the SAD aperture. The high-excitation objective lens, which was not fitted with the improved coil cooling of later instruments, was operated at constant excitation using the z-motion of the stage to bring the specimen to the beam focus. This resulted in improved specimen drift characteristics due to the use of a virtually constant objective lens power and also produced a constant value of post-specimen angular compression of the electron beam. This angular compression greatly reduced the beam diameter at the first of the three PSLs and consequently, similar to TEM projector lenses, greatly reduced the image distortion of the PSL system. As seen in Figure 23, the PSLs were inserted between the objective lens and the original specimen chamber so that variable camera length diffraction patterns could be viewed on the usual DPOS screen in this chamber. The PSLs were designed to produce a fixed source distance for the energy-loss analyzer. The re-engineering of the optical column that was needed to insert the PSL system was relatively straightforward and added considerable retro- spective justification to the original decision to design the microscope with the gun at the base of the system. The high-excitation lens, the double condenser, and VOA (Section 5.2.3) became standard in the HB5 and HB501 models, although not the z-motion of the stage or the PSL system.

8.2. The HB501A A number of VGM’s field emission instruments were designed to perform Auger analysis. Approximately half of these were 60-kV instruments designed as UHV SEMs and designated HB50A; the remainder were 100-kV STEM instruments designated HB501A. The HB501A was designed for high-resolution STEM operation with the specimen in the usual position, immersed in the objective lens, and for Auger analysis in a second position in the chamber above the objective lens. Figure 40 illustrates these two positions. The Auger spectrometer, a standard VG Scientific product, was a hemispherical analyzer and was located in a vacuum vessel attached to the side of the specimen chamber. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 277

Energy loss spectrometer

Auger input lens Specimen airlock

Auger spectrometer Specimen Objective lens preparation chamber (option)

Possible specimen positions Primary electron beam

FIGURE 40 Schematic diagram of HB501A showing the Auger and high-resolution specimen positions and the Auger spectrometer and lens.

Electrons emitted from the specimen in the ‘‘SEM’’ position passed into the analyzer through an electrostatic input lens, which focused them onto the analyzer’s entrance aperture. The lens voltage was scanned synchronously with the analyzer voltages to produce an Auger spectrum. Addi- tional pumping was provided for the specimen chamber of these microscopes such that a routine pressure of 3 10 8 Pa was achieved. A specially designed transfer system and specimen holder (Figure 41) enabled specimens to be moved between the Auger and STEM positions. As can be seen the mechanism was extremely complicated and the design consumed considerable effort. In the STEM position all the usual analyses could be performed and in the Auger position STEM imaging with reduced resolution could be performed, thus enabling a given area to be identified in both positions. There were, however, limitations with the Auger spectroscopy at low energy because of stray magnetic fields escaping from the objective lens. These stray fields could be significantly reduced using a coil that was wound around the outside of the column support tube; this can be seen on the HB501A column that is illustrated in Figure 42. A further field reduction could be obtained with an appropriate shift excitation of the microdiffraction coils. However, the field compensation was never perfect and, as the energy of the analyzed electrons was reduced, the transmission 278 Ian R. M. Wardell and Peter E. Bovey

FIGURE 41 Component parts of the mechanism, which enabled the transfer of specimens between the upper and lower specimen positions in the HB501A diagrammed in Figure 40. At center right is the vertically oriented tube, which holds a specimen capsule at its lower end. This device, which picks up and releases the capsule in the fashion of a ballpoint pen, is maneuvered up and down and swung over the airlock to effect specimen transfers. through the electrostatic lens reduced to a premature cut-off at 30 eV. Figure 43 shows a typical Auger spectrum from a copper sample using a 30-kV incident beam and illustrates the fall-off in transmission at low energy. The Auger analyzer is visible in Figure 42 just forward of the non- dispersive X-ray detector on the left, and on the right-hand side of the optical column can be seen special UHV airlock facilities for handling, preparing, and transferring specimens. These facilities would vary between instruments depending on the intended application.

9. THE HB601

By 1991 VGM had built and delivered the first of their 300-kV STEMs, the HB603, which is described in detail by Sebastian von Harrach elsewhere in this volume. As part of the HB603 development a new-generation microscope digital-control system was designed that was also suitable A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 279

FIGURE 42 An HB501A with an Auger analyzer next to a non-dispersive X-ray detector on the extreme left. Below the selected area diffraction aperture and wound around the outside of the column support tube can be seen the coil, which was used to neutralize the specimen chamber DC fields that prevented low-energy electrons from reaching the Auger analyzer.

15 215 415 615 815 1015 KE (eV)

FIGURE 43 A typical HB501A Auger spectrum from a copper sample. The sudden fall in signal at about 30 eV is caused by residual magnetic fields in the specimen chamber. 280 Ian R. M. Wardell and Peter E. Bovey

FIGURE 44 The first HB601, with digital electronics, on exhibition at the 50th Annual Meeting of the Electron Microscope Society of America (EMSA) in Boston, Massachusetts, USA. for driving the 100-kV instruments. In 1992 the integration of these digital electronics with the HB501 column was completed. The first of these instruments (designated HB601) went on display that year at the 50th Annual Meeting of the Electron Microscope Society of America (EMSA) in Boston (Figure 44). This instrument is currently at the University of Illinois in Chicago, where it is one of several instruments that comprise the Electron Microscopy Research Resource Center directed by former VGM product specialist, Dr. A. W. Nicholls. In total, seven HB601 instruments were delivered.

10. POSTSCRIPT

In 1995 Fisons sold its Scientific Instrument Division, of which VG Micro- scopes was a part, and shortly afterward, on May 13, 1996, the new owners decided that VG Microscopes would cease trading. Existing microscope orders were completed and the last instrument, an HB601UX, was delivered to Japan. Figure 45 records that event in September 1996 at the Japanese Research and Development Corporation (JRDC), Nagoya. The customer, Kenji Kaneko, is photographed with installation engineers, Alan Nicholls and David Linnell. This is believed to have been the 70th successful STEM installation by VG Microscopes. This number includes four 300-kV HB603 instruments. An additional fourteen 60-kV cold field-emission SEMs with Auger were also delivered in the 22 year period from 1974 to 1996. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 281

FIGURE 45 The last VG Microscopes’ installation at JRDC, Japan in September 1996. The customer Kenji Kaneko (right) is photographed with installation engineers Alan Nicholls (left) and David Linnell.

Of course, many HB501s, and maybe some HB5s, are still in operation. The original Cavendish HB5 is certainly no longer in operation, having been neatly sectioned and turned into the exhibit in the Cavendish Laboratory Museum (Figure 46). Many HB5s and HB501s were modified to further push back scientific frontiers and this original Cavendish microscope was no exception. An experimental energy analyzer can be seen on top of the specimen chamber, and the middle section was stretched to provide space for some experimental quadrupoles and octupoles introduced by Ondrej Krivanek and colleagues. Thirty years after Deltrap’s initial investigations at the Cavendish Laboratory (Deltrap, 1964) this instrument became the test bed for proving that spherical aberration correction really could be made to work (Krivanek et al., 1997, 1998). Much of the remainder of the exhibit, including the all- important cold field-emission electron gun, is the original HB5 as produced by VG Microscopes in 1974. Worldwide, another six HB5, HB501, and HB601 instruments were modified with spherical aberration correctors manufactured by the Nion company established by Krivanek, taking their resolution capabilities to the 1-A˚ level (Batson, Delby, and Krivanek, 2002). This includes another Cavendish HB501 (Figure 47), which was renamed SuperSTEM1 and moved to Daresbury, UK, as part of the UK SuperSTEM project. 282 Ian R. M. Wardell and Peter E. Bovey

FIGURE 46 The second HB5, which was delivered to the Cavendish Laboratory in November 1974, is now an exhibit in the Cavendish museum. Sectioning by the Laboratory workshop reveals the inner workings of the microscope. The working parts are those originally delivered with additions and innovations created by two decades of experimentalists. (Photograph courtesy of Cavendish Laboratory.)

ACKNOWLEDGMENTS

For any inaccuracies in the previous text, please accept the authors’ apologies. Documenta- tion to support what has been written on VG Microscopes’ history is now sparse and what does exit is very distributed. The result is that some of that written herein has inevitably been based on memory, which the authors realize with regret becomes a little more inaccurate the farther back in time they go. Much of this chapter would not have been written without the help of several people who have been pestered for their memories and records going back nearly 40 years. In particular, we would wish to thank former VG colleagues Andy Waye, John Morphew, John Colling, Alan Nicholls, Sebastian von Harrach, and Bryan Barnard. We are grateful to Ian Vatter at the National Nuclear Laboratory, Windscale, for providing us with a copy of an HB501 operating manual, which we found most useful. We thank Ondrej Krivanek for sifting the recesses of his mind, and Ron Burge, Alan Craven, and John Venables and his former colleague, Chris Harland, for searching through old publications. Also, we thank Mick Brown and Jon Rickard of the Cavendish Laboratory for their help in furnishing us with details that we had forgotten or of which we were unaware. We are particularly grateful to them for the photographs of their HB5 before, during, and after it was prepared as a museum exhibit. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 283

FIGURE 47 SuperSTEM 1 at Daresbury is one of the Cambridge HB501s retrofitted with a Nion Mark 2 quadrupole-octupole spherical aberration corrector. It produces bright- field and high-angle dark-field images with 1-A˚ resolution. (Photograph courtesy of Mhairi Gass, SuperSTEM, Daresbury Laboratory, UK.)

REFERENCES

Andrews, S. B., and Leapman, R. D. (1989). Performance of the cryotransfer/cold stage for a VG Microscopes HB501 STEM. Microbeam Anal. 85–88. Batson, P. E., and Craven, A. J. (1979). Extended fine structure on the carbon core-ionization edge obtained from nanometer-sized areas with electron-energy-loss spectroscopy. Phys. Rev. Lett. 42(14), 893–896. Batson, P. E., Dellby, N., and Krivanek, O. L. (2002). Sub-A˚ ngstrom resolution using aberration corrected electron optics. Nature 418, 617–620. Bishop, H. E., Coad, J. P., and Rivie`re, J. C. (1972/3). A design for a cylindrical mirror analyser for use in Auger spectroscopy. J. Electron Spectrosc. 1(4), 389–401. Bishop, H. E., Rivie`re, J. C., and Coad, C. P. (1971). Auger spectroscopy of titanium. Surf. Sci. 24(1), 1–17. Browne, M. T., Lackovic, S., and Burge, R. E. (1975). Instrumentation and recording for the Vacuum Generators HB5 STEM instrument. In ‘‘Proc. Inst. Phys. Meeting, Bristol,’’ (J. A. Venables ed.), 27–30. Brundle, C. R., and Roberts, M. W. (1972). Some observations on the surface sensitivity of photoelectron spectroscopy. Proc. Roy. Soc. Lond. A 331, 383–394. 284 Ian R. M. Wardell and Peter E. Bovey

Brundle, C. R., Roberts, M. W., Latham, D., and Yates, K. (1974). An ultra-high vacuum spectrometer for surface studies. J. Electron Spectros. 3(4), 241–261. Butler, J. W. (1966). Digital computer techniques in electron microscopy. In ‘‘Proc. 6th Int. Cong. for Electron Microscopy (Kyoto),’’ 1, 191–192. Colliex, C., and Trebbia, P. (1982). Performance and applications of electron energy loss spectroscopy in STEM. Ultramicroscopy 9(3), 259–266. Cowley, J. M. (1969). Image contrast in a transmission scanning electron microscope. Appl. Phys. Lett. 15(2), 58–59. Craven, A. J., and Buggy, T. W. (1981). Design considerations and performance of an analytical STEM. Ultramicroscopy 7, 27–37. Crewe, A. V., and Wall, J. (1970). A scanning microscope with 5 A˚ resolution. J. Mol. Biol. 48, 375–393. Crewe, A. V., Isaacson, M., and Johnson, D. (1969). A simple scanning electron microscope. Rev. Sci. Instrum. 40(2), 241–246. Crewe, A. V., Isaacson, M., and Johnson, D. (1971). A high resolution electron spectrometer for use in transmission scanning electron microscopy. Rev. Sci. Instrum. 42(4), 411–420. Crewe, A. V., Wall, J., and Langmore, J. (1970). Visibility of single atoms. Science 168, 1338–1340. Crewe, A. V., Eggenburger, D. N., Wall, J., and Welter, L. M. (1968). Electron gun using a field emission source. Rev. Sci. Instrum. 39(4), 576–583. Deltrap, J. H. M. (1964). Correction of spherical aberration with combined quadrupole- octupole units. In ‘‘Proc. 3rd Regional Conf. on Electron Microscopy,’’ Prague, 45–46. Denbigh, P. N., and Grigson, C. W. B. (1965). Scanning electron diffraction with energy analysis. J. Sci. Instrum. 42, 305–311. Engel, A., Dubochet, J., and Kellenburger, E. (1976). Some progress in the use of a scanning transmission electron microscope for the observation of biomacromolecules. J. Ultra- struct. Res. 57, 322–330. Everhart, T. E., and Thornley, R. F. M. (1960). Wide-band detector for micro-microampere low energy electron currents. J. Sci. Instrum. 37, 246–248. Griffiths, B. W., Jones, A. V., and Wardell, I. R. M. (1973). An ultra-high vacuum scanning electron microscope with Auger analysis facilities. In ‘‘Proc. Conf. Scanning Electron Microscopy, Systems and Applications,’’ Newcastle. Institute of Physics, London, 42–45. Grigson, C. W. B. (1961). High-speed direct-recording system for electron diffraction. Nature 192, 647–648. Isaacson, M., and Scheinfein, M. (1983). A high performance electron energy loss spectrometer for use with a dedicated STEM. J. Vac. Sci. Technol. B 1(4), 1338–1343. Jeanguillaume, C., Krivanek, O. L., and Colliex, C. (1981). Optimum design and use of homogenous magnetic field spectrometers in EELS. Inst. Phys. Conf. Series 61, 189–192. Krivanek, O. L., Dellby, N., Spence, A. L., and Brown, L. M. (1998). Spherical aberration correction in dedicated STEM. In ‘‘Electron Microscopy, ICEM14 Cancun, Mexico,’’ (H. A. Caldero´n and M. Jose´ Yacana´n, M., eds.), Bristol: IOP Publishing, pp. 55–56. Krivanek, O. L., Dellby, N., Spence, A. L., Camps, R. A., and Brown, L. M. (1997). Aberration correction in the STEM, EMAG97. Inst. Phys. Conf. Series 153, 35–40. Lilburne, M. T., Herd, J. S., and Stuart, L. E. H. (1970). A pulse control circuit for use in the preparation of field emitter tips. J. Phys. E: Sci. Instrum. 3, 936–937. Parker, N. W., Utlaut, M., and Isaacson, M. S. (1978). Design of magnetic spectrometers with second-order aberrations corrected. 1 Theory. Optik 51, 333–351. Pennycook, S. J. (1989). High-resolution imaging with large-angle elastically scattered electrons. EMSA Bulletin 19(1), 67–73. Pennycook, S. J. (1992). Atomic scale imaging of materials by Z-contrast scanning transmission electron microscopy. Anal. Chem. 64(4)A, 263–272. A History of Vacuum Generators’ 100-kV Scanning Transmission Electron Microscope 285

Pennycook, S. J., and Boatner, L. A. (1988). Chemically sensitive structure-imaging with a scanning transmission electron microscope. Nature 336, 565–567. Pennycook, S. J., and Jesson, D. E. (1990). High-resolution incoherent imaging of crystals. Phys. Rev. Lett. 64(8), 938–942. Pennycook, S. J., Jesson, D. E., and Chisholm, M. F. (1989). High-resolution imaging of semiconductor interfaces by Z-contrast STEM. Inst. Phys. Conf. Series 100, 51–58. Reid, R. J., and Mykura, H. (1969). Low-energy electron diffraction patterns from faceted metal surfaces. J. Phys. D. Appl. Phys. 2, 145–146. Venables, J. A., and Archer, G. D. (1980). On the design of FEG-SEM columns. Proc. Eur. Reg. Conf. Electron Microsc. 1, 54–55. Venables, J. A., and Janssen, A. P. (1980a). The electron-optical performance of a FEG-SEM column. Inst. Phys. Conf. Series 52, 65–68. Venables, J. A., and Janssen, A. P. (1980b). Performance of a field emission gun scanning electron microscope column. Ultramicroscopy 5, 297–315. Venables, J. A., Cowley, J. M., and von Harrach, H. S. (1987). A field-emission STEM for surface studies, EMAG87, Manchester. Inst. Phys. Conf. Series 90, 85–88. von Harrach, H. S., and Colling, J. A. (1989). A two-dimensional detection system for energy loss spectroscopy. EMAG-MICRO 89. Inst. Phys. Conf. Series 98, 67–70. Wardell, I. R. M., Morphew, J., and Bovey, P. E. (1973). Results and Performance of a High Resolution STEM. Proc. Conf. Scanning Electron Microscopy, Systems and Applications, Institute of Physics, London 182–185. Wardell, I. R. M. (1981). Some considerations concerning a STEM probe forming system. Ultramicroscopy 7, 39–44. Wilson, C. J., Batson, P. E., Craven, A. J., and Brown, L. M. (1977). Differentiated energy loss spectroscopy in STEM. Inst. Phys. Conf. Series 36, 365–369. Xu, P., Kirkland, E. J., Silcox, J., and Keyse, R. (1990). High-resolution imaging of silicon (111) using a 100-kV STEM. Ultramicroscopy 32(2), 93–102. Chapter 7

Development of the 300-kV Vacuum Generator STEM (1985–1996)

H. S. von Harrach

Contents 1. Prelude in Oxford (1973–1975) 288 2. The MIDAS Project (1985–1988) 288 2.1. System 289 2.2. Gun Lens 289 2.3. Objective Lens 290 2.4. Side-Entry Stage 291 2.5. MIDAS Performance 291 3. The HB603 300-kV STEM instruments 292 3.1. The Prototype Design Phase 293 4. MIT and ANL Designs 305 5. Oak Ridge Design 307 6. Lehigh Design 310 7. Testing, Testing 311 8. Record-Breaking Results 316 8.1. Source Brightness 316 8.2. Energy Spread 316 8.3. ADF Resolution 316 8.4. X-Ray Microanalysis 317 9. Conclusions 320 Acknowledgments 321 References 322

FEI Electron Optics, Eindhoven, The Netherlands

287 288 H. S. von Harrach

1. PRELUDE IN OXFORD (1973–1975)

Project 6 was a brave attempt by the Oxford Metallurgy Department to build an ultrahigh vacuum (UHV) scanning transmission electron microscope (STEM) with cold-field emission gun (CFEG), similar to Albert Crew’s pioneering instrument (Crewe and Wall, 1970). Led by an inspired researcher, David Joy, as the generator of novel ideas, and aided by a competent technician, the project lacked only an engineer experienced in building such instruments. Some major components were bought from microscope companies, such as the gun chamber from VG and the high tension (HT) tank from Associated Electrical Industries (AEI), but we had no experience of system integration. Fortunately, with the help of the Head of Department, Sir Peter Hirsch, we were able to get advice from Mike Haine, who had recently retired from AEI’s Electron Microscopy division. Under Haine’s tutelage, we learned how to diagnose instabilities in the STEM image, using techniques that are still used today, such as measuring the sensitivity of a given deflection coil (in terms of nanometers of deflection per milliampere of coil current) and calculating the current stability required to achieve a given image resolution. So the instrument was gradually improved in STEM resolution. David Joy came up with ideas for high-resolution microanalysis using the high brightness capability of the CFEG. As it happened, Ray Egerton was developing the electron energy-loss spectroscopy (EELS) technique at Oxford at the time and saw the potential of the instrument. In 1974 Charles Lyman joined us to develop the X-ray detector system for the instrument. I presented the first results at a meeting in Oxford, but a demonstration of the STEM had to be canceled at the last minute because the field emission tip blew up in a high-voltage breakdown. There was an embarrassing moment when Dr. Cosslett from Cavendish arrived at the door of the lab, only to be told by a student he could not enter, until Roger Booker intervened and he was allowed to see the instrument. Afterward, he diplomatically offered help from his department—which of course was never taken up. The STEM resolution of the instrument (von Harrach et al., 1974) was still no better than 10 A˚ when I left Oxford at the end of 1975 and Ed Boyes took over the running of the project. (Boyes et al., 1977)

2. THE MIDAS PROJECT (1985–1988)

After some years of working with electron microscopes as a metallurgist I joined VG Scientific in 1985 to develop new STEM instrumentation, following on the success of the HB5. VG had just received an order from Arizona State University (ASU) for a UHV STEM with an Auger detection Development of the 300-kV VG STEM (1985–1996) 289 system, code-named MIDAS (Microscope for Imaging, Diffraction Analy- sis of Surfaces). John Venables at Sussex University acted as coordinator and immediate sparring partner for me. While he and colleagues at ASU designed the through-the-lens Auger system, I was tasked with developing a new side-entry stage, objective lens, and projection system. Given the problem of adding space for Auger detectors both above and below the objective lens, I eventually redesigned the optics of the whole column, fortunately guided by Ian Wardell who had many years’ experience of optical design. In many ways this instrument turned out to be a precursor of the later 300-kV STEM.

2.1. System Since Auger electron spectroscopy was the main aim of the instrument, the entire system was designed for UHV at the 10–8 Pa level—hence, bakeable to 200 C using metal vacuum seals only. A large specimen preparation chamber (Figure 1) with ports for different deposition and surface analysis stations was incorporated into the microscope. Sample holders (stubs) were loaded into the prep chamber via an air lock and samples were prepared and transferred in UHV onto the specimen goniometer.

2.2. Gun Lens The HB5 gun always had a weak point in its magnetic shielding at the bench level where the gun isolation valve was positioned. The result was strong 50-Hz interference from environmental magnetic fields on the

FIGURE 1 MIDAS specimen preparation system attached to the microscope column for specimen transfer in ultrahigh vacuum. (From VG Microscopes brochure HB501S.) 290 H. S. von Harrach beam that limited the resolution and probe current, since the interference had to be demagnified more than was otherwise necessary. So John Morphew, the mechanical design leader, devised a rather ingenious mechanical solution that allowed him to add more multiple overlapping mu-metal shields in this area. We also wanted to improve the magnetic gun lens, which had been John Venables’ idea (Venables and Archer, 1980; Venables et al., 1983) some years earlier. The lens was an unusual design as it had to project the magnetic field to the FEG tip region (at 60-mm object distance) to reduce the gun aberrations. This had led to a 3 increase in probe current at small probe sizes relative to the electrostatic gun lens in the HB5. The magnetic lens had a large-diameter lower pole just above the gun chamber. On the HB5 this pole was fixed and led to source misalignment problems in several systems. My solution was to make the lower pole mechanically alignable to move the source accurately on to the objective lens axis. The lens operated at a gun-side Cs value of 8 mm. It meant that the gun lens aberrations were not significant at small probe sizes.

2.3. Objective Lens For a side-entry stage geometry the symmetrical condenser-objective lens design was commonly used by other manufacturers (Riecke, 1962). How- ever, the MIDAS system required Auger electrons to be collected from the objective lens and transferred to the Auger analyzer efficiently. By moving all the objective lens coils to the upper side of the pole pieces, this could be achieved at the lower (electron entrance) side by reducing the distance from the specimen to the analyzer. It required only a relatively weak lens below the pole pieces to contain the Auger electrons by a so-called magnetic parallelizer, which transferred practically all the Auger electrons in a 12 cone (Kruit and Venables, 1988). The Auger electrons were then deflected by 15 by a Wien filter designed to have minimum effect on the primary beam. The Auger electrons were deflected by an additional 75 in a cylindrical mirror analyzer, followed by a concentric hemispherical analyzer for high-energy resolution (Figure 2). It is with some embarrassment that I remember switching on the objective lens for the first time after the microscope had been assembled and baked-out. As soon as the lens current was switched on there was an audible click—and then another when it was switched off. After much head scratching we realized that it must have come from the pole pieces— upper and lower poles joined to a nonmagnetic former—jumping up to the upper yoke when energized. We had failed to bolt it down, and the half-millimeter gap designed to reduce the field asymmetry made room for such a jump. Development of the 300-kV VG STEM (1985–1996) 291

Upper analysis CMA chamber sector

Upper parallelizer Specimen Objective lens

Lower parallelizer Wien filter Lower analysis chamber

CHA

FIGURE 2 MIDAS objective lens with Auger detectors below and above. (From Venables and Hembree, 1991. Reproduced by permission from the Institute of Physics.)

2.4. Side-Entry Stage George Brown was an extraordinary British engineer who had worked on the HB5 top-entry stage for VG in the 1980s, but his proudest achievement, soon after the Second World War, had been the design of an engine for a car that ran 70 miles to the (Imperial) gallon of petrol—astounding for its time! He was reluctant to take on a new side-entry stage design, as he was already ailing, but he did come up with the concepts of what later became the HB603 stage. Brown recognized the advantages of the cartridge system used in the HB5 top-entry stages, that is, the fact that only the stage drive mechanisms were outside vacuum, whereas the cartridge was completely in vacuum, isolated from the environment. He adapted the side-entry specimen rod concept by shortening the rod so that it was completely in vacuum (Figure 3) and used a transfer mechanism to insert it into the stage. The transfer mechanism was mounted in an air lock that could be fitted with a heater (ultraviolet lamp) or ion source for cleaning the sample.

2.5. MIDAS Performance The instrument (Figure 4) was completed in 1988 and installed at ASU. The design features were published in a number of papers (Hembree et al., 1989; Venables et al., 1987). 292 H. S. von Harrach

FIGURE 3 Detachable sample holder with double-tilt gimbal. (From VG Microscopes brochure HB501S.)

This ‘‘through-the-lens’’ design produced an enormous increase in Auger detection efficiency compared with other instruments at the time. To optimize the efficiency of the Auger detection, the specimen was biased to –500 V. This led to a resolution improvement of the Auger images from some 10 nm to below 1 nm. In the case of Ag particles, fewer than 20 atoms could be detected (Figure 5)(Liu et al., 1992). The results also show very high secondary electron (SE) collection efficiency at < 2-nm probe size.

3. THE HB603 300-KV STEM INSTRUMENTS

VG had received development orders for a 300-kV STEM from MIT and Argonne National Lab (ANL) in 1985 but did not have the capacity to start the project until 1987 when MIDAS was near completion. New VG Scien- tific management had attempted to cancel the order but retracted under pressure from the U.S. Department of Energy, the funding agency for the ANL project. A further order received from Steve Pennycook at Oak Ridge National Labs helped to convince the VG management to go ahead. Indeed, Development of the 300-kV VG STEM (1985–1996) 293

FIGURE 4 The MIDAS instrument: 100-kV STEM with through-the-lens Auger detectors. (From VG Microscopes brochure HB501S.) the HB5 had been so successful with record sales in 1988 that it was decided to split off the Microscopes group once more as a separate company; the new VG Microscopes had John Colling as General Manager, Peter Bovey as Marketing Manager, and myself as Technical Manager.

3.1. The Prototype Design Phase The 300 kV instrument was, on the face of it, just a task of scaling up from 100 kV. But in fact, it involved far more than that. VG had no computer- controlled microscopes at the time, so a group of three software engineers, later led by Steve Elliot, was formed to develop the instrument control 294 H. S. von Harrach

(a) (b)

(c)

5nm

FIGURE 5 (a) Auger AgMNN, (b) secondary electron, and (c) ADF STEM images of silver particles on alumina. (From Liu et al.. 1992; reproduced by permission from Elsevier Science BV.) starting from a blank sheet of paper. Similarly, the FEG and HT supply had to be redesigned completely—with the help of John Collins (who had designed the 100-kV HT supply and much of the STEM electronics for the HB5). Given the high materials cost of the instrument, VG could not afford to build a prototype, so we had to design to ‘‘first-time-right’’ principles (i.e., to conservative design rules).

3.1.1. System/Pumping Near-UHV system specifications for the specimen region (except for ANL specs) led to the idea of differentially pumped double O-ring seals. The fact that Viton O-rings are permeable to gases is not a problem if the intermediate vacuum is even as high as 100 Pa, (i.e., pre–vacuum pump level), since the leak rate is still reduced by a factor of 1000. Combined with baking the whole column to 150 C it is not difficult to reach pressures of 10–7 Pa. Following the VG tradition, the pumping system was based on oil- diffusion pumps with an efficient cold trap to eliminate back-diffusion of oil into the column. The exception was the ANL system, which was designed entirely around ion getter pumps. The electron source region was rather different since the cold FEG had to operate at below 5 10–9 Pa to keep stable emission. To achieve that, only ion pumps and titanium sublimation pumps were considered suitable, and a higher bake-out temperature of about 250 C was needed to get the base pressure to 2 10–9 Pa. Development of the 300-kV VG STEM (1985–1996) 295

Extensive discussions ensued about the possibility of designing an ion pump for the source region, floating at 300 kV. This was favored by Colling, but it entailed the complication of a competitor’s patent for a similar design. In the end, the decision was made against the floating pump because it required considerable extra power fed via the high-voltage generator. The downside was that a second accelerator tube would be needed to achieve sufficient pumping speed at the field emission tip. The design concept (Figure 6) was probably quite innovative and we drafted a patent for it that was never submitted because it was unlikely that anyone would be foolish enough to copy it (this seems to have been confirmed). Here, I must introduce the unsung mechanical genius in the team—John Morphew. Morphew had worked at AEI previously and had joined VG to design the HB5 column, but with the HB603 he reached the peak of his creativity. Essentially, the whole instrument, except the stage, started on his drawing board and every part drawing produced by the Drawing Office, still working without computer-aided design systems, was scrutinized by him and errors corrected. It is mainly due to John Morphew that a superbly stable machine was designed, even though we felt at the time that his robust designs (a column diameter of 300 mm) were more appropriate for tanks than microscopes. It is incidentally also the reason why the later addition of spherical aberration correctors and improvements to sub-A˚ ngstrom resolution became possible without lengthening the column.

Interface pumping Isolation valve Condenser 1 lens Condenser stigmator Virtual OBJ. aperture

Accelerator (11 stages) HT tank Field-emission gun Gun chamber Gun pumping

FIGURE 6 Schematic of gun system. The HT tank is attached via a bus bar to the gun, which is positioned between two accelerator stacks with an ion pump and Ti sublimation pump at the bottom. (From author’s archive.) 296 H. S. von Harrach

3.1.2. HT Tank-Gun and Accelerator The above considerations led to the system with the gun placed symmetrically between two 11-stage accelerator tubes (see Figure 6). After looking for a supplier of 300-kV generator and cable (Haefeli was already in cooperation with Philips and would not even talk to us!), we decided to do without a high-tension cable and connect the HT tank via a bus-bar directly to the gun. This concept had been used on high-voltage microscopes previously with the HT system at the top of the column. For our system, with the gun at the bottom, it was relatively easy to design the HT generator mechanically coupled to the column but still retractable for servicing. John Colling developed a generator design using an oil-filled tank, rather than a sulfur hexa- fluoride SF6 gas pressure vessel, principally because of the thermal stability achieved by oil enveloping and efficiently cooling the electronics at high voltage. He came up with the concept of a control system for the generator, consisting of a microprocessor at ground potential that was fiber optically coupled to a microprocessor at high voltage. This neatly avoided the interference problems commonly experienced with other control systems and helped to achieve the 1-ppm HT stability required. Finding suitable, bakeable accelerator tubes was another challenge. We were fortunate to find a U.S. company (National Electrostatics Corporation) that had been making just the right tubes for particle accelerators. The tubes were made of alumina rings that were bonded to titanium electrodes under high pressure and temperature and could be baked to 400 C. We were initially concerned about the fragility of these tubes, but when their sales manager dropped a spanner on the ceramic to make the point, we saw how strong these tubes actually were, even for the 11-stage units we used in the end. For the gun chamber we chose SF6 as insulating medium at 5-bar pressure. We had no experience with pressure vessels or the –11 problems of vacuum seals with 5 bar of SF6 on one side and 10 mbar (10–9Pa) on the other. We chose to use gold wire seals, rather than copper gaskets, since they required smaller flanges on the accelerators, but they had not been used before on such large diameters (125 mm) and proved to leak after baking to 250 C. In fact, the first time we baked the accelerators there was a rather nasty smell of burning material. It turned out that someone had fitted the cold junction of the ther- mocouple for monitoring the temperature in a hot region so that the accelerator actually reached 370 C! After much experimentation, we found a solution that ensured enough elastic deformation of the flanges to keep the gold seals under compression at all stages of the bake-out cycles. Development of the 300-kV VG STEM (1985–1996) 297

3.1.3. Gun Electron Optics The design of the magnetic gun lens on the HB501 had been successful in obtaining the highest probe current into a small probe. To copy that principle on the 300-kV system with a multistage accelerator tube of some 220-mm length was obviously not possible. The idea of incorporating a magnetic lens around the field emission tip was rejected on the basis of the large amount of power required to supply a short focal length lens via the HT generator. A permanent magnet lens, on the other hand, could not operate over the whole range of 50 kV to 300 kV. This left us with the option of an electrostatic gun lens, following the concepts of Veneklassen (1972), with a crossover at a fixed point in the accelerator and therefore constant magnification. Robert Keyes moved from the Applications group to join the project and work on the gun design. He simulated the gun performance with the programs of Eric Munro (1971), aiming to reduce the gun lens aberrations to a minimum. The importance of minimizing the gun aberrations is seen from Eq. 7.1. For a source magnification, ms, the effective spherical aberration coefficient of the illumination system is

Cs ¼ðCso þ Csc þ CsgÞ; (7.1) ¼ þ 4 where Cso is the objective lens spherical aberration, Csc (1 mc) Csgo is the condenser lens aberration with magnification mc from condenser to ¼ 4 3/2 specimen plane, and Csg ms (Vi/Vo) Csgo is the object-side gun spherical aberration at the gun object voltage Vo (equal to the extraction voltage) and acceleration voltage Vi (Csxo denotes the Cs value of lens x at infinite conjugate). < Because Cso 1 mm for the objective lens, Csg must be 0.1 mm to have negligible effect on the total Cs value. The CFEG source diameter is 5 nm so that the probe is normally demagnified for high-resolution << conditions (i.e., ms 1). For the large probe conditions, the gun lens Csg becomes dominant if

> ; ; =ð ð 4ð = Þ3=2Þ > : Csg Cso that is Csgo Cso ms Vi Vo 1 (7.2) ¼ > This condition occurs for Csgo 10 mm at ms 0.1. Similarly, for ¼ 2 3/2 chromatic aberration, the gun Cc contribution Ccgo ms (Vi/Vo) so Ccg > becomes larger than the objective Cc value at ms 0.02, although the total chromatic aberration contribution to the probe size does not become > dominant until ms 0.2 (see Section 3.1.5). We chose a tetrode gun design with a decelerating final lens or focus electrode to produce a crossover near the differential pumping aperture (DPA) in the accelerator. The DPA was placed in the third accelerator stage from the gun, thereby limiting the volume at gun vacuum level (2 10–9 Pa) and providing a crossover at a useful distance from the first condenser lens. 298 H. S. von Harrach

3.1.4. Column Electron Optics The chief object of the column design was to achieve the highest brightness, and consequently, the highest current in a small probe for analytical and high-resolution STEM. Obviously, condenser lenses are necessary to allow the probe size to vary, and here the HB5 approach of two condenser lenses was used. In fact, the Argonne specification had a further requirement for 10-nm parallel illumination, which led to the addition of a third condenser lens close to the objective lens. Figure 7 shows the theoretical probe current versus probe diameter predicted for the design at 300 kV, based on the geometric optics of the column (see, e.g., Venables and Janssen, 1980). The graph shows the probe size for a wide-gap objective pole piece with Cs value of 4.3 mm at a theoretical brightness of 3.9109 A/cm2/sr. The curve shows the influ- ¼ ence of the gun lens chromatic aberration (Cc 9.5 mm) from around 0.5 nm probe size upward (where the slope in the curve changes). Data published from the Lehigh HB603 (Watanabe et al., 2006) suggest that the probe current is overestimated by a factor of 1.6 (see Figure 7).

3.1.5. Objective Lens with Analytical and High-Resolution Pole Pieces The two aims of highest imaging and highest analytical resolution are not entirely compatible because of the geometric requirements for the energy- dispersive X-ray (EDX) detectors. Two types of objective pole pieces were

Calculated probe size vs probe current at brightness = 3.9 ϫ 109 A/cm2/sr, 300 kV, objective Cs = 4.3 mm, energy spread 0.6 eV 10

Cs = 4.3 mm Lehigh data 0.1 Probe current (nA)

0.01 0.1 110 Probe size FWHM (nm)

FIGURE 7 Calculated probe current versus probe size curve for a source brightness of 9 2 3.9 10 A/cm /sr at objective Cs¼ 4.3 mm, Cc ¼ 4.0 mm, gun Cs ¼ 13 mm, Cc ¼ 9.5 mm. (Graph redrawn from author’s archive; Lehigh data from Watanabe et al., 2006.) Development of the 300-kV VG STEM (1985–1996) 299 therefore developed: an analytical version with wide-gap pole pieces and a high-resolution column with minimum gap and Cs value. Both required the use of a material with high-saturation magnetic field, such as an iron- cobalt alloy. Previously only pure iron pole pieces had been used by VG and these were prone to give rise to high astigmatism, probably as a result of inhomogeneities in the material. I contacted a scientist at the VG Mass Spectrometer company and found that they produced Fe-Co magnets with 1-ppm field uniformity. In fact, the material turned out to be easier to machine than soft iron to produce poles less than 1-m out-of-round- ness and with low astigmatism. The objective lens was designed as a symmetrical condenser-objective lens (Rieke, 1962) with a wide enough gap to allow an X-ray detector to be inserted to a distance of 9.5mm from the specimen, resulting in a collection angle of 0.3 srad. The EDX performance would clearly suffer from high background signals due to back-scattered electrons generating X- rays from the pole pieces (Williams et al., 1986), so it was decided to cover both pole faces with beryllium shields, which Nestor Zaluzec (ANL) calculated to need at least 0.5-mm thickness. The analytical objective lens design ended up with a 14-mm gap (13 mm between Be shields) and a rather high 4.3-mm Cs value for the specimen at mid-gap position (Figure 8). The large gap allowed us to design the double-tilt holder with 60 of tilt about the eucentric axis. However, the STEM resolution was still theoretically 0.2 nm at 300 kV and, more importantly for microanalysis, a STEM probe current of 0.5 nA was obtained at 1.1-nm full width at tenth maximum (FWTM) probe diameter (Watanabe et al., 2006).

FIGURE 8 Schematic of analytical objective lens with Be shields on the pole faces and bores. The X-ray detector is shown on incident electron side. (From Lyman et al., 1994; reproduced by permission from the Royal Microscopy Society.) 300 H. S. von Harrach

3.1.6. Specimen Stage We were fortunate in saving a small workshop from the company policy of subcontracting everything. It was really thanks to the great skill of Dave Mitchell and Terry Manders in making stages and pole pieces that the key parts of the microscope worked well. These two could make the smallest parts to watchmaker precision, exactly what was needed for the stage parts. The problem of differential expansion on baking the microscopes to 180 C was particularly difficult to cope with for moving stage parts. George Brown’s solution was to coat sliding surfaces with molybdenum disulfide power, which was ‘‘burnished’’ into the surfaces in hours of tedious labor until the parts moved perfectly without backlash. The problem of course with this approach is that the MoS2 particles wear with use and the surfaces roughen or seize up in UHV. We gradually changed to rollers spring-loaded by a constant force against the moving parts, and this helped to keep the stages working. The stage concept involved the use of a large-diameter disk with ruby ball bearings running in a V-groove for eucentric rotation of the rod. The y and z motions were coupled in a manner to tilt the rod within a spherical bearing at the center of the disk. The rod was located in a cup at the front end and that cup formed part of the x-motion unit with a central beta tilt drive pin that entered a plunger at the center of the rod nose (Figure 9). Both hot and cold stages were designed, in addition to the double-tilt holders with Be gimbals. For specimen cooling, the nose of the holder was cooled via multiple leaf springs that maintained adequate thermal contact while the holder was rotated about the eucentric axis; the springs were in turn connected by a copper braid (see Figure 9)toa

Tilt unit

Pb ring

Pb shields

X-motion unit

FIGURE 9 HB603 specimen stage in objective lens chamber with pole pieces mounted. y-z–Tilt unit (top center), x-motion unit with braid for cooling (bottom center); note large lead ring and shielding blocks are all in ultrahigh vacuum. (From author’s archive.) Development of the 300-kV VG STEM (1985–1996) 301 liquid nitrogen–cooled block. A single-tilt heating holder (for Argonne) was designed for up to 1100 C and used a furnace cup in the specimen cartridge. George Brown did not live to see the final specimen stage on the HB603. He would not have been disappointed with the result: a stage with the lowest drift performance of any microscope at the time (<0.1 nm per minute) and ultrahigh stability (10 pm vibration in a lab environment).

3.1.7. Projector System The projector system is not very critical for STEM operation since the STEM resolution is determined entirely by the illumination system. However, the camera length must be variable for optimum matching of the collection angle of the detectors (ADF and EELS), and for imaging of diffraction patterns. The customers specified a camera length of up to 2000 mm and down to 20 range for microdiffraction. For ADF STEM, an inner collection semi-angle of the ADF detector up to 50 mrad (i.e., a camera length of 50 mm) was required. In practice, this meant a three-lens system, including a strong final projector for large camera lengths (Figure 10). As usual, Argonne had special requirements, especially for TEM at up to 400,000 magnification. That meant a fourth projector lens was designed for Argonne only. Steve Pennycook at Oak Ridge, on the other hand, wanted only high- resolution STEM capability without the long camera lengths, so his system needed only a single projector lens to be able to vary the inner angle of the ADF detector (more correctly, this should be called the Howie detector after its inventor, Archie Howie, 1979) and to view diffraction patterns at wide angular range.

3.1.8. Detectors for STEM and EELS The wide range of detectors for the four instruments required a new approach that was capable of allowing for different combinations of scintillators. Since the detectors were more than 2.5 meters above floor level, John Morphew came up with the idea of a motorized carriage that could be moved into position for the different scintillators, much like the aperture mechanisms. The YAG scintillators were thinned to 50 m and fitted with 45-degree mirrors for viewing diffraction patterns with a charge-coupled device (CCD) camera or collecting light into a photomultiplier for an ADF signal. All the scintillators (and mirrors) had central holes of 2-mm and 4-mm diameter so that the bright-field signal could enter an EELS spectrometer. For all the other HB603s, a serial EELS system was designed and scaled up from the HB5 100-kV design. Scaling up did include adding water cooling to the magnet and vast amounts of lead shielding all around (Figure 11). 302 H. S. von Harrach

Electron energy loss spectrometer Collector aperture Annular dark field detector Diffraction pattern CCD camera YAG screen

P3 lens

P2 lens

P1 lens

Post specimen scan Objective lens Specimen stage objective aperture

X-ray detector Secondary electron detector Selected area aperture Main scan coils

C2 lens

Vacuum isolation valve C1 lens

Virtual objective aperture Accelerator

Field-emission gun

FIGURE 10 Schematic diagram of HB603 column with three projector lenses. (From author’s archive.)

3.1.9. Software Architecture The operating systems available on personal computers (PCs) at the time were not real-time, multitasking except for OS/2. Although it was already a minority system, OS/2 was chosen. As Figure 12 shows, the control system was based on two PCs—one for controlling the instrument and one for image acquisition and display. The supervisory PC communi- cated with the column electronics via opto-isolated RS232 serial interfaces. Two frame stores were used to acquire images—bright-field and Development of the 300-kV VG STEM (1985–1996) 303

FIGURE 11 HB603 EELS system (top), screen mechanism (left), and diffraction camera (right). ADF STEM detector is opposite the camera. (From author’s archive.)

FIGURE 12 HB603 Control system with opto-isolated serial interfaces. (From von Harrach, 1994; reproduced by permission from Elsevier Science BV.)

ADF—simultaneously with up to 4096 4096 pixels and display them on two monitors (Figure 13). A third monitor displayed the user interface, which could have been used to control all the functions of the instrument. There were extensive debates on the balance between controls to be made available by pushbuttons or on the monitor via mouse clicks. The opinion prevailed that 304 H. S. von Harrach

FIGURE 13 (a) HB603 Control panels and monitors. (b) Example of user interface for HT control. (From VG Microscopes brochure HB603.) direct control by pushbuttons was essential in many cases, so we ended up with three panels of pushbuttons (see Figure 13). The many options for STEM and TEM operation and alignment made the design of the user interface a mammoth task that Steve Elliot and his small team worked on with great persistence, especially with the ever-increasing changes requested by customers. So, Nestor Zaluzec wanted to have three levels of users requiring different levels of authorization in a multi-user environment. Steve Pennycook wanted the ADF detector to operate in pulse mode for the lowest signal levels, David Williams from Lehigh wanted to have beam blanking, and so the list went on. Development of the 300-kV VG STEM (1985–1996) 305

4. MIT AND ANL DESIGNS

Whereas the MIT instrument (Figure 14) was designed for highest- resolution microanalysis and 2.0-A˚ STEM resolution, the ANL machine had many additional requirements, as specified in a 43-page development contract, including a UHV preparation chamber and sample transfer system to the microscope stage. Nestor Zaluzec, the Argonne customer, became a frequent visitor at VG to discuss the details of the design as it progressed. He contributed significantly to the design of the EDX geometry for 0.3-srad collection angle of the detector and to the Be shielding—designs from which all the other HB603 customers benefited. Zaluzec insisted on having electrically isolated aperture mechanisms with eight positions, and for the virtual objective aperture (in front of the first condenser lens) he proposed the best design for reducing stray X-ray signals: a Be disk in front of the Pt beam-limiting aperture, and 1-cm

FIGURE 14 The MIT HB603 column with EDX and serial EELS systems. (From VG Micro- scopes brochure HB603.) 306 H. S. von Harrach thickness of Pt behind the aperture to absorb the X-rays generated by the aperture as much as possible. Over the period of 2 to 3 years he filled some dozen U.S. government regulation notebooks with design notes, which probably contain the most complete instructions existing on how to build a UHV STEM instrument. We worked with Link Systems (later part of Oxford Instruments) to develop a windowless detector with mechanical shutter (Lyman et al., 1994), similar to detectors they had already supplied for the HB5. Argonne had also specified a through-the-lens Auger system, similar to the MIDAS design. A cylindrical mirror analyzer (CMA)-type spectrometer was designed to fit a chamber below the objective lens, as well as an SE detector (Figure 15). The list of attachments to the Argonne preparation chamber (Figure 16) was rather like reading the catalog of VG surface science equipment available: It included an argon ion gun, a film deposition station with four sources, secondary ion mass spectroscopy (SIMS) system with Ga liquid metal ion source, film thickness monitor, gas reaction cell, reverse-view low-energy electron diffraction (LEED) system, and vacuum transfer vessel with ion pump. The idea was to be able to make or modify specimens under UHV, deposit films, check the surface

FIGURE 15 Argonne column (middle part), with Auger spectrometer (front) and specimen transfer chamber (right). (From author’s archive.) Development of the 300-kV VG STEM (1985–1996) 307

FIGURE 16 Argonne preparation chamber. (From author’s archive.) cleanliness with LEED, and analyze the chemistry of surface atoms with SIMS. This was all standard equipment, but having it attached to the microscope to be able to transfer specimens in UHV was a new concept that filled me with horror: hang all that equipment on the side of the column and still expect high-resolution performance? Well, I suppose we had done just that on the MIDAS instrument.

5. OAK RIDGE DESIGN

The objective lens pole pieces for the Oak Ridge instrument were designed purely for the highest resolution, compatible with the space needed for the specimen holder. The tilt range of the holder was reduced to 12 to minimize the pole piece gap. Robert Keyes designed the pole pieces with a 4-mm gap and a calculated Cs value of 1.0 mm, which gave a theoretical STEM resolution of 0.125 nm, as shown in the wave optical calculation of the transfer function (Figure 17). Simulations of the probe profile and the Si(110) image (Figure 18) showed that the 0.136-nm dumbbells could be resolved at a narrow range of defocus values. However, this 308 H. S. von Harrach

Incoherent 1.5

0.5 Scherzer 0 OTF -0.5

-1

-1.5 0 0.2 0.4 0.6 0.8 1 -1 Spatial frequency (Å ) Df=-438 Å a = 9.36 mrad

FIGURE 17 Calculated ADF STEM optical transfer function (OTF, top), showing 0.125-nm resolution at Scherzer defocus. (From von Harrach et al., 1993; reproduced with permission from IOP Publishing Ltd.)

−2.5 2.50 −2.5 2.50 −2.5 2.50 −2.5 2.50 −2.5 2.50 −300 −400 −500 −600 −700 Defocus (Å)

FIGURE 18 Calculated probe profiles at defocus values of –30 to –70 nm (top) and Si (110)-simulated images (bottom) at corresponding defocus values (von Harrach et al., 1993; reproduced with permission from IOP Publishing Ltd.) left very little room (20 pm) for incoherent effects as a result of power supply instabilities and environmental interference (AC magnetic fields, acoustic noise, and floor vibration). The specifications for the instrument stabilities were actually quite relaxed—3-ppm HT stability, 2-ppm objective lens current stability—which would have resulted in up to 75-pm incoherent Development of the 300-kV VG STEM (1985–1996) 309 probe broadening. In fact, the crucial powers supplies, especially the beam deflectors, were designed to sub-ppm stability over a wide frequency range (von Harrach, 1994) to achieve the lowest amount of probe blurring. The Oak Ridge system, HB603U, which had no EELS spectrometer at that time, was fitted with a photomultiplier straight above a 50-m thick YAG scintillator to transmit the maximum light to the ADF signal (Figure 19). For the bright-field STEM signal, a small scintillator with a thin fiber-optic light guide was designed to cast the minimum shadow onto the ADF scintillator. To view diffraction patterns, a motor-driven mirror was rotated into position below the ADF photomultiplier and an intensified CCD camera viewed the scintillator via the 45 mirror.

ADF photo− multiplier

Intensified CCD camera

BF photo multiplier

YAG scintillators

Projector lens

Specimen

Incident probe

FIGURE 19 Oak Ridge detector system. (From von Harrach et al., 1993; reproduced with permission from IOP Publishing Ltd.) 310 H. S. von Harrach

6. LEHIGH DESIGN

EDX pulse-processing electronics uses a pulse pileup rejection circuit to avoid counting two photons arriving at the detector at short intervals as one photon of twice the energy. This gives rise to a dead time that limited the maximum output count rate of the Oxford detector to about 8 kcps at short pulse-processing time. A method of reducing the dead time was proposed by Jaklevic et al. (1972) to blank the beam whenever a photon was detected, thereby preventing pulse pileup. This method was implemented by conjugate blanking of the beam on the selected area (SA) aperture (Figure 20) and theoretically should achieve an increase in throughput by 3 to 4 times (Statham et al., 1974). The optics had to be adjusted quite critically for conjugate blanking— that is, to obtain a crossover at the center of the blanking plates so that the probe did not sweep across the specimen while being blanked. Using the SA aperture for blanking the beam meant positioning the crossover before the SA aperture, rather than at the aperture as normal, such that the beam was still completely cut off during blanking. Lehigh was the only HB603 customer to have two EDX detectors, one Si (Li) with 0.3-sr and one intrinsic Ge detector with 0.2-sr collection angle, fitted at right angles to each other and to the two stage tilt axes. The intrinsic Ge detector had higher sensitivity at higher X-ray energies (>10 keV) and slightly better resolution (115 eV compared with 135 eVat Mn- Ka), but was otherwise not very advantageous in view of the lower collection angle and the fact that it could not be blanked simultaneously with the Si (Li) detector.

Specimen

Unblanked beam Selected area diffraction aperture

Blanked beam

(lenses omitted)

Electrostatic blanking plates

Electron gun

FIGURE 20 Schematic of beam-blanking scheme. (From Lyman, 1994; reproduced with permission from the Royal Microscopical Society.) Development of the 300-kV VG STEM (1985–1996) 311

7. TESTING, TESTING

After nearly two years of design work the parts for the MIT system were arriving in the summer of 1990. The microscope was assembled by the production team, baked, and ready for testing from October 1990. Given that this was the first prototype system, things went quite well, but the first major problem was the high voltage breaking down at around 225 kV in the lower accelerator. When we queried this with the accelerator manufacturers, they pointed out that the design required electrode inserts that considerably reduced the free diameter for pumping the gun. Fortu- nately, with the electrodes fitted, the gun pressure still reached 2 10–9 Pa and the HT could be increased gradually to the full conditioning voltage of 345 kV. It was during this period of high-voltage problems that I went into Ian Polke’s office one lunchtime and asked his roommate where Ian was. ‘‘He’s just there under his desk, meditating’’ was the answer, as though that was quite normal. Ian is one of the calmest persons I have come across, excellent at high-voltage design, but he needed to be able to keep calm in all the turmoil of that time! The ‘‘teething problems’’ kept us busy for several months. We found that the mu-metal shielding at the level of the bench plate was inadequate for attenuating 50-Hz magnetic fields in the room adequately, so a rather ugly box was devised to cover the gap. Furthermore, we found serious X-ray leaks at the top of the accelerator due to the fact that up to 1 A of beam current could be focused onto the virtual objective aperture (VOA). A software solution was found by limiting the range of gun lens voltages to produce less than 0.5 A on the VOA. Tony Garrett-Read (MIT) came to East Grinstead for the acceptance test in March 1991. With Alan Nicholls’ skillful operation, the microscope achieved the specified resolution, 0.204-nm lattice fringes with Au(200)— no ADF STEM resolution was specified!—and the EDX performance (<1% hole count and peak to background (P/B) ratio > 4000 with Cr film specimen) and 0.6-eV EELS energy resolution. So the system, as shown in Figure 21, was shipped in April 1991. The Argonne column was built up in parallel with the MIT instrument, but with all the extra features it took another year to complete the testing. The baking of the whole instrument to 200 C created problems with the stage and transfer mechanisms that tended to seize up after a period of operating in UHV in the 10–8 Pa range. The climax was a 10-day acceptance test period with Nestor Zaluzec, which you could call day-and-night testing! It became a habit to break for a late-night pizza delivered to the factory and consumed on the console of the next HB603 customer (Oak Ridge, in this case). 312 H. S. von Harrach

FIGURE 21 MIT HB603 instrument. (From VG Microscopes brochure HB603.)

We did achieve the STEM resolution of 0.2 nm, 0.4-eV EELS energy resolution, EDX hole count below 0.1%, and all the other specifications except TEM resolution. Zaluzec accepted the system in April 1992. The Lehigh HB603 and the Oak Ridge HB603U (Figure 22)bothbene- fited from the experience gained from the first two systems and were improved in both mechanics and electronics. Nevertheless, there were still teething problems. The Oak Ridge objective lens required more power than the others (15 A at 150 V), and because it was water-cooled the power supply had so much condensation on its circuit boards one wet English summer day that the power transistors shorted out and caught fire; fortunately, several people were present and it was quickly extinguished. In trying to reach higher STEM resolution we found that the environmental instabilities, not the instabilities of the microscope electronics, became the limiting factors. These are summarized in Table 1. In fact, the acoustic noise in the room, partly due to cooling fans, resulted in stage vibration as the limiting factor. The bellows on the stage drives were very sensitive to acoustic noise, mainly generated by the HB603 electronics itself. We diagnosed the vibration, using the method of placing the focused probe on the edge of a gold cluster or a silicon atom column, and connecting the ADF signal to a frequency analyzer. The relative movement of Development of the 300-kV VG STEM (1985–1996) 313

FIGURE 22 Oak Ridge HB603U in test. (From author’s archive.) the probe and specimen modulates the ADF signal, which is recorded by the frequency analyzer as a number of peaks at certain frequencies. Sharp peaks at 50- to 60-Hz and harmonics were obviously due to mains frequency electronics interference or ground loops; broader peaks were a result of mechanical vibration. This gave us at least a qualitative idea of the main frequencies limiting the STEM resolution. It was then a question of finding the sources of the interference and eliminating them one by one—like peeling an onion, we worked from the large disturbances down to the smaller and smaller ones. One unusual problem was an occasional beam deflection of about 1 A˚ . By measuring the deflection for different crossover positions we eventually established that the cause was situated between the first and second condenser lens. That is about the worst possible position since the column needed to be dismantled from either the top or the bottom to reach it. Furthermore, Robert Keyes, who was brilliant at this sort of troubleshoot- ing, had just left VG and I was the only person able to do the work. I decided to start at the top. In five days of solid work I examined every part close to the beam for the possible cause of the charging and found nothing. I then started dismantling the gun from the bottom, working up to the condenser. Eventually, I found that the beam-blanking plate TABLE 1 Environmental Instabilities

Frequency Effect on Instability range Maximum amplitude image Source Remedy

EM ﬁelds 50/60 Hz 50/90 mG/cm beam 10-pm Mains, cables, Scan lock to 50/60 RF path distortion, transformers, Hz, FR and LF blurring RF generators shielding Ground loops 50/60 Hz Depends on system 10-pm Mains earthing Separate ground distortion connection, low impedance Acoustic noise 0–2 kHz Depends on stage 10 pm Cooling fans, Acoustic damping, design transformers, remote etc. operation Ambient Minutes to <0.1 K/hr depending 10 pm/min Air conditioning Low-speed fans, temperature hours on column and curtains stage design Coolant Minutes to <0.1 K/hr depending 10 pm/min Closed-circuit Coolant temperature 2 kHz on objective lens chillers, pipe temperature and and cooling joints pressure control turbulence

EM, electron microscopy; RF, radiofrequency; LF, low-frequency.From Von Harrach (1995). Reproduced by permission from Elsevier Science BV. Development of the 300-kV VG STEM (1985–1996) 315 above the first condenser lens was cemented with a ceramic that could be in line of some scattered electrons and charging up. We removed the ceramic and found that the problem disappeared. Another unusual effect observed was an occasional vibration that coincided with a ticking sound. This turned out to be due to the diffusion pump oil, which produced an occasional bubble and the vibration was picked up by the stage, causing specimen vibration. It was cured by reducing the pump heater power and eliminating the bubble formation. We eventually saw the silicon (110) dumbbells (Figure 23) but still had occasional vibration problems. When Steve Pennycook came for the acceptance tests, Alan Nicholls was demonstrating the instrument. Pen- nycook was not really happy with the vibration problems until Alan Nicholls decided to drape his heavy woolen sweater over the stage drive mechanism—that damped the vibration adequately, the dumbbells were the clearest ever, and Pennycook signed the acceptance document! The Lehigh HB603 was the last of the four instruments to be ordered— the order had originally been lost to Philips—but the fastest to be built and tested. We had improved the design in several areas and upgraded many of the electronics boards by then. Obviously the experience of building the MIT instrument helped since it was very similar. The special feature of the Lehigh instrument was the beam-blanking system for EDX acquisition. The blanking electronics had been failing at high speed, delaying the acceptance tests, but when it worked it did

(a) (b)

0.136 nm

FIGURE 23 The ‘‘holy grail’’ achieved: Si 110 dumbells of 0.136-nm spacing. (a) Incoherent STEM image of Si <110> at 300 kV, 20-sec exposure. (b) Processed image. (From von Harrach, 1994; reproduced by permission of Elsevier Science BV.) 316 H. S. von Harrach produce the expected increase in throughput. When I called Charles Lyman on the phone to tell him the throughput was about 3 times faster, he said immediately, ‘‘We’re coming over.’’ So Jo Goldstein, David Wil- liams, and Charles Lyman spent a week taking the instrument through its paces in the factory, signing the acceptance in March 1993.

8. RECORD-BREAKING RESULTS

8.1. Source Brightness The chief advantage of a cold-field emission source is the high brightness and low energy spread of the source compared with a Schottky FEG. The brightness has always been taken to be 109 A/cm2/sr at 100 kV and was therefore expected to reach 3 109 at 300 kV. The highest brightness was undoubtedly achieved with the Oak Ridge HB603, where the value can be estimated from the experimental values at 300 kV of probe current, I ¼ 120 pA, convergence angle, a ¼ 14 mr, and probe diameter, d ¼ 0.136 nm to resolve Si(110) dumbbells. In the absence of other records, if we assume that the demagnified source diameter was equal to the aberration and diffraction components of the probe (i.e., about 0.08 nm), then the brightness was 4 109 A/cm2/sr. The high probe current should remain constant over the exposure time of an image. Argonne therefore specified that the beam current should not vary more than 2%/100 sec and 10%/1000 sec. Both these specifications were fulfilled on the HB603 at 5-A emission current from the source.

8.2. Energy Spread The energy spread of the cold field emission W(310) source has been measured in the past at around 0.3 eV on 100-kV systems (Morin and Fink, 1994). Since the instability of the HT generator at 300 kV was designed to be below 1 ppm/min, we expected about 0.6-eV total energy spread for a 5-minute exposure on a high-resolution EELS system. We were pleased to obtain 0.4-eV full width half maximum (FWHM) as shown in the EELS spectrum (Figure 24).

8.3. ADF Resolution The Oak Ridge group produced many high-resolution STEM images with their HB603U, one of the most impressive being an ADF image of GaAs (110) (Nellist and Pennycook, 1999), showing not only the 0.14-nm spacing of the dumbbells, but also a difference in intensity for the Ga and As atom columns, as expected from Z-contrast images (Figure 25). Development of the 300-kV VG STEM (1985–1996) 317

0.4 eV

015eV

FIGURE 24 Low-loss spectrum of Al film, showing 0.4-eV FWHM resolution. (From von Harrach, 1994; reproduced by permission from Elsevier Science BV.)

(a)

(b) Ga As

FIGURE 25 ADF STEM image of GaAs (110) showing (a) the 0.14-nm dumbbell spacing and (b) a line-scan across a row of atoms showing the higher intensity of As columns. (From Nellist and Pennycook, 1999; reproduced by permission from Elsevier Science BV.)

The instrument was retrofitted in 2003 with a Nion Cs corrector and produced ADF STEM images of Si (112) showing 78-pm spacings (Nellist et al., 2004), proving again the superb stability of the HB603 design.

8.4. X-Ray Microanalysis The Lehigh HB603 was designed for maximum X-ray detection efficiency. It was the first STEM to use two EDX detectors and the beam-blanking technique. The gain in throughput by beam blanking can be seen in 318 H. S. von Harrach

30,000 Cu grid bar Si(Li) + beam blanking

20,000

Counts out (cps) 10,000 Normal Si(Li)

0 0 20,000 40,000 60,000 Counts in (cps)

FIGURE 26 Input-output curves for acquisition with and without beam blanking at a processing time of 14s(Lyman et al., 1994; reproduced by permission from the Royal Microscopical Society.)

Figure 26: the output count rate (OCR) is increased by up to a factor of 3.5 compared with normal acquisition. At the highest input count rates, the beam is blanked for much of the time and a peak OCR is reached at 29 kcps. The HB603 system was designed to reach the highest possible (P/B) ratio in the spectra. This was a problem for most previous electron microscopes at 200 and 300 kV (see Williams and Steel, 1987) where the P/B ratio actually decreased at higher acceleration voltages due to increased scattering. Figure 27 shows that the P/B ratio for the HB603 increases with kilovoltage, as it should theoretically (Zaluzec, 1979); it did not quite reach the theoretical P/B value of 7000 at 300 kV. Putting the two design features of maximum detection efficiency and highest P/B ratio together, Lehigh achieved the highest possible mass detectability or minimum mass fraction (MMF). This is because the MMF (P. P/B)–1/2 (Ziebold, 1967); that is, the higher the count rate P for a given element, and the higher the P/B ratio, the better the detection sensitivity. This gave a sensitivity equivalent to detecting two to three atoms in a thin Cu alloy specimen (Watanabe and Williams, 1999), as shown in Figure 28. The other aim of the Lehigh group was, of course, high spatial-resolution X-ray microanalysis. The HB603 produced a probe current of 0.5 nA at a FWTM of 1.1 nm (Watanabe et al., 2006). This probe size does not equal the X-ray resolution achievable because of beam broadening, except in very thin films. The advantage of operating at 300 kV was therefore both the Development of the 300-kV VG STEM (1985–1996) 319

7000 Specimen: NIST cr film

6000 IG detector

5000 Si(Li) detector Peak-to-background 4000

3000 50 100 150 200 250 300 350 Accelerating voltage (kV)

FIGURE 27 P/B ratio for NIST Cr film at 100–300 kV with Si(Li) and intrinsic Ge detectors. (From Lyman et al., 1994; reproduced with permission from the Royal Microscopical Society.)

Corresponding number of Mn atoms 1 2 5 10 50 100 250 t = 500 s

Cs-corrected 10.0 HB 603 Single atom

Conventional HB 603 1.0 Detectable limit (below 1 is not detectable) Signal above detection limit

0.1 0 20 40 60 80 100 120 Thickness, t (nm)

FIGURE 28 Detection limit of Mn atoms in Cu-Mn alloy thin specimen at 300 kV for the ‘‘conventional’’ HB603 and Cs-corrected HB603. (From Watanabe et al., 2006; reproduced by permission from Microscopy Society of America.) 320 H. S. von Harrach

10 m m Ni in Fe, 30 kV EPMA (Goldstein and 1m m Yakowitz, 1975)

Mn in Cu, Ni in Fe, 120 kV AEM 100 kV AEM 100 nm (Williams, 1987) (Roming and Goldstein, 1979)

NI in Fe, 10 nm 100 nm 100 kV FEG–AEM (Lyman, 1987) Spatial resolution 10 nm Mn in Cu, 300 kV FEG–AEM 1nm Cs–corrected (Watanabe and IV FEG–AEM Williames, 1999s)

1Å 0.001 0.01 0.1 1.0 Minimum mass fraction (wt%)

FIGURE 29 X-ray microanalysis performance of various instruments plotted as spatial resolution versus minimum mass fraction (From Watanabe et al., 2006; reproduced with permission from Microscopy Society of America.)

reduced broadening compared with 10 0kV and the higher probe current at small probe sizes. It is therefore to be hoped that with the addition of a spherical aberration corrector, the dream of analyzing single atoms in an X-ray map can finally be achieved. This is illustrated in Figure 29 (Watanabe et al., 2006), but to date the final goal has not yet been reached.

9. CONCLUSIONS

This was a period of incredible creativity that still amazes me: How did we get all this done in about 40 man-years? Considering all the special features of the different instruments, the challenge of designing a software architecture and electronic system from scratch, the variety of tasks for a team of some 12 people, it was an incredible achievement. In looking back, I think there are some key factors that helped to make this possible. First, a team of motivated, highly trained engineers who took up the challenge of making this the best STEM in the world. Second, the environment of a small company with a minimum of procedures and red tape. And third, and most importantly, a group of customers who had the confidence that this product would be success. Development of the 300-kV VG STEM (1985–1996) 321

Trend in EDX and STEM resolution over the last 60 years 10000 EDX resolution Castaing’s microprobe 1000 STEM resolution Duncumb’s EMMA 100

AEM 10 Cs HB603 correctors 1 Crewe’s FEG VG HB5

Spatial resolution (nm) HB603 0.1

0.01 1950 1960 1970 1980 1990 2000 2010 Year

FIGURE 30 Overview of X-ray and STEM resolution in the past 60 years.

It took other manufacturers another 10 years before they achieved the same level of STEM performance. Today, 22 years after the start of the development, two of the four microscopes are still in operation: the HB603U at Oak Ridge and the Lehigh HB603, both retrofitted with Cs aberration correctors and producing results at the sub-A˚ ngstrom STEM resolution and almost-atomic analytical resolution, respectively. The history of analytical electron microscopy is encapsulated in the graph of a spatial resolution over the years (Figure 30). Here are two worlds, that of high-resolution imaging and of analytical resolution— rather like Moore’s law in the semiconductor industry with its size shrinkage for microprocessor and for memory chips—where the progress in improving analytical resolution has been much faster than for image resolution in the past. The two are now converging on the atomic level, maybe their ultimate best.

ACKNOWLEDGMENTS

I would like to thank John Colling, John Morphew, Peter Bovey, Alan Nicholls, Robert Keyes, Ian Polke, Steve Jones, Steve Elliott, and the rest of the development team at VG Microscopes without whose enthusiasm these instruments would never have been successful. My thanks go also to Ian Wardell for his careful review of the manuscript, and, above all, to my wife for her patience with my passion for electron microscopy. 322 H. S. von Harrach

REFERENCES

Boyes, E. D., Lyman, C. E., Verney, G. E., and Walker, A. R. (1977). A field-emission gun STEM and microanalysis. EMAG., pp. 61–64. Crewe, A. V., and Wall, J. (1970). A scanning microscope with 5 A resolution. J. Mol. Biol. 48, 375–393. Hembree, G. G., Crozier, P. A., Drucker, J. S., Krishnamuthy, M., Venables, J. A., and Cowley, J. M. (1989). Biased secondary imaging in a UHV-STEM. Ultramicroscopy 31, 111–115. Howie, A. (1979). Image contrast and localised signal selection techniques. J. Micros. 117, 11–23. Jaklevic, J. M., Goulding, F. S., and Landis, D. A. (1972). High rate X-ray fluorescence analysis by pulsed excitation. IEEE Trans. Nucl. Sci. 3, 392–395. Kruit, P., and Venables, J. A. (1988). High spatial resolution surface-sensitive electron spectroscopy using a magnetic parallelizer. Ultramicroscopy 25, 183–193. Liu, J., Hembree, G. G., Sinnler, G. E., and Venables, J. A. (1992). High resolution Auger electron spectroscopy and microscopy of a supported metal catalyst. Surf. Sci. Lett. L 111– 117. Lyman, C. E., Goldstein, J. I., Williams, D. B., Ackland, D. W., von Harrach, S., Nicholls, A. W., and Statham, P. J. (1994). High-performance X-ray detection in a new analytical electron microscope. J. Micross. 176, 85–98. Morin, R., and Fink, H. (1994). Highly monochromatic electron point-source beams. Appl. Phys. Lett. 65(18), 2362–2364. Munro, E. (1971). Computer aided design methods in electron optics. Cambridge: Dissertation. Nellist, P. D., and Pennycook, S. J. (1999). Incoherent imaging using dynamically scattered coherent electrons. Ultramiscroscopy 78, 111–124. Nellist, P. D., Chisholm, M. F., Dellby, N., Krivanek, O. L., Murfitt, M. F., Szilagyi, Z.S, Lupini, A. R., Borisevich, A., Sides, W. H. Jr., and Pennycook, S. J. (2004). Direct sub- Angstrom imaging of a crystal lattice. Science 305, 1741. Riecke, W. D. (1962). Feinstrahl-Elektronenbeugung mit dreistufigem Kondensor und langbrennweitiger letzter Kondensorstufe. Optik 19, 81–116. Statham, P. J., Long, J. V. P, White, G., and Kandiah, K. (1974). Quantitative analysis with an energy-dispersive detector using a pulsed electron probe and active signal processing. X-ray Spectrometry 3, 153–158. Venables, J. A., and Janssen, A. P. (1980). Performance of a field emission gun scanning electron microscope column. Ultramicroscopy 5, 297–315. Venables, J. A., and Archer, G. D. (1980). On the design of FEG SEM columns. Proc. 7th Eur. Congr. on Electr. Microsc. 1, 54–55. Venables, J. A., and Hembree, G. G. (1991). Secondary and Auger Eelectron Imaging in UHV- SEM and STEM. Inst. Phys. Conf. Ser. 119, 33–38. Venables, J. A., Cowley, J. M., and von Harrach, H. S. (1987). A field-emission STEM for surface studies. Inst. Phys. Conf. Ser. 90, 85–86. Venables, J.A, Spiller, G. D. T., Fathers, D. J., Harland, D. J., and Hanbuecken, M. (1983). UHV-SEM studies of surface processes—recent progress. Ultramicroscopy 11, 149–156. Veneklassen, L. H. (1972). Some general considerations concerning the optics of the field emission illumination system. Optik 36, 410–433. von Harrach, H., Joy, D. C., Verney, G. E., and Walker, A. R. (1974). Ein Hochauflo¨sungs- Raster-Elektronenmikroskop, Beitr. Elektronenmikroskop. Direktabb. Oberfl. 7, 139–147. von Harrach, H. S. (1994). Medium-voltage field-emission STEM—the ultimate AEM. Microsc. Microanal. Microstruct. 5, 153–164. Development of the 300-kV VG STEM (1985–1996) 323

von Harrach, H. S. (1995). Instrumental factors in high-resolution FEG STEM. Ultramicroscopy 58, 1–5. von Harrach, H. S., Nicholls, A. W., Jesson, D. E., and Pennycook, S. J. (1993). First results of a 300 kV high resolution field-emission STEM. EMAG 93, Inst. Phys. Conf.Ser. 138, 499–502. Watanabe, M., Ackland, D. W., Burrows, A., Kiely, C. J., Williams, D. B., Krivanek, O. L., Dellby, N., Murfitt, M. F., and Szilagyi, Z. (2006). Improvements in the X-ray analytical capabilities of a scanning transmission electron microscope by a spherical aberration corrector. Microsc. Microanal. 12, 515–526. Watanabe, M., and Williams, D. B. (1999). Atomic-level detection by X-ray microanalysis on the analytical electron microscope. Ultrmicroscopy 78, 89–101. Williams, D. B., Goldstein, J. I., and Fiori, C. E. (1986). Principles of X-ray energy-dispersive spectroscopy in the analytical electron microscope. In ‘‘Principles of Analytical Electron Microscopy’’ (D. C. Joy, A. D. Romig Jr., and J. I. Goldstein, eds.), pp. 123–152. New York & London: Plenum. Williams, D. B., and Steel, E. B. (1987). In ‘‘A standard Cr thin-film specimen to measure the X-ray peak-to-background ratio in analytical electron microscopy’’. Analytical Electron Microscopy 1987. (D. C. Joy, ed.), pp. 228–230. San Francisco Press: San Francisco. Zaluzec, N. J. (1979). Quantitative X-ray microanalysis: instrumental considerations and applications to materials science. In ‘‘Introduction to Analytical Electron Micrsocopy’’ ( J. J. Hren, J. J. Goldstein, and D. C. Joy, eds.), pp. 121–167. New York: Plenum. Ziebold, T. O. (1967). Precision and sensitivity in electron microprobe analysis. Anal.Chem. 39, 859–861. Chapter 8

On the High-Voltage STEM Project in Toulouse (MEBATH)

Bernard Jouffrey

Contents 1. Introduction 325 2. The Building 328 3. Generator 333 4. The Column 336 4.1. The Source 336 4.2. Accelerating Tube 342 4.3. Lenses 346 4.4. Spectrometers 348 5. Suspension of the Platform and the Microscope 349 6. Recording of The Signal 351 7. Conclusions 352 Acknowledgments 352 References 353

1. INTRODUCTION

The name of this project comes from the French title ‘‘microscope e´lectronique a` balayage a` tre`s haute tension’’ (MEBATH). It was launched in 1972, or so, and lasted until 1986. It was the subject of many discussions inside and outside the laboratory. In fact, the request for funds to begin the construction of this microscope is dated 1977.

Laboratoire de Structures, Sols et Mate´riaux (LMSS-Mat), E´ cole Centrale Paris, Unite´Mixte de Recherche (UMR), National Center for Scientific Research (CNRS) 8579, Chaˆtenay-Malabry, France

325 326 Bernard Jouffrey

Since the work of Crewe (1970, 1974), the advantages of conventional transmission electron microscopy (CTEM) and scanning transmission electron microscopy (STEM) have been extensively discussed (Hawkes, 1983). Before starting this undertaking, the principle of the project was presented to a number of researchers to seek their advice; the CNRS and I wanted to have their input. The project was also discussed in the CNRS national committee, which is composed of elected and nominated members of the research community. Instrumentation has always been the source of arguments between people who think it is too risky to undertake and a smaller number of people who think it is essential to discover new things, thanks to new instruments. It is clear to all that instrument development is a difficult task in a laboratory. Finally, after my arrival in Toulouse in 1971, it was decided to undertake this project. The accelerating voltage of 1.6 MeV had been chosen for two reasons. At the time we were conducting irradiation studies in Orsay—first with ions, and then we intended to also use electrons (Ruault and Jouffrey, 1972, 1975). The threshold of the electron defects in gold is 1.3 MeV and it is 1.5 MeV for uranium. The second reason was related to the first Toulouse high-voltage electron microscope, known as the ‘‘Boule’’ because of the great spherical housing in which it was situated; the voltage for this microscope was calculated for 1.5 MeV and worked at 1.2 MeV. By choosing 1.6 MeV, it was thought that the new instrument would work correctly above 1 MeV. On my second visit (1966) from Orsay to the laboratory to conduct irradiation experiments on aluminum, I observed—what people knew already—that point defects were created and produced small loops of point defects. As we had studied irradiation in Orsay, I thought it would be a good domain for development in Toulouse. I had come another time, in 1967 or ’68, to study the interaction of point defects with preexisting defects. These were vacancy loops obtained by rapid quenching of aluminum foils. Some images had been published by Andre´ Rocher in the book on the Perros-Guirec summer school, which was published by the French Electron Microscopy Society (SFME) ( Jouffrey and Rocher, 1972). When I visited the laboratory in 1970 and confirmed my decision to accept the responsibility as director, I discussed my plans with researchers. I observed that we needed a project for this laboratory, which had, thanks to Gaston Dupouy, a reputation in high-voltage electron microscopy (HVEM) (Dupouy, Perrier, and Durrieu, 1960; Dupouy et al., 1969a,b). The first images, with the 3-MeV microscope (Dupouy, 1973; Jouffrey, 1972), were being obtained, little by little, first at 1 MeV and then the energy was slightly increased. In 1973, for a study made with Rocher, it was possible to obtain an image at 3150 keV. On the High-Voltage STEM Project in Toulouse (MEBATH) 327

At the time, the question was which project(s) were suitable for this big laboratory—which included about 130 people, among whom was a large group of about 80 engineers and technicians? I personally decided to continue the work I had impulsed and started on electron energy-loss spectroscopy (EELS) in Orsay during the thesis of my second student, Christian Colliex, but at high energy, say at 1 MeV, as a minimum. I discussed this point in the laboratory and soon found that Jean Se´vely, who wanted, after his thesis work on plate sensitivity at high voltage with Gaston Dupouy as a supervisor, to move to another subject. He agreed with enthusiasm to work in this new field. The work, in Orsay, on a pure magnetic filter had shown that such a filter had to be symmetric (Senoussi, 1971).And it was a good goal to aim for in our new work. Two assistants from the Paul Sabatier University came to work in the field. They had no thesis subject and were happy to have this proposal, which they accepted very rapidly. Another point encouraged us to consider a scanning project. In the International Congress on Electron Microscopy, held in Grenoble in 1970, Albert Crewe had a real success. His results, obtained with STEM, showed the wonderful pictures and movie on single atoms and were fascinating. Another point was not negligible. I came to Toulouse twice in 1968 and 1969 to test the 1-MeV microscope (‘‘La Boule’’). The images were good even if the resolution was limited to 0.7 nm. I observed the formation of radiation damage, as already mentioned above; I was really interested in this, as my thesis work was on radiation damage but that due to ion bombardment. Also, Marie-Odile Ruault (1981; Bernas et al.,1971) prepared her thesis with me on this subject. The first question was to understand the mechanism at the origin of the formation of loops in aluminum and the tetrahedra of stacking faults in gold under irradiation. Was the precipitation homogeneous or heterogeneous? It would be of interest to use a fixed small probe to observe how the defects gather to form loops or tetrahedra. We also were strongly involved—with L. Kubin at Orsay—with plastic deformation in body-centered cubic (BCC) materials, in fact niobium essentially. Pierre Petroff, now at Santa Barbara, was in my group in Orsay during his military service and we had prepared and observed lithium, which is also BCC at room temperature. It was for this reason that the Li K-edge was observed. Petroff was looking at the gliding geometry of dislocations in lithium and was involved in the use of the samples in the EELS work we had done at the same time. It also was clear that a scanning microscope would be a good approach to EELS. So in all these domains numerous discussions were intitiated both within and outside the laboratory. This idea was rather well accepted by the researchers. Technicians were more enthusiastic. French researchers, involved in materials science, were contacted to determine their feeling for such a project. The result of the CNRS and De´le´gation Ge´ne´rale a` la Recherche Scientifique et Technique (DGRST) poll 328 Bernard Jouffrey was positive. The project name was MEBATH (microscopie a` balayage a` tre`s haute tension), as mentioned previously. I decided to launch several independent projects in the laboratory that could be useful to the new MEBATH project but could also provide experience and be sufficient themselves for theses and new orientations in the laboratory. When we started, we had no experience in field emission. In Orsay we had decided— long before the move to Toulouse—to conduct experiments on field emission and we had obtained a contract to work in this domain. So when I arrived in Toulouse, we started to fabricate a field emission gun (FEG). It was easy to get started because people such as F. Sonier already knew how to make tips and had worked on thermal emission with tips as cathodes of electron guns. We knew theoretically that this type of gun, as we had discussed often with Castaing in Orsay (also on ion field emission), would be important for microscopy. And now, from the work of the group with Crewe, we had the proof. Other groups, in France for instance, were also embarking on the adventure (Pitaval, 1977; Troyon, 1977). To begin we adapted such a gun to a Siemens Elmiskop I on which we mounted a Castaing–Henry system (Denizart et al., 1981a,b, 1983). The domains on which we worked were as follows: Calculations of different triode and tetrode FEGs. The fabrication of a FEG (triode) and its power supply adapted to a Siemens Elmiskop I equipped with a Castaing–Henry filter from Orsay. The study of a magnetic sector that was placed under the column of the 1.2-MeV electron microscope. Another one was later located under the 3-MeV microscope. Moreover, the omega filter project was conducted for the 1.2-MeV microscope a few years later. In situ experiments for plastic deformations, electron irradiations also, were largely developed on both high-voltage microscopes. The same specimen holders could be either at low temperature or high temperature, at will, on the two microscopes. In situ ion radiation damage was also a domain we developed. I thought these projects would be useful to the MEBATH project but they were also treated independently as new orientations in the laboratory. Figure 1 shows a drawings of the diagram of the microscope made for the proposal to request funds to fabricate the microscope.

2. THE BUILDING

The laboratory had extensive experience in HVEM. We know that the first electron microscope working above 1 MeV (at 1.2 MeV to be exact) was constructed under the guidance of Gaston Dupouy and Franz Perrier On the High-Voltage STEM Project in Toulouse (MEBATH) 329

FIGURE 1 This drawing represents the diagram proposed in the request for funding in 1977.

(Dupouy, Perrier, and Durrieu, 1960). It was located at Toulouse, close to the Paul Sabatier University but on a CNRS campus, at the Laboratoire d’Optique Electronique in the Rangueil area. The ‘‘Canal du Midi,’’ constructed by Paul Ricquet (1604–1680), was separated from the laboratory by just a narrow path along the canal. When the proposal was made to me to come to Toulouse to take charge of the Electron Optics Laboratory, Gaston Dupouy was about 70 years old (he was born in 1900). It was Raimond Castaing who first proposed this transition to me. The suggestion was first made by Hubert Curien, who was then Director-General of 330 Bernard Jouffrey

CNRS; he had discussed the question with Raimond Castaing and Gaston Dupouy. I already knew Hubert Curien and Gaston Duouy slightly. Gaston Dupouy was my ‘‘sponsor’’ when I entered the CNRS (in 1961) and while I was working on my thesis. His role was actually small but he had to send a report every year on my work. My supervisor was Raimond Castaing. I had met Gaston Dupouy for the first time in February 1962 at Toulouse on the occasion of the third meeting of the SFME, where with Raimond Castaing we presented the work on the effect of an ion bombardment on gold. These were in situ experiments. On another hand, Hubert Curien (who became Ministre de la Recherche et de la Technologie) had asked me, by the end of 1967, to take charge of the reproduction of the Toulouse 1.2-MeV microscope. The idea was to have several microscopes in France at the same time; Hitachi, JEOL, and AEI in Great Britain were developing commercial high-voltage microscopes. The task was difficult for two reasons: first, I had to learn about HVEM problems, and second, Gaston Dupouy considered the microscope as his own child. The essential goal was to render the 1.2-MeV Toulouse electron microscope available to materials scientists. And so, for instance, we had to change the system to correct the astigmatism and replace Hillier’s screws with coils to make possible the introduction of tilting specimen holders. When it became clear that I would indeed go to Toulouse, the decision was made (with Robert Chabbal, Scientific Director of the ‘‘Mathe´ma- tiques et Physique de base’’ department at CNRS) to introduce materials science in Toulouse and hence to change progressively the specialty of the laboratory but to still keep a part devoted to electron optics. At the same time, I asked Jean Cantacuze`ne, the director of the chemistry department, to ask X-ray experts from Toulouse and some chemists to come. This request was refused for reasons I shall not go into here. However, at this time it was clear that use electron microscopy would be used in Toulouse for materials science. Fortunately, when I arrived an important part of my Orsay group also came to Toulouse. Ladislas Kubin was finishing his thesis on plastic deformation of niobium at low temperature (Kubin and Jouffrey, 1971). Jean-Luc Martin, who worked at ONERA (the French aerospace lab where I was ‘‘collaborateur scientifique’’), came a little later, in 1975, to work with Kubin to develop in situ plastic deformations with HVEM (Martin and Kubin, 1979), as Toru Imura was doing in Nagoya. Andre´ Rocher, who first did a nice study in Orsay on the contrast of intersecting stacking faults in face-centered cubic materials, started to adapt dynamical theory to HVEM. The work was carried out first with Claudie Mory. She stayed in Orsay and commuted for a while to complete her ‘‘third cycle thesis,’’ a work which at that time followed a DEA (Diploˆme d’E´ tudes Approfondies) exam before embarking on the main thesis. In her case, she passed her DEA of Solid-State Physics, which was On the High-Voltage STEM Project in Toulouse (MEBATH) 331 under the guidance of Jacques Friedel and Andre´ Guinier. Her subject of research was on the contrast of stacking faults at high voltage (Mory, Rocher, and Jouffrey, 1975). Following this work, Andre´ Rocher studied essentially, by that time, two subjects: electron penetration in different materials as a function of the accelerating voltage, and the critical voltage in copper. It was in the framework of this last work (in 1972) that the image obtained at the highest energy with the 3-MeV microscope was obtained (3150 MeV). Other researchers who had prepared their theses with Gaston Dupouy—Rene´ Ayroles and Annie Mazel—were also working on the problem of contrast at high voltage. Marie-Odile Ruault, also from the DEA of Solid-State Physics in Orsay, was working on ion irradiations. In Toulouse, she did both electron irradiations and ion in situ experiments adapting a mass spectrometer to a Philips EM300 microscope. Thus the activities of the laboratory became very concerned with material sciences. For the same purpose, Jean Se´vely accepted an offer to develop a group working on EELS. Some ‘‘assistants’’ came from the university to prepare a thesis, as mentioned above. And so Jose´-Philippe Pe´rez prepared a thesis on the understanding of energy-loss behavior until 1.2 MeV for thin and thick materials. The first results were published in 1973 and presented at the International Congress on HVEM in Oxford. At the same time, Gerald Zanchi was working on the calculation of the omega filter. It was constructed and adapted to the column of the 1.2-MeV microscope in 1976. The first image was a ‘‘flash’’—we saw an image for just a fraction of a second. A component of the high-voltage power supply exploded. Criticisms ran strong in the laboratory and some suggested that we should give up this work on the omega filter, but we did not quit. It took six months to get a new piece, and after 1977 the filter worked perfectly for 20 years. I remember that H. Hashimoto, who visited us to see this filter, told me in my office that they would like to make the same device not only at high voltage but at 200 keV. He was accompanied by someone from JEOL. I suppose this visit was partly at the origin of the present filter with some differences that were well defined and published by Tsuno (1999). At high voltage, an omega filter was recently installed at the University of Kyushu, and many 200-keV systems have been installed through the world. We knew the weak points of the 3-MeV microscope. Its point-to point- resolution was 0.4 nm. It was calculated for .09 nm. However, it was possible to observe Ba single atoms located by chemists on a molecule. The distance between the pairs of these heavy atoms was 1.6 nm. The pictures taken by Dominique Dorignac (Dorignac and Jouffrey, 1975; Jouffrey, Dorignac, and Tanaka, 1978) were quite convincing. In the years 1969–1970, I visited the laboratory of Gottfried Mo¨llenstedt in Tu¨ bingen. Hannes Lichte showed me the installation of 332 Bernard Jouffrey high-resolution holography and I realized all the precautions they had used to avoid vibrations, particularly those related to water cooling. The need to avoid all sources of vibrations was quite apparent. I was convinced that we had to study this important question of vibrations in detail. In addition, Walter Riecke came to the laboratory for a time and conducted some experiments to detect the reactions of the 3-MeV microscope to mechanical shocks. We needed different sources of funds to accomplish the goals for the project. CNRS, the DGRST (the organization that played the role of the Ministry of Research in those years), and later the Re´gion Midi-Pyre´ne´es (the smallest contribution) participated in the financial aspect of the project. In fact, the DGRST made the highest contribution. The request for funds from the DGRST was made early in 1977. The CNRS had already promised its participation. This request came after numerous discussions in France over a period of several years. A report later at a meeting in the United States on the future of electron microscopy was written by Marilyn Farley in 1972. One conclusion concerned the interest of high-voltage STEM. Articles by John Cowley, Albert Crewe, and Elmar Zeitler reached the same conclusion. Because there was no room large enough to lodge this microscope and its high-voltage power supply, a building had been erected on the side (Figure 2) of the 3-MeV ‘‘Le briquet’’ (cigarette lighter). It was constructed

FIGURE 2 MEBATH building near the 3-MeV microscope. On the High-Voltage STEM Project in Toulouse (MEBATH) 333 by the building department of CNRS. This building has been studied and prepared in detail by Robert Cathelinaud and Andre´ Se´gue´la, Jacques Trinquier (joint director of the laboratory), and others in the laboratory after a series of 31 meetings between the beginning of September 1979 and the end of May 1980. About 10 companies were involved, including one company that specialized in Faraday cages for the upper level of the building and another for the suspension of the microscope. All this work was completed under the auspices of CNRS with Mr. Masdupuy and the architect, Mr. Simon. Mrs. Arlette Blanc balanced the budget, in collaboration with Mr. A. Le´andri and Isabelle Massip. The building was divided into three main parts. The most impressive were the rooms where the microscope was located and the observation room. The third room, upstairs, was where the two tanks were installed on the platform. This room, as mentioned, was a true Faraday cage in copper with a very good earth, with no earth circuit more or less as a loop. The origin of this important precaution with the earth was a difficulty we observed with the 3-MeV building and its own earth. The power supplies of the 3-MeV microscope were sensitive to a TV transmitter (FR3) that was located at a few kilometers from the laboratory, on the heights of Ramon- ville-Saint-Agne, a village close to Rangueil, the name of the area where our campus was located, beyond the University Paul Sabatier. The tests we made on the role of the earth were very clear. The oscillations we observed were perfectly in phase with the emissions of FR3 Toulouse. The microscope worked better at midnight to around 6 am. The building was completed in July 1980. During this same year, the elements of the microscope were practically calculated. In particular, the program necessary to understand how the junction of the gun to the accelerating tube could be made was nearly ready.

3. GENERATOR

The two stainless steel tanks (Figures 3, 4 and 5) were installed at this time. They were linked to the dielectric SF6 gas installation of the 3-MeV microscope. The MEBATH and the 3-MeV building were in close proximity so they could share the SF6 installation; it was one way to save an important sum of money. The generator was constructed by the Haefely company of Basel (today Haefely Test AG). The first offer from Haefely was dated October 1973, the second was received in November, and the final proposal in July 1974. Other companies were contacted; we also had an interesting proposal from Delta-Ray company in 1973. The nominal current of the rectifier in permanent service was 500 mA. The voltage was continuously adjustable between 160 and 1600 kV. 334 Bernard Jouffrey

C D F E

f=1,40m H B

400

J 250

A K L

2000 M ,80 m

N 1 O

FIGURE 3 Schematic for tanks and the microscope.

There were nine levels in cascade, and the pressure of the dielectric gas, ¼ SF6, was 5 obsolute bar (4.08 Atu¨ , 1 Atu¨ 0.981 absolute bar). Thanks to the use of a dielectric gas under pressure, the dimensions of the system were substantially smaller than those of the 1.2-MeV microscope generator working in air. The total height of the rectifier was only 1.86 m and the diameter was 0.80 m. The frequency of the power supply at the bottom of the generator was 7 kHz at maximum. The scheme was classical, like that of the 3-MeV microscope. The generator and the accelerator had their own tanks connected by a transfer resistance. The two-tank system had been tested with the 3-MeV generator and accelerator and worked quite well. The length of this resistance was 2 m. The system was a Greinacher type. This high voltage has been tested and worked well. The only point that required adjustment was related to the residual current. On the High-Voltage STEM Project in Toulouse (MEBATH) 335

FIGURE 4 The top of the accelerator, the transfer resistance, the generator, and the tanks, during mounting, on the damped platform of the microscope.

FIGURE 5 The tanks have been locked in position. 336 Bernard Jouffrey

4. THE COLUMN

When the project was discussed, it was rapidly apparent that the structure of the column was a true difficulty. In fact, the most time-consuming task was related to the accelerating tube and its connection to the column and the suspension. We discussed the problem with several specialists in vibrations. It was a busy time. I recently found a paper where I gave the concluding remarks at a workshop organized in Eindhoven. I don’t remember the exact date; it was around 1978. I presented our project, which was not completely finalized on paper. But one idea was to have two positions for the object. One would be for high resolution, say close to 0.1 nm (theoretically, it was possible at high voltage). The other position would be for 2 to 3 nm in resolution but a large volume around the object for in situ experiments in particular (Figure 6). The other point was in favor of convertible microscopes, CTEM-STEM systems. However, our principal effort was STEM. There were two projects for the column. The simplest one was intended to get as small a probe as possible. It was calculated for 0.1 nm. The recording of the scattered electrons was thought to be by the classical way concerning the small-angle scattering and large angle. We rather suspected that we would have X-ray emission, which could disturb the real signal. But we decided to postpone this question until later. The other version included a more sophisticated column (see Figure 6). The detection of characteristic X-rays at the second level of the object was included. We discussed this point and the deflection coils with CAMECA. This version was on paper in July 1980. It was reported in a regular meeting about the scanning microscope in Toulouse on July 30, 1980. The other version was simpler and can be seen on Figure 15.

4.1. The Source We intended to have a cold-field emission gun (CFEG). We obviously knew about the nice work of Albert Crewe, and at first we calculated for the same kind of gun but also considered other solutions. The details of these calculations are in the publications by Denizart et al. (1981a,b), and Roques et al., 1983a,b. The calculations were achieved by several approaches, in particular the treatment of Munro (1971). The presence of Peter Hawkes in the laboratory from 1975 was a good help in resolving all these problems of electron optics (Hawkes and Kasper, 1989) and computing. Other approaches were proposed by Kern (1978), but it was shown by Kasper (1979) that the different methods yielded similar results. On the High-Voltage STEM Project in Toulouse (MEBATH) 337

FIGURE 6 The column project; the two arms are not necessary.

The conditions are obviously different for CTEM and STEM. The calculations on the FEGs were essentially those of Sylvie Roques. The point is to keep a stable position of the image of the source (zi) for a large range of beam intensity or electron energy. We considered two cases as follows: (i) The entrance pupil is defined by one of the anodes. The spherical aberration is the most important contribution to the size of the beam; therefore, the lowest spherical aberration is required, just as with CTEM. (ii) An aperture is located beyond the second anode in such a way that it is the conjugate of the entrance a pupil, and the angle 0 (Figure 7 see also figure 8 for the tetrode gun.) 338 Bernard Jouffrey

V0 Vi 0 a a f 0 i A1 S0 Si r r 0 i

Entrance pupil Exit pupil A1A1 A2 D e A1A2 plane plane

FIGURE 7 Triode gun and parameters.

A1 A2 A3

(vex) (vi) (vac) DCA

f f f 1 b 2 b 3 1 3

DA A D 1 2 A2A3

v1 v2 v3

e1 e2 e3

zЈ z S0 0 Si

FIGURE 8 Tetrode gun and parameters.

a should be the optimal angle opt. The spherical aberration, in that case, can be of the same order of magnitude as the diffraction limit (Venables and Janssen, 1980; Wells, 1974). In the case of the triode gun, we studied in detail the influence of the b geometrical parameters. A1 is the first anode, the angle parameter of the cone in the anodes, e the thickness of the first anode, f the diameter of the cylindrical hole in the first anode, and the distance between the r r two anodes is DA1A2 .If 0 is the size of the emitting area, i is the size of the r image of 0,andzi the position of this image. Like most of the calculations in the literature, the total size of the image is calculated by the mean square r summation, in the Gaussian plane, of the Gaussian radius G , the spherical r r i aberration term S , the chromatic contribution C , and the diffraction limit r i i Di . The radius of this blurred image is, in this approximation, denoted as On the High-Voltage STEM Project in Toulouse (MEBATH) 339

1=2 r ¼ r2 þ r2 þ r2 þ r2 ; i Gi Si Ci Di (8.1) with r ¼ r Gi ML 0, ML being the magnification r ¼ a3 Si CSi r ¼ aD = D Ci CCi V V with V the variation of the applied voltage as usual, and r ¼ : l =a: Di 0 61 i

At this level, there are no relativistic corrections aside from the expres- l sion for i, calculated for the energy at the image level eVi, where e is the charge of the electron. It is convenient to refer back to the level of the source (object space). r In this case, the apparent size of the source ap is given by hi r 1=2 r ¼ i ¼ r2 þ a3 2 þ ðÞa D = 2 þð : l =a Þ2 : ap 0 CS0 0 CC0 0 V V0 0 61 0 0 (8.2) ML

So, the brightness of the source is given by

I V B ¼ i : (8.3) p2r2 a2 ap 0 V0

From this expression (8.3), we notice that the brightness increases with the accelerating voltage. We know also that the theoretical maximum brightness attainable is given by the Langmuir formula. At very high voltages, there is a relativistic term. For instance, there is 2 a term, 1 þ eVi=2m0c , that appears ( Jouffrey, 1977); so, at high voltage the brightness is much higher. ¼ An important parameter is the ratio k Vacc/Vex,Vacc, the accelerating voltage Vex, the voltage between the tip and the first anode (extraction voltage) in a triode gun. The right position of the source—zi—depends r r only on this parameter k. But ap, i, and the brightness are functions of Vacc for a given k. Figure 9 and 10 show some results on the position of the source and the variation of the spherical aberration as a function of k1, the equivalent of k in the case of a tetrode gun. As far as possible, the optics of the system must be insensitive to the emitted current and the voltage between the two anodes. Two configurations have been computed in detail (Roques, Dernizart, and Sonier, 1983a,b), one with three electrodes (triode gun) and another with four electrodes (tetrode gun). 340 Bernard Jouffrey

(a) zi (mm)

k =3 100 1

k1 =1 k1 =0 0 10 20 k2 D =5mm CA1 f =5mm −50 A2

k1 =1 −100 k1 =3

(b) zi (mm)

100 k1 = 2.5

50 k1 = 7.5

k1 = 12.5 k1 =0

0 25 50 k2 D =10mm CA1 f =3mm A2

FIGURE 9 Position of the image of the source as a function of k parameters (tetrode gun).

We first tested a homemade CFEG that was adapted to a Siemens Elmiskop I, equipped with a Castaing–Henry filter (Castaing and Henry, 1962). The source was a triode gun (the tip plus two anodes). The voltage applied to the first anode was continuously variable from 0 to 12 kV and reversible for the remolding of the tip from 0 to 8 kV. The power supply was set in a tank where the insulation was made of pres- surized Freon 12 gas at 50 psi. We tested a remote control through optical fibers, which worked correctly. The mounting was not easy, and I remember that some components in the high-voltage power supply often flashed. The electronic components (Pinna et al., 1981) finally were strong enough and the vacuum in the gun sufficient to take micrographs and On the High-Voltage STEM Project in Toulouse (MEBATH) 341

CSi (mm) k1 = 12.5

k1 = 7.5

5000 k1 =25

2500

k1 =0

0 25 50 k2 D =5mm CA1 f =5mm A2

FIGURE 10 Spherical aberration as a function of k parameters (tetrode gun). study the contrast of Fresnel fringes either in the elastic (zero-loss) mode or in plasmon-filtered images (15 eV for the plasmon loss in aluminum). The apparatus worked, and some results were obtained on Fresnel fringes in filtered images. The purpose of the experiment was to reproduce the original experiments of Castaing and Henry on the structure of the inelastic Fresnel fringes using the plasmon-loss–filtered image (Castaing and Henry, 1964). We wanted to know whether the lack of clear interference was fundamental or due to a lack of coherence of the incident beam. The experimental results were clear and showed the lack of strong interference is fundamental. This demonstrated the incoherence of the inelastic interactions, as was claimed by Castaing and Henry. I think this was the first time an energy filter was used with a CFEG in CTEM (Denizart et al., 1983). The residual pressure in the gun was better than 10 10 torr. Between the gun and the column of the microscope we placed a differential pumping chamber with a pressure of 2.0 10 8 torr. At the level of the microscope, close to the differential chamber, the pressure was 1.5 10 6 torr. An air lock was therefore built to separate the gun from the column of the microscope. We had many difficulties with the stability of emission and had to open the power supply tank often. The system we used is described in the reference by Pinna et al. (1981). Following our calculations with the triode gun, we thought that a tetrode might perhaps be better adapted to our scanning microscope. So we calculated the properties with different geometries but three anodes and the tip. 342 Bernard Jouffrey

For the tetrode gun, there are two key parameters, k1 ¼ V2=V1 and k2 ¼ V3=V1. This work showed that the crossover will remain in much the same position for Vi from 30 to 80 kV, the working voltage of the gun itself, above the accelerating tube. It was shown also by Sylvie Roques (in 1980, 1981), using the method of Munro, for the calculation of the optical properties of gun plus the accelerator, from 400 to 1600 kV, that the crossover at the bottom of the accelerator would be of the order of 50 nm in diameter with a divergence of the beam of 10 3 rad. The gun itself, for the STEM, had been constructed. It was fabricated in 1980 and tested in the new MEBATH building on a special bench outside the column. After a few months, the results were quite impressive. The vacuum was excellent—better than 10 10 torr. The emission was very stable, much more than on the Siemens microscope. We benefited from our experience with the previous gun, but the most difficult problem was to adapt this gun to the accelerator. From a practical point of view, a delicate question was to imagine a correct solution to the problem of piloting the gun in the tanks, at the top of the accelerating tube, during the observations as the high voltage is applied. As in the case of the Siemens power supply, we wanted to use optical fibers to accomplish this. We also knew that we surely needed to intervene often, at least at the beginning, to change the tip, for instance.

4.2. Accelerating Tube The most important difficulty concerned the level of the vacuum. In 1975 we very seriously considered the structure, dimensions, and their relation to the gun and the microscope itself. As for the guns themselves, the calculations were made by Sylvie Roques still using the Munro program. Peter Hawkes came to the laboratory by this time and he facilitated the access to this program; his assistance was very useful as already mentioned. The calculations were essentially carried out for 400 and 1600 kV. The results showed that the distance from the gun to the entrance of the tube was not so important; it could be flexible. Practically, it was possible to increase the distance between the gun and the accelerating tube without an alteration of the size of the crossover at the end of the accelerating tube. At the bottom, the crossover was 50 nm, with an acceptance angle at this level of 10 3 rad. I remember our discussions about the size of the crossover to get a probe of 0.1 nm. Figure 11 shows the accelerating tube, and Figures 12, 13, and 14 show the mounting of electrodes in the tube. The essential difficulty for this tube concerned the vacuum. It was in 1975 when we thought in detail about the structure of the accelerating tube and its connection with the column of the microscope. On one On the High-Voltage STEM Project in Toulouse (MEBATH) 343

FIGURE 11 Accelerating tube with an extra block of electrodes.

FIGURE 12 Top electrode of a sector. 344 Bernard Jouffrey

FIGURE 13 Mounting of electrodes. Bernard Raynaud (left), and Pierre Vignaud (right).

FIGURE 14 Series of electrode in one sector of the accelerating tube. On the High-Voltage STEM Project in Toulouse (MEBATH) 345 side, the vibration modes must be controlled. A low-frequency filter will protect all the apparatus against vibrations from the ground or the air. A second system, filtering higher frequencies, will protect the tube and the microscope because part of the sources of vibrations is due to the tanks themselves and the transformer at the bottom of the generator in the tank. Several solutions have been studied. The final structure of the system is shown in Figures 1 and 15. The completed accelerating tube arrived in the laboratory on December 13, 1981. The first frustum in ceramics was tested within the fortnight. The residual vacuum was 10 10 torr. It was clear that our problems will come from the total tube. To achieve good vacuum for the entire length of the

FIGURE 15 Simple column. 346 Bernard Jouffrey accelerating tube, we had to heat it to outgas the walls of the tube. Finally, a leakage occurred due to problems of local stresses and stopped the project because we had no money to repair the system. A first trial had been made previously with an accelerating tube made only of one global piece; it did not work as well. Moreover, the company we had chosen to fabricate the tube later closed, which was more than unfortunate for our project. 4.3. Lenses The delicate question of the connection of the gun and the accelerator to the objective lens was considered in detail as I discussed above. Clearly, the maximum number of electrons had to be collected. Also, the conjugate image of the crossover at the bottom of the accelerating tube would be reduced in size by two lenses. Originally, we planned to use the type of lenses used previously in the laboratory. We realized that it could be possible to make anisotropic lenses that offered the advantage of being about three times lighter (Balladore et al., 1981). However, the use of such a lens did not greatly affect the calculations. With this objective in mind, we considered two lenses ( Jouffrey, Trinquier, and Balladore, 1979) that had been used for the 1.2-MeV and 3-MeV microscopes, respectively. The pole pieces were made of Fe-Co 50%. A reasonable value—15 mm—was finally chosen for the gap and the bore. We studied two lenses a and b (Table 1) with this geometry; the discussion here relates to a working voltage of 1.2 MeV. The first lens had a maximum axial magnetic field of 2.04 T (excitation of 35,000 ampere-turns [A-turn]). A parallel incident beam had a single focus at F1, where the sample is set. The beam aperture is defined by the semi- a angle 1. The question was to know the combination that would yield the best signal, that is, the maximum current. The magnetic induction is concentrated in a confined volume defined by the gap and the bores. In the 3-MeV lens, the bore and the gap had been set to 12 mm and the induction could reach 2.6 T, which corresponds to 51,500 A-turn. The focal length at 3 MeV was 10.5 mm. This strong lens can produce at 1.6 MeV a second focus that could be useful for the STEM. For instance, at 1.2 MeV for a 300-nm gold sample, 56% of electrons are scattered outside 5.10 2 rad. With a collection angle of 1.5 10 1 rad, we lose only 10%. Therefore, we considered using a more powerful lens in order to have a second focus, where the electrons scattered at the first focus are gathered in a smaller angle (Figure 16). As we know from Abbe’s law, the size of the probe would be increased at this position. Resolution is improved if a powerful lens is used. The field reaches 2.5 T for NI ¼ 59,500 A-turns (table 1). In this scenario, we have pffiffiffiffiffiffiffiffi D ¼ : l; Z0 1 2 CS the Scherzer parameter. TABLE 1 Optical characteristics of two lenses

a0 a0 Dz0 3 4 3 4 NI (A.t.) Cs (mm) (10 rad) Dz0 (10 mm) rm (A˚) NI (A.t) Cs (mm) (10 rad) (10 mm) rm (A˚) 10,000 35.10 3.57 2.10 2.34 42,500 2.59 6.85 0.57 1.22 15,000 9.47 4.96 1.09 1.68 46,750 2.50 6.91 0.56 1.21 20,000 5.50 5.67 0.89 1.47 51,000 2.52 6.90 0.56 1.21 25,000 4.08 6.11 0.72 1.36 55,250 2.62 6.83 0.57 1.22 30,000 3.39 6.41 0.65 1.31 59,500 2.84 6.69 0.60 1.25

A.t., Ampere-turns. 348 Bernard Jouffrey

(a) (b)

BM BM

a a 1 1 Ј Ј a z F z z 2 z 1 F2 F1

FIGURE 16 Double focalization.

a ¼ : ðl= Þ1=4 ¼ : l3=4 1=4 ¼ : l=a 0 1 6 CS and rm 0 6 CS 0 96 0. It would have been possible to use a strong lens to have a second focus. Finally, we adopted a solution with two lenses (see Figure 6). Three coils constituted the first lens; 1.9–2 kW at 10 A gives 33,000—35,000 A-turn. The second objective with one coil gives 9,000 to 10,000 A-turn.

4.4. Spectrometers This is the domain where we obtained results rather rapidly, independent of the MEBATH project. But the final goal was to use it, in part, for local chemical analysis. At the Grenoble Congress in 1970, where Albert Crewe presented his pioneering results on STEM, a presentation was also made on energy-loss spectrometry at high voltage, 600 kV, by Cosslett’s group at Cambridge. For reason(s) I never understood, they did not obtain energy-loss values in the good range. Something was odd in their results; it is the only publication of this group on the subject. Nevertheless, it was clear that work in this field—EELS at high voltage—was beginning. In our case, the first results were obtained with a 90 magnetic sector located under the column (Se´vely, Pe´rez, and Jouffrey, 1973). Several other papers were published on this geometry (see for instance, Pe´rez et al., 1975). The specifications of the magnetic sector inserted under the column were rather good. At 1 MeV, the analyzer was calculated for a radius of 20 cm and a magnetic field of 235 Gauss. In these conditions, the energy dispersion is 1.35 mm/eV. The full width at half maximum of the zero-loss peak was experimentally found as 3eV. This energy resolution is limited by the dispersion caused by instabilities of the electron gun, which was determined by Dupouy and colleagues (Dupouy, Perrier, Se´gue´la, 1966). At 3 MeV, the radius of the sector is 32 cm and the magnetic field is 358 Gauss. The experiments were pursued up to a voltage of 2.5 MeV. On the High-Voltage STEM Project in Toulouse (MEBATH) 349

As is well known, the second-order aberrations (aperture and dispersion aberrations) can be corrected by curving the entrance and exit faces of the magnetic sector. Curving the edges at the boundaries of the magnet is similar to using a sextupole-like aberration corrector (Pe´rez, Se´vely, and Jouffrey, 1981). In 1976, we knew that the use of high voltage is favorable for the observation of edges ( Jouffrey and Se´vely, 1976). The behavior of the plasmon mean free path was understood both theoretically (Ashley and Ritchie, 1970) and experimentally (Se´vely, Pe´rez, and Jouffrey, 1974). Before I left Orsay, the study and the fabrication of our omega system, a pure magnetic filter, was discussed with Castaing, Henry, and Senoussi as a system to be built. The work of Senoussi (1971) showed that we had to use symmetrical optics in an in-column filter. This work followed that of Cotte (1938) and later Hennequin (1960) and that of Castaing and Henry as already mentioned. Our omega filter (Zanchi, Pe´rez, and Se´vely, 1975; Zanchi, Se´vely, and Jouffrey, 1977a)(Figure 17) worked in 1976 (Zanchi, Se´vely, and Jouffrey, 1977b; Se´vely et al., 1977) as mentioned previously.

5. SUSPENSION OF THE PLATFORM AND THE MICROSCOPE

At the beginning of the 1970s, a city project required construction of a highway along the towpath of the ‘‘Canal du Midi.’’ We contacted those in charge of this project, the Prefet, the mayor, Dominique Baudis, and others in the municipality to explain the risk of vibrations, which could affect the performance of the microscopes. This risk was real, in particular for all the electron microscopes of the laboratory. Finally the highway project was abandoned. At this time, we started to study the vibration mode of the 3-MeV microscope (Mainy and Jouffrey, 1980; Mainy et al., 1980). The results of the various tests in 1979–1980 to determine and understand the effect of ground vibration on the image quality obtained by the 1.2-MeV and 3-MeV microscopes have been used to design the suspension system of the microscope and the tanks that contain the generator and accelerating tube. The different elements of this suspension and the dimensions of the cross-beam supporting system of the platform were defined in detail in collaboration with the ‘‘Laboratoire de Me´canique et d’Acoustique de Marseille’’ (Director M. Nayrolles). The natural frequencies were first calculated and then measured when the tanks were installed on the platform. The total system was calculated to eliminate most vertical and longitudinal vibrations. Vertical vibrations were filtered by air dampers. Horizontal vibrations were eliminated through ‘‘gapecs,’’ which were composed of stacks of rubber membranes. In addition, a 350 Bernard Jouffrey

Accelerating tube Ion pumping

Condensors

Objective lens First intermediate lens

Magnetic filter Second intermediate lens

Projector

Magnetic sector Counting device

FIGURE 17 1.2-MeV electron microscope and the omega filter. The magnetic sector is under the column to avoid artefacts from X-ray emission in the column. soil with sand from Fontainebleau, famous for its constant size, completed the antivibration mounting (see Figure 1). The proper frequencies of the damping system have been tested and were found to be as follows: Vertical 1.6 Hz Transversal 0.6 Hz Longitudinal 0.6 Hz On the High-Voltage STEM Project in Toulouse (MEBATH) 351

6. RECORDING OF THE SIGNAL

At the time of this project (1970–1980), the recording of a signal in electron microscopy and its digitization was not completely obvious. Micrographs were then recorded on glass plates for high-voltage microscopes. The same commercial emulsions were used to record EELS spectra. Precau- tions were needed to conduct quantitative experiments owing to the different scales of intensities on the same spectrum; the difference in intensity can be about six orders of magnitude. Then we used a micro- densitometer to extract the intensity profiles. The same type of difficulties occurred in diffraction. Obviously, the problem was more or less worri- some depending on the thickness of the samples. The difficulty was more pronounced with thin samples because of the no-loss peak. Another difficulty related to artefacts due to X-ray photons, and the situation was also delicate with the development of counting systems. Once the origin of this problem had been detected, we searched for the best way to avoid the participation of photons in the recording of the electron signal. With the development of high-voltage electron microscopy it was particularly complicated because of the hard X-ray emission along the column and the plates themselves. The emission caused by interactions with the slit to select an energy window was also a difficulty in filtered images. An in- column system worked better than an under-column magnetic sector. The solid angle for the emission is higher in this last situation. Several methods are available to overcome this difficulty. The first is independent of the type of recording. It consists of using an in-column system (such as the omega filter) and adding a second deviator under the column. We tested this solution with the 1.2-MeV microscope (this prompted the drawings shown in Figures 6 and 17); this geometry is very efficient. Another approach is to separate electronically the X-ray pulses from those caused by electrons. This can be accomplished by an amplitude analysis of the output signal of the solid-state counting device (Kihn et al., 1980). From the beginning of the project, when the approval of our proposal by DGRST was confirmed, we bought a computer for the project. We chose a CII MITRA 125 (Bull Company). The decision came after somewhat difficult discussions; the MITRA was in competition with a PDP system. One reason for the choice was that it was developed in Toulouse, at least partly. The transfer (2 sec) from the microscope to the MITRA computer was made through a Triade 80 system of Sintra-Alcatel-Thomson (December 1981). Later a different system was fabricated for the 1. 2-MeV microscope with a PDP 11/05 and a Tracor-Northern TN-11, which piloted the deflection of the electron beam, the necessary changes of intensity (a dynamics of 106), in a process of recording energy loss spectra for every elementary area of the sample and recording energy filtered images as well (Pinna et al., 1978). 352 Bernard Jouffrey

To test a directly numerized recording, I proposed to Henri Pinna that he use the 1.2-MeV microscope, which was equipped with the omega filter, and place an Si detector under the screen. A very small hole was made through the screen of the microscope. And so we displaced the image through deflectors that were in the projector. The images were numerically recorded. We were certain that the laboratory could manage the digitalization of the images ( Jouffrey, 1977).

7. CONCLUSIONS

The MEBATH project was extremely motivating to some people (Engi- neers and Technicians principally) of the laboratory and to me. But, like all difficult projects, it also was the source of difficult discussions and times. The omega filter project was submitted to the same logic. When we tested it the first time, a component of the high-voltage supply flashed off as I described above. Many people in the laboratory urged me to give up. I understand their reasons. It took six months to repair it. When the high voltage was on again, IrememberthejoyofGeraldZanchiandallotherswho,fromtimetotime, remembered this good time. Everyone was therefore in favor of the project. Soon after this time, when I left Toulouse to join the E´cole Centrale Paris to leave the personnel and the new director free to make their own decisions, the high-voltage microscopes were halted as in many other places. The column of the 1.2-MeV microscope was sold by the director of the Center for Material Elaboration & Structural Studies (CEMES) for the price of the kilogram of iron. The 3-MeV microscope will perhaps become a museum. Nevertheless, the memories of the people who worked with such enthusiasm on all these projects and experiments—for instance, in situ experiments with the 3-MeV microscope on plastic deformations, field ion guns, or on critical voltage, irradiations, the Golgi apparatus, the moon samples—are an important part of our common past and souvenirs of the impressive results that we achieved.

ACKNOWLEDGMENTS

I am indebted to many people—for discussions, participation in the work on this project, and other related research that helped us all very much. Unfortunately, some of these persons are now dead: Andre´ Se´gue´la was the engineer responsible for this project; Robert Cathelinaud was responsible for the workshop staff and wrote many of the meeting reports on the different stages, and was very active; and another engineer, Jean-Claude Lacaze, was extremely motivated to find a satisfactory structure for the column. Henri Pinna was very active involved in the search for solutions to the electronics and recording problems. Peter Hawkes gave me the opportunity to write these lines and helped us in different aspects. Mrs. Arlette Blanc helped us in the funding problem. My thanks are also due to Isabelle Massip, who helped me untiringly. She also kept many On the High-Voltage STEM Project in Toulouse (MEBATH) 353

documents concerning this project in particular. Without them, I would have been unable to write this paper. The names of the people who appear in the reports, for instance, on the minutes of the meeting of November 30, 1981, are J. Trinquier, J. L. Balladore, R. Cathelinaud, M. Denizart, M. Fournie´, P. Hawkes, J. Cl. Lacaze, H. Pinna, A. Se´gue´la, J. Se´vely, B. Raynaud, F. Sonier, J. Spiesser—I hope that no one has been forgotten. Many thanks to all.

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Jouffrey, B. (Ed.) (1972). In ‘‘Me´thodes et techniques nouvelles d’observation en me´tallurgie physique’’, Perros-Guirec, September 1969, Socie´te´ Française de Microscopie E´ lectronique: Paris. Jouffrey, B. (1977). Inelastic scattering and electron spectroscopy. Inst. Phys. Conf. Ser. No 36 Chap 9, 351. Jouffrey, B., Dorignac, D., and Tanaka, M. (1978). Atomic level electron microscopy as a function of accelerating voltage. Chem. Scripta 14, 63–73. Jouffrey, B., and Rocher, A. (1972). Microscopie a` Haute Tension, In ‘‘Me´thodes et Techniques nouvelles d’observation en me´tallurgie physique’’, (Jouffrey, B. ed.), Ecole d’e´te´ de Perros- Guirec-1969. Jouffrey, B., and Se´vely, J. (1976). Utilisation en microscopie e´lectronique des pertes d’eńergie des electrons pour l’analyse chimique locale. Revue Phys. Appl. 11, 101–111. Jouffrey, B., Trinquier, J., and Balladore, J. L. (1979). Double focus electron probe forming lens for STEM. J. Microsc. Spectrosc. Electron. 4, 89–93. Kasper, E. (1979). On the numerical field calculation in field emission devices. Optik 54, 135. Kern, D. (1978). Theoretische Untersuchungen an rotationsymmetrischen Strahlerzeugungssyste- men mit Feldemissionsquelle.Tu¨ bingen: Dissertation. Kihn, Y., Pe´rez, J. Ph., Se´vely, J., Zanchi, G., and Jouffrey, B. (1980). Data collection problems in high voltage electron energy loss spectroscopy. Electron Microsc. 4, 42–44. Kubin, L. P., and Jouffrey, B. (1971). On low temperature plastic instability in pure Niobium single crystal. Phil. Mag 24, 437–449. Mainy, D., and Jouffrey, B. (1980). E´ tude du comportement vibratoire d’un microscope e´lectronique a` haute tension. J. Microsc. Spectrosc. Electron. 5, 1–12. Mainy, D., Cathelinaud, R., Pinna, H., Pottier, M., Corsain, G., and Jouffrey, B. (1980). E´ tude du comportement d’un microscope e´lectronique a` haute tension, Deuxie`me partie : etude expe´rimentale. J. Microsc. Spectrosc. Electron. 5, 13–21. Martin, J. L., and Kubin, L. P. (1979). Optimum conditions for straining experiments in the HVEM. Phys. Sat. Sol. A 56, 487–494. Mory, C., Rocher, A., and Jouffrey, B. (1975). Observation de de´fauts d’empilement sous tre`s haute tension. J. Phys. 36, 163–170. Munro, E. (1971). Computer-aided-design in electron optics. Thesis: University of Cambridge. Pe´rez, J. Ph., Se´vely, J., and Jouffrey, B. (1981). Definition of a corrected magnetic prism spectrometer for electron microscopy. Nucl. Instrum. Methods 187, 237–239. Pe´rez, J. Ph., Zanchi, G., Se´vely, J., and Jouffrey, B. (1975). Discussion on a magnetic energy analyser used for very high voltage electron microscopy (1 MV and 3 MV). Optik 43, 487–494. Pinna, H., Kihn, Y., Se´vely, J., and Jouffrey, B. (1978). Comptage des electrons par un de´tecteur Si(Li). Application a` l’enregistrement des spectres de pertes ’eńergie d’e´lectrons en microscopie e´lectronique a` haute tension. J. Microsc. Spectrosc. Electron. 3, 1–14. Pinna, H., Liang, K., Denizart, M., and Jouffrey, B. (1981). Electronic environment for a field emission gun in electron microscopy. Rev. Phys. Appl. 18, 659–665. Pitaval, M. (1977). Observations de de´fauts cristallins en miccroscopie e´lectronique a` balayage. Thesis. France: Lyon. Roques, S., Denizart, M., and Sonier, F. (1983a). Tetrode field emission guns for electron microscopy. Optik 64(1), 51–66. Roques, S., Denizart, M., and Sonier, F. (1983b). Optical characteristics of the electron source given by a tetrode film emission gun. Optik 64(4), 299–312. Ruault, M. O., and Jouffrey, B. (1972). Contribution to the study of irradiation defects created in gold by electrons. C. R. Acad. Sci. Paris B 275, 451–454. Ruault, M. O., and Jouffrey, B. (1975). Studies of the nature of clusters in gold obtained with 2.5 MeV electrons. J. Appl. Cryst. 8, 228. On the High-Voltage STEM Project in Toulouse (MEBATH) 355

Ruault, M. O. (1981). Pheńome`nes d’irradiations dans les me´taux, In ‘‘Microscopie e´lectronique en sciences des mate´riaux’’, (Jouffrey, B., Bourret, A., and Colliex, C., ed.), p. 351. Ecole d’e´te´ du CNRS: Bombannes. Senoussi, S. (1971). E´tude d’un dispositif de filtrage des vitesses purement magne´tique adaptable a` un microscope a` tre`s haute tension. Orsay: Third cycle thesis. Se`vely, J., Pe´rez, J. Ph., and Jouffrey, B. (1973). Reálisation d’un analyseur de pertes d’eńergie des electrons a` tre`s haute tension. C. R. Acad. Sci. 276, 515–518. Se´vely, J., Pe´rez, J. Ph., and Jouffrey, B. (1974). High Voltage Electron Microscopy. Academic Press, Oxford 32. Se´vely, J., Zanchi, G., Pe´rez, J. Ph., and Jouffrey, B. (1977). Optics of a magnetic energy filter for CTEM. Inst. Phys. Conf. Ser. No. 36 45–48. Chap. 1. Troyon, M. (1977). E´ tude et realisation de canons a` emission de champ en microscopie e´lectronique par transmission. Ame´lioration du transfert du contraste de phase et de la resolution en holographie de Fraunhofer en ligne. France: Thesis. Reims. Tsuno, K. (1999). Optical design of electron microscope lenses and energy filters. J. Electr. Microsc. 48(6), 801–820. Venables, J. A., and Jansen, A. P. (1980). Performance of a field emission gun scanning electron microscope column. Ultramicroscopy 5, 297–315. Wells, O. C. (1974). Scanning Electron Microscopy. McGraw-Hill, New York 70. Zanchi, G., Pe´rez, J. Ph., and Se´vely, J. (1975). Adaptation of a magnetic filtering device on a one megavolt electron microscope. Optik 43 (5), 495–501. Zanchi, G., Se´vely, J., and Jouffrey, B. (1977a). Second order image aberration of a one megavolt magnetic filter. Optik 48(2), 173–192. Zanchi, G., Se´vely, J., and Jouffrey, B. (1977b). An energy filter for high voltage electron microscopy. J. Microsc. Spectrosc. Electron. 2, 95–104. Chapter 9

Scanning Transmission Electron Microscopy: Biological Applications

Andreas Engel

Contents 1. Introduction 358 2. Image Formation 359 2.1. Electron-Sample Interactions 359 2.2. The Optical System 360 2.3. Detectors 360 2.4. Single-Electron Counting 361 2.5. Imaging Modes 363 3. Imaging Thin Sections 363 4. Imaging Negatively Stained Samples 365 5. Mass Measurements Using the Basel STEM 366 5.1. Principle 366 5.2. Estimate of the Statistical Error 369 5.3. Mass Determination of Biological Samples 371 6. Specific Examples of STEM Imaging and Mass Measurements 373 7. High-Throughput Visual Proteomics 378 8. Conclusions and Perspectives 380 Acknowledgments 382 References 382

M.E. Mu¨ ller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, CH-4056 Switzerland, and Department of Pharmacology, Case Western Reserve University, 10900 Euclid Avenue, Wood Bldg 321D, Cleveland, Ohio 44106, USA

357 358 Andreas Engel

ABBREVIATIONS s e elastic scattering cross section s i inelastic scattering cross section e collection efficiency of the detector e average elastic scattering cross section of the irradiated atoms fraction of electrons scattered onto the annular detector A cross section of a spherical protein complex r radius of a spherical protein complex D incident electron dose (electrons per unit area) M mass average atomic mass of biological matter N number of atoms SAD annular detector signal Sbac background (i.e., total number of electrons scattered onto the annular detector by the carbon film) Sprot total number of electrons scattered onto the annular detector by the protein Stot the total signal, Sprot þ Sbac SD standard deviation SDbac standard deviation of Sbac SDtot standard deviation of Stot AD annular detector cryo-EM cryo-electron microscopy PMT photomultiplier tube SP spectrometer STEM scanning transmission electron microscope/microscopy TEM transmission electron microscope/microscopy

~~1. INTRODUCTION~~

In the early days of electron microscopy, Manfred von Ardenne built a special electron microscope that raster-scanned a small probe over an object to map its local properties (von Ardenne, 1938). Three decades later Albert Crewe and collaborators took up this approach at the Enrico Fermi Institute in Chicago. By using a field emission electron source they produced an electron probe a few A˚ ngstroms in diameter and were thus able to visualize single heavy atoms with superb contrast (Crewe, Wall, and Langmore, 1970). This progress with the scanning transmission electron microscope (STEM) encouraged Michael Beer and colleagues in Baltimore to use this instrument for sequencing DNA, but electron beam– induced motion of heavy atom labels attached to specific nucleotides Scanning Transmission Electron Microscopy 359 prevented a breakthrough by this approach (Cole, Wiggins, and Beer, 1977). Nevertheless, the STEM found its application in biology not only because the images it produces are of great clarity but also because of its analytical power and retains its importance today (Mu¨ ller and Engel, 2006). In particular, the STEM turned out to be the ideal instrument to measure the mass of single biomacromolecules based on their electron scattering (Engel, 1978; Wall, 1979). Despite these attractive possibilities amazingly few STEMs are used in biology, but this instrument is widely applied in the material sciences. The few dedicated STEMs used in biology have contributed invaluable new information over several decades. Two groups have routinely used STEM for mass measurements: one in the Brookhaven National Laboratory (see chapter 3 by Wall) and the other at the University of Basel in Switzerland. The coupling of image and mass affords them the power to answer questions not resolvable by ultracentrifugation or mass spectrometry. This chapter discusses the use of the STEM both as an imaging tool and to measure mass, and to examine the uncertainties to be expected in STEM mass datasets.

2. IMAGE FORMATION 2.1. Electron-Sample Interactions Electrons probe the structure of the sample studied. They carry the information after being scattered elastically by the nuclei of the sample atoms and inelastically by their electron shells. These interactions are s s quantified by the scattering cross sections e and i, which depend on the atomic number Z: 4 3=2 2 se ¼ 1:5 10 Z =b (9.1)

~~2 2 2 2 2 b ¼ðn=cÞ ¼ 1 ½meoc =ðmeoc þ EÞ (9.2)~~

~~4 1=2 2 si ¼ 1:5 10 Z lnð2=yEÞ=b (9.3)~~

~~2 2 yE ¼ DE=ðb ðmeoc þ EÞÞ (9.4)~~

Here c is the velocity of light, meo the rest mass of an electron with 2 y D meoc ¼ 511 keV, E the angle of inelastic scattering, E the energy loss, and E the acceleration energy (Langmore et al., 1973; Wall, Isaacson, and Langmore, 1974b). Because scattered elastically electrons interact just with nuclei whose diameters are of order 10–15 m, they are scattered into an angular interval 360 Andreas Engel of up to a few degrees and carry the high-resolution information on the location of the sample’s atoms. Inelastic interactions are produced by the collision of impinging electrons with the shell electrons whose orbits have a diameter of several A˚ ngstroms. Thus, inelastic events are delocalized compared to elastic ones (Isaacson et al., 1974; Reichelt and Engel, 1986). In this process shell or valence electrons are pushed out of the atom, leaving it in an ionized state and taking the energy DE from the impinging electron. Inelastically scattered electrons carry interesting chemical information, but the inelastic scattering event is directly related to beam- induced specimen damage.

2.2. The Optical System Transmission electron microscopes (TEMs) can depict a relatively thin, but nevertheless three-dimensional (3D) structure, whose image is a true projection of the 3D object if all the atoms of the latter are within the depth of focus D of the objective lens. To a first approximation, D is given by

D d2=l; (9.5) where l is the electron wavelength and d is the probe’s diameter. When the spherical aberration Cs of the probe-forming lens is optimally compensated by a small underfocus Df, the Scherzer equation yields the following (Reimer, 1989):

~~1=4 3=4 d Cs l : (9.6)~~

2.3. Detectors Three types of detectors in the STEM acquire the information from the cylindrical volume of diameter d irradiated by the probe: (i) annular detectors (ADs) that collect a large fraction of the elastically scattered electrons over different angular intervals ranging from typically 10– 200 mrad, (ii) the axial bright-field detector that assesses the amount of unscattered electrons, which interfere with elastic electrons scattered at low angles, typically 0–10 mrad, and (iii) the electron spectrometer (SP) that analyzes the inelastically scattered electrons, which are predomi- nantly scattered at low angles. Signals (i) and (iii) from scattered electrons are both linearly related to the number of atoms within the irradiated cylinder, thereby providing information about the sample mass at resolution d for elastic electrons. However, since electrons interact strongly with matter, multiple scattering concurrent with loss of signal and image blurring imposes further limitations when imaging thick objects Scanning Transmission Electron Microscopy 361

~~(b) (a) SPAD 0.6 Polystyrene AD AD-signal SP-signal E1 =10eV E2 =80eV 0.4 No~~

~~NSC SP~~

~~0.2~~

~~0 Ratio 0 100 200 nm~~

FIGURE 1 The image of a polystyrene sphere of 300-nm diameter formed by elastic and inelastic electrons illustrates the effects of multiple scattering (Reichelt and Engel, 1986). (a) At 80 kV the annular detector (AD) signal increases steadily, whereas the inelastic signal reveals a contrast inversion because of multiply scattered electrons that are not captured by the spectrometer (SP). The ratio AD/SP exhibits a nonlinear brightness versus thickness increase. (b) AD and SP signals recorded at 80 kV (solid circles and squares) are scaled to 100 kV (open circles and squares) for comparison with a Monte Carlo simulation of multiple scattering in polystyrene (Reichelt and Engel, 1984).

(Reichelt and Engel, 1986). For samples with a thickness close to or larger than the mean free path L, multiple scattering increases the effective probe diameter, and the number of scattered electrons is not linearly related to the sample thickness. As shown in Figure 1 the AD signal is less sensitive to multiple scattering effects than the signal produced by inelastic electrons, because the latter is acquired by an SP that has a much smaller collection angle than the AD.

2.4. Single-Electron Counting As a result of inelastic scattering impinging electrons destroy biological materials. Therefore it is important to extract as much information as possible from all scattered electrons; that is, most of them need to be collected, and all those collected need to be counted. The AD can do both: (i) It collects elastically scattered electrons over a large angular range, measuring 60% to 80% of all elastic events, and (ii) and it counts single electrons. The latter is achieved by using a scintillator that converts the energy of the impinging electron into a light pulse shorter than 15 ns, an efficient optical coupling arrangement, a photomultiplier tube (PMT), a discriminator to eliminate PMT noise, and finally a counter. The PMT needs to exhibit noise that is essentially limited to single photoelectron 362 Andreas Engel

~~(a) (b) 108 107~~

~~CaF2 NE 110 NE 905 106 Vth-2 photoelectron equivalents 107 Counts/s Maximum count rate 105~~

~~106 1.65 1.9 2.25 0.1 23456 ms PMT voltage (kV) Time (c) (d)~~

~~1.1 DQE~~

~~1.0~~

~~20 mV/U 0.9~~

~~100 kV 0.1 1 10 Count rate (MHz) 5 nS/U~~

FIGURE 2 Essentials of single-electron counting using a photomultiplier tube (PMT) (Engel, Christen, and Michel, 1981). (a) PMTs are capable of amplifying signals produced by a small number of photons, but as the gain increases the count rate drops. (b) When hit by many photons, a PMT produces significant noise that decreases with two different time constants, as illustrated by measurements with various scintillators. (c) The pulse-height distribution produced by the PMT 8850 (RCA) when 100-keV electrons impinge on an NE 905 plastic scintillator reveals a large number of pulses corresponding to single photoelectron events (arrow), a small number of two- and three-photoelectron events (arrowheads) and a distinct signal composed of 4 photoelectron events (*). The latter is the result of single 100-keV electrons hitting the scintillator and can be discriminated from the noise by suppressing all events corresponding to 3 photoelectrons. (d) The detection quantum efficiency DQE ¼ 2 2 (S/Nexp) /(S/Ntheory) achieved by the combination of the plastic scintillator NE 905, the PMT 8850 by RCA, and a LeCroy 100-MHz discriminator depends on the count rate, but it is close to unity for count rates up to 10 MHz. The ability to discriminate noise is most important because noise pulses outnumber signal pulses under normal operation conditions. Scanning Transmission Electron Microscopy 363 pulses, thereby allowing noise to be discriminated efficiently. This technology was developed in the 1960s by the high-energy physicists and has been adapted to achieve single-electron counting with the AD of the Basel STEM (Engel, Christen, and Michel, 1981)(Figure 2). Although axial bright-field and energy-loss detectors can collect only a fraction of the elastically and inelastically scattered electrons, single-electron counting can be achieved as well.

2.5. Imaging Modes It is of interest to compare different modes of TEM by a numerical simulation of the image formation (Engel, Wiggins, and Woodruff, 1974). In Figure 3 the most widely used coherent axial bright-field mode and the phase plate mode (taking Unwin’s spider web phase plate as an example; Unwin, 1971) are compared with the AD mode of the STEM. The simulation assumed a perfectly coherent illumination, neglected inelastic events, and did not include radiation damage and noise. Haase’s single-atom scattering theory (Haase, 1970) was used to describe the specimen, a one-dimensional model of a thin carbon film carrying six osmium atoms of which through- focus series were calculated. In all cases, the best image is obtained at the Scherzer focus, which is about 50 nm for Cs ¼ 1 mm and 100 kV acceleration voltage. Whereas interference phenomena make direct interpretation of the image details difficult, the structural information can be extracted with appropriate processing in the axial bright-field (Figure 3a) and phase plate (Figure 3b) modes. Although the test object consists of mainly high spatial frequencies, the phase plate mode exhibits a distinctly better contrast than the axial bright-field mode. In the STEM annular dark-field images, however, interference phenomena are not predominant and such images can be directly interpreted (Figure 3c). It is important to note that in the STEM annular dark-field mode the information cannot be retrieved from out-of-focus images as easily as in the two other modes.

~~3. IMAGING THIN SECTIONS~~

Because electrons interact strongly with matter, only thin samples can be visualized in the electron microscope. Most cells and all tissue samples are too thick for TEM. Electron microscopists have thus developed methods to stabilize the biological matter in its close-to-native state and to cut thin sections thereof (Armbruster et al., 1982; Carlemalm et al., 1985; Kellenberger et al., 1987). Although the ultimate sample preparation method entails high-pressure freezing and cryo-sectioning (Al-Amoudi et al., 2004), more conventional approaches use plastic embedding and sectioning at room temperature. In the latter approach, heavy metals are (a) Δf = 2000 Å

~~1500 Å~~

~~1000 Å~~

~~500 Å~~

0Å

~~−500 Å~~

~~−1000 Å~~

~~0 50 100 150 Å (b)~~

~~Δf = 2000 Å 1500 Å 1000 Å 500 Å 0Å −500 Å −1000 Å~~

~~0 50 100 150 Å (c)~~

~~Δf = 2000 Å 1500 Å 1000 Å 500 Å 0Å −500 Å −1000 Å~~

~~0 50 100 150 Å~~

FIGURE 3 Numerical simulation of TEM modes (Engel, Wiggins, and Woodruff, 1974). Six Os atoms were placed on a thin 140-A˚–wide carbon film and through-focus series were calculated for (a) coherent axial bright-field, (b) phase plate, and (c) STEM annular detector dark-field imaging modes. The simulation assumed a coherent 100-kV electron source, Cs ¼ 1 mm, and neglected inelastic scattering. The relative contrast of heavy atoms compared with the carbon film increases from (a) to (c). Whereas the atoms are most distinct in the Scherzer focus (Df ¼ 500 A˚) in (a) and (c), the phase plate needs to be operated at Df ¼ 0A˚. Despite a coherent electron source, STEM dark-field images shown in (c) exhibit no interference fringes, thus providing heavy atom images of superb clarity. Scanning Transmission Electron Microscopy 365 often used to specifically stain proteins, lipids, and nucleic acids to produce contrast in the TEM. For imaging sections the STEM offers interesting advantages. First, it is capable of producing sharp images even of relatively thick samples because multiple scattering has a smaller influence than in the TEM, whose postsample optical system introduces chromatic aberration. Second, the STEM offers the possibility to divide the elastic by the inelastic signal and thus achieving Z-contrast, as suggested by Eqs. (9.1) and (9.3). Although this simple model is not correct for thick samples because of multiple scattering (see Figure 1; Egerton, 1982), the ratio signal provides excellent contrast in this case as well (Kellenberger et al., 1986; Reichelt and Engel, 1986). Several examples have been presented (Carlemalm and Kellenberger, 1982; Jones and Leonard, 1978) and STEM imaging of thick sections has recently been discussed (Sousa et al., 2009). Even without heavy metal stain, plastic-embedded tissues exhibit sufficient contrast to visualize molecular details as illustrated by the section of a septate junction displayed in Figure 4. Whereas the STEM annular dark-filed image exhibits significant contrast, depicting the membrane as a dark grey band (Figure 4a), molecular details (Figure 4b) emerge only after dividing this image with the concomitantly acquired inelastic image.

~~4. IMAGING NEGATIVELY STAINED SAMPLES~~

Negative staining is of advantage in imaging single complexes despite its possible adversary effect on the protein preparation (Bremer et al., 1992). Because heavy atom stains scatter electrons elastically almost two orders of magnitude more than the light atoms of biological matter, the stain provides most of the signal. The high contrast delivered by the dark-field detector system (Engel, Wiggins, and Woodruff, 1974) can be exploited both in negative as well as positive stain microscopy (Mu¨ ller et al., 1998; Wall et al., 1974a). These images frequently provide information only otherwise visible after averaging some hundred projections recorded with a TEM. They thus make it possible to document the presence of distinct protein conformations, information that would be lost by averaging techniques. Annular dark-field images of negatively stained samples recorded by the Basel STEM over many years have given tremendous insight into biological questions of interest (Bremer et al., 1994, 1991; Broz et al., 2007; Burghout et al., 2004; Ducret et al., 1998; Engel, Dubochet, and Kellenberger, 1976; Hohn et al., 1979; Kohler et al., 2004; Martin et al., 1994; Mueller et al., 1998, 2005; Nouwen et al., 1999; Remigy et al., 1999; Rohrwild et al., 1997; Steinmetz et al., 1997, 1998; Takamori et al., 2006). The examples in Figure 5 demonstrate the power of the STEM AD dark-field mode. 366 Andreas Engel

~~(a)~~

~~(b)~~

FIGURE 4 Images of a thin unstained section acquired by STEM dark-field modes reveal a septate junction ring (Carlemalm and Kellenberger, 1982). (a) The annular dark-field image (AD) shows the septate junction as dark band without distinct proteins. (b) Simultaneous acquisition of the inelastic image via the spectrometer (SP) and calculation of the AD/SP ratio yields an image that unveils the proteins forming the septate junction. Ratio images are often referred to as Z-contrast images (see Eqs. 9.1 and 9.3), but for thick samples such as thin sections nonlinear effects related to multiple scattering need to be accounted for (see Figure 1). Scale bar: 100 nm.

5. MASS MEASUREMENTS USING THE BASEL STEM 5.1. Principle As the focused electron beam is raster-scanned over the sample each cylindrical volume element (voxel) is irradiated by a known number of electrons. Scattered electrons are counted, and the ratio of scattered to impinging electrons is calculated for every raster point (pixel), providing a number that is proportional to the mass of every voxel (Engel, 1978). An image comprising n n pixels is thus the collection of n2 scattering measurements, and represents the mass map of the sample (Engel, Baumeister, and Saxton, 1982; Sosinsky et al., 1992; Wall and Hainfeld, 1986). Although this principle is simple, a number of parameters need to be tightly controlled to achieve mass measurements that are reproduced Scanning Transmission Electron Microscopy 367

~~(a) (b) (c)~~

~~(d) (e)~~

~~(f)~~

~~(g)~~

FIGURE 5 Negatively stained samples are depicted by the STEM annular detector dark- field mode with outstanding quality, which has allowed a diversity of questions in structural biology to be addressed. Panel (a) shows images of GroEL-GroES and GroEL- GroES2 complexes (Engel et al., 1995) and the binding of antibodies to GroEL (Martin et al., 1994). The sevenfold symmetry of these complexes is evident from single top views, as well as the layered structure of the GroEL cylinder from side views. Antibodies were raised against a peptide located at the rim of the cylindrical complex, and the binding site is well resolved. Scale bars: 15 nm. Panel (b) displays antibodies (inset, top left) and Fab fragments. To help interpretation of these annular dark-field images the projection of an antibody conformation at atomic resolution is shown at twice the magnification. Scale bars: 15 nm and 5 nm (inset). (c) Top view of a negatively stained secretin complex PulD-PulS (Nouwen et al., 1999) imaged by STEM (top left). The floppy protrusions have been identified as PulS; their arrangement suggests the complex to exhibit 12-fold rotational symmetry (bottom left, cartoon). PulS cannot by detected in single side views (bottom right), probably because this flexible structure collapses during sample preparation. Class averages from vitrified PulD revealed arms and small pro- truding domains to which PulS is thought to bind (top right) (Chami et al., 2005). Scale bar: 15 nm. Panel (d) shows the hook structure of an E. coli flagellum (scale bar: 15 nm). Two different bacterial surface layers are shown in panel (e). The hexagonally packed intermediate layer of Deinococcus radiodurans (Engel, Baumeister, and Saxton, 1982) has 368 Andreas Engel at absolute accuracy of a few percent (Mu¨ ller et al., 1992). Given the ability to count each electron collected by the AD, the error introduced here is simply of statistical nature. Nevertheless, the single-electron counting system needs to be regularly calibrated. To provide an accurate value of the electron dose, both probe current and magnification need to be monitored as precisely as possible. Since the probe current is a predetermined fraction of the objective aperture current, the latter provides an appropriate relative signal. Although the magnification can be calibrated, it depends on the focus. Therefore, the objective lens current is another useful parameter to acquire (Mu¨ ller et al., 1992). Because the objective lens in the VG STEM HB-5 used in Basel has a weak postspecimen field, the detector geometry variations are negligible for the focus range used under normal operating conditions. Alternatively, the objective lens current is kept constant and the specimen is moved along the z-axis until the probe is focused on the sample. The latter approach is technically more complicated than the former one, but it has the advantage that the post- specimen field—and thus the collection geometry of the AD—remain constant. Thus, it might be the only solution for highly excited objective lenses. Keeping a STEM well calibrated for mass measurements is a technical investment that might be circumvented by co-preparing a suitable standard such a tobacco mosaic virus (TMV). Although this trick works in many cases, it is not useful with ‘‘sticky’’ proteins (e.g., detergent- solubilized membrane proteins; Mu¨ ller and Engel, 1998) that easily interact with TMV, leading to a significant mass increase. Although such phenomena can directly be recognized by the appearance of the TMV, the absolute calibration of the measurement is then not possible.

a center-to-center distance between hexamers of 18 nm. The tetragonal S-layer of B. brevis exhibits a unit cell size of 13 nm (Aebi et al., 1973). Both S-layers are held together by elongated domains, which emanate from the cores to form a tight contact. These double arms are directly discernable in the STEM annular dark-field images. Scale bar: 15 nm. Panel (f) displays needles that are part of the Y. enterocolitica injectisome (Mueller et al., 2005). The tip complex was shown to be assembled from LcrV proteins by antibody labeling (top). This complex has the shape of a hat and exhibits a central channel (bottom and insets, bottom right). These features are seen directly in images of single complexes but become particularly clear after averaging a few complexes (middle right). Scale bars: 20 nm and 5 nm (average). Panel (g) reveals three filamentous assemblies. Left: the helical filament observed upon overexpression of PulG (Kohler et al., 2004); middle: a short stretch of an actin filament revealing single actin molecules (Steinmetz et al., 1997); right: the pilus of Streptococcus pneumoniae, a very thin adhesive structure produced by several Gram-positive pathogens (Telford et al., 2006). Scale bar: 10 nm. Scanning Transmission Electron Microscopy 369

5.2. Estimate of the Statistical Error Approximately 70% of all the electrons elastically scattered by each voxel are collected by the annular dark-field detector system capable of single-electron counting. Combining these n2 intensities yields a dark-field image of the sample that can be used for mass analysis. For thin samples, the annular detector signal, SAD, is directly proportional to the number of atoms, N, irradiated by the STEM probe, weighted by their average elastic scattering ~~e cross section, e , and the collection efficiency of the detector, :~~

SAD ¼ N e < se > D; (9.7) where D is the incident electron dose determined by the probe current, dwelling time, and pixel area. Monitoring SAD allows N and thus the corresponding mass, M, to be calculated:

M ¼ N < Ma > ¼ SAD < Ma >=ðe < se > DÞ (9.8) < > where Ma is the average atomic mass of biological matter. Theoretical data on elastic scattering are sufficiently precise to permit the calculation of the absolute mass scale for proteins, making a fully calibrated instrument independent of mass standards. Thus the error of the measurement is given by the statistics of single electrons counted by the AD and by the cleanliness of the carbon film. The statistical noise for spherical protein complexes of mass M can be r ˚ 3 < > estimated from its density ¼ 0.82 Da/A and the average mass Ma ¼ 13.42 Da of its atoms that have an average elastic scattering cross section ~~˚ 2 e ¼ 0.0102 A for 80-kV electrons (Engel, 1978). The radius r and cross section A of such proteins (taking the mass in Daltons (Da) and the length in A˚ ngstroms (A˚ )) are:~~

~~= = r ¼f3M=ð4prÞg1 3 ¼ 0:66 M1 3 (9.9)~~

= A ¼ pr2 ¼ 1:38 M2 3 (9.10) The fraction of the incident electrons scattered onto the AD (e ¼ 0.69 < > in the Basel STEM (Engel, 1978)byN ¼ M/ Ma atoms of the protein is 2=3 4 1=3 ¼ Me < se >=ð< Ma > 1:38 M Þ¼3:80 10 M : (9.11)

~~Thus the total number of electrons Sprot counted by the AD when that protein is irradiated by dose D (in electrons/A˚ 2) amounts to 4 Sprot ¼ DA ¼ MDe < se >= ¼ 5:24 10 MD (9.12)~~

From Eq. (9.12) the mass M of a protein complex can be calculated from the relative signal Sprot/D. For mass measurements, samples are 370 Andreas Engel adsorbed to thin carbon films and must necessarily be left unstained. To minimize background, all traces of nonvolatile salts are subsequently removed by a series of washes with quartz double-distilled water or, where instability of the sample demands it, with volatile buffer solution (e.g., 10 to 100 mM ammonium acetate) (Mu¨ ller et al., 1992). This step is critical because the background can be the largest source of random errors and can thus significantly increase the scatter of the measurements. Freeze-drying generally follows to prevent structural collapse during dehydration. Images are recorded at low electron dose, typically 3 to 4 electrons/A˚ 2, to minimize beam-induced mass loss. When measurements are made at ambient temperature, the small mass-loss correction necessary is experimentally determined. It should be noted that cooling the sample with liquid nitrogen greatly reduces the beam-induced mass loss (Wall and Hainfeld, 1986), allowing measurements to be executed at a higher dose, hence reducing the statistical error. Nevertheless, mass histograms from measurements at room temperature with the Basel STEM have provided results comparable to those obtained with the Brookhaven STEM at the temperature of liquid nitrogen (Wall and Hainfeld, 1986). Image evaluation requires summation over all picture elements covered by the structure of interest, followed by normalization to the recording dose and subtraction of the background (i.e., the carbon film scattering). This allows the mass of particles, the mass-per-length of filamentous structures, and the mass-per-area of layers to be calculated, which can efficiently be achieved by the MASDET software package (Krzyzanek et al., 2009). The statistical error for a homogeneous preparation of a spherical protein complex on a thin clean carbon film is given by the standard deviation, SD, where: 2 2 1=2 1=2 1=2: SD ¼ðSDtot þ SDbac Þ with SDtot ¼ Stot and SDbac ¼ Sbac (9.13)

Stot is the total signal, that is, the sum of the protein signal Sprot [Eq. (9.12)] and the background, Sbac, from the carbon film. Ideally, the latter should be adapted precisely to the cross section of the protein [Eq. (9.10)], but in practice, particularly in the case of membrane proteins, which remain associated with detergent, the evaluation boxes can be twice as large, increasing Sbac by a factor of four. Taking this into account, the Sbac for carbon films that are typically 50 A˚ thick and scatter 3% into the AD is denoted as : 2=3: Sbac ¼ 0 166 DM (9.14) From this, typical SD values were calculated for a protein complexes with mass of 100 kDa to 10 MDa that are imaged at doses between 2 and 8 electrons/A˚ 2 (Table 1). As a result of additional sources of error (contaminants on the carbon film, dissociation of the protein complex, Scanning Transmission Electron Microscopy 371

~~TABLE 1 Theoretical Statistical Errors of Mass Measurements by STEM~~

~~Dose (e/A˚ 2)2 4 8 Mass (kDa) SD (kDa) SD (kDa) SD (kDa)~~

100.0 37.4 26.4 18.7 200.0 47.5 33.6 23.8 300.0 54.7 38.7 27.4 400.0 60.5 42.8 30.3 500.0 65.5 46.3 32.7 600.0 69.8 49.4 34.9 700.0 73.7 52.1 36.9 1000.0 83.7 59.2 41.8 2000.0 107.3 75.8 53.6 4000.0 138.0 97.6 69.0 10000.0 193.9 137.1 97.0 variable amounts of detergent bound per membrane protein complex), the experimental SD tends to be larger. Nevertheless, under ideal conditions, such SDs have been achieved (see Figures 6 and 7). Thus only a relatively small number of particles, in general a few hundred, need to be measured to obtain a standard error of the mean that is better than the instrument’s calibration (typically 2%). Although the precision of STEM cannot match that of the mass spectrometer, its capability to measure an enormously wide mass range, its independence from shape assumptions and the presence of an image make the STEM an invaluable tool.

5.3. Mass Determination of Biological Samples Proteins as small as 100 kDa and larger than 100 MDa have been successfully examined independent of their shape (Baschong et al., 1991; Engel, 1978; Reichelt et al., 1990; Takamori et al., 2006; Wall, 1979; for reviews see Mu¨ ller and Engel, 1998, 2001, 2006; Thomas et al., 1994)). As demonstrated in Figure 6, the STEM is the only technique that not only allows the total mass of a large protein complex to be directly determined, but also the mass-per-length of filamentous structures and the mass-per-area of pla- nar structures such as surface layers or membrane assemblies. The mass- per-length is a useful, if not essential, parameter defining the structure of filaments, indicating the number of strands present and in the case of helices, restricting the number of possible assembly rules. A wealth of information has been collected with the Basel STEM to provide a basis for initial and refined models of intermediate filaments (Aebi et al., 1988; Herrmann et al., 1996, 2000), amyloid fibrils (Goldsbury et al., 1997, 2000, 372 Andreas Engel

~~(a) (b) 30 Top Side 50 25 40 20 30 15~~

~~20 10 Number of particles Number of particles 10 5~~

~~0 0 0.6 0.8 1.0 1.2 1.4 0.6 0.8 1.0 1.2 1.4 Mass/MDa (d) Mass/MDa (c) 132.9 kDa/nm 20 136 kDa/nm 134.2 kDa/nm 136.6 kDa/nm 15 135.6 kDa/nm~~

~~Number of segments 5~~

~~0 120 130 140 150 MPL (kDa/nm)~~

~~(e) (f) (g)~~

FIGURE 6 Imaging unstained freeze-dried protein complexes and mass measurements. (a) Selected top views and side views from a heterogeneous mixture of GroEL, GroEL- GroES, and GroEL-GroES2 complexes (Engel et al., 1995). The top views are round and easy to discriminate from the side views; the side views shown exhibit the football-like shape of the GroEL-GroES2 complex. Scale bar: 20 nm. (b) Left panel: Mass analysis of top views reveals a skewed histogram that is fitted with two major Gaussians centered at 850 and 950 kDa, representing GroEL and GroEL-GroES complexes. Right panel: Mass analysis of all side views present yields a complex histogram that is fitted with three major Gaussians centered at 850, 950, and 1120 kDa, representing GroEL, GroEL-GroES, and GroEL-GroES2 complexes. (c) Mass analysis of a single tobacco mosaic virus (TMV). User-selected boxes are automatically evaluated by the MASDET software (Krzyzanek et al., 2009) to yield mass-per-length (MPL) values and radial mass profiles (Steven et al., 1984). The central channel of the TMV rod is revealed as a small dip in the profile. Scale bar: 50 nm. (d) The mass histogram of a calibration measurement from 58 boxes giving a Gaussian distribution centered at 135 kDa/nm with a full width at half maximum height of 6 kDa/nm (SE ¼0.4 kDa/nm). Mass measurements are scaled Scanning Transmission Electron Microscopy 373

2005; Kammerer et al., 2004; White et al., 2009), and a variety of helical assemblies (Bremer et al., 1991, 1994; Di Capua et al., 1982; Hahn et al., 2002; Kohler et al., 2004; Steinmetz et al., 1998). Similarly, the mass-per- area defines the unit cell stoichiometry in 2D crystals (Walz et al., 1994)or the number of layers in a sheetlike structure (Schenk et al., 2005), the latter information being otherwise accessible only by chance at their edges or by atomic force microscopy. Further, in conjunction with a lipid assay by analytical ultracentrifugation, mass-per-area measurements can be used to confirm the predicted packing of membrane proteins in 2D crystalline arrays (Mu¨ ller and Engel, 1998). Finally, the presence of an image allows the mass of specific, well-defined regions of intact complexes of any shape to be determined and a mass map to be generated (Engel, Baumeister, and Saxton, 1982; Sosinsky et al., 1992; Wall and Hainfeld, 1986). When combined with sodium dodecyl sulfate–gel electrophoresis and, where available, sequence information, STEM mass measurements serve to define protein stoichiometry. The methodology of mass spectrometry has developed to a high level of sophistication over the past decade, allowing the proteins in hetero- oligomeric complexes to be identified. In combination with electron microscopy, in particular with STEM as an initial step, such progress opens an important new avenue that could result in the full structural definition of protein complexes. STEM measurements provide the first link in this chain, as the total mass of individual protein complexes is determined. In combination, STEM measurements would both reveal whether the proteins identified by the bulk mass spectrometry technique were part of a single complex or from a number of complexes that collectively have the determined composition. In the former case, STEM measurements would provide a basis to estimate their stoichiometry.

~~6. SPECIFIC EXAMPLES OF STEM IMAGING AND MASS MEASUREMENTS~~

Over the years both dark-field imaging and mass measurement by STEM have frequently been used to address specific questions not directly accessible by other techniques. Some results obtained in Basel are with such calibration measurements, circumventing the requirement to co-prepare TMV with the sample to be studied. (e) A hexagonally packed intermediate (HPI) layer sheet recorded at 0.37 e–/A˚ 2 does not show the HPI hexamers clearly. Scale bar: 60 nm. (f) The second scan taken at a 10 times higher dose reveals doughnut-like hexamers, connecting arms, and some adsorbed contaminants. (g) The correlation average was calculated from image (e), using the unit cell coordinates obtained from image (f). This mass map has a spatial resolution of 3 nm (Engel, Baumeister, and Saxton, 1982). 374 Andreas Engel

~~(a) (b) 400 1200 160~~

~~300 120 800 200 80 400 100 40 Number of particles Number of particles~~

~~0 0 0 0 500 1000 1500 0 1.0 2.0 3.0 4.0 Mass MDa Mass MDa (c) (d) STEM TEM STEM TEM~~

~~Before After~~

FIGURE 7 The PulD and YscC secretins. (a) Histogram showing the mass distributions measured for the PulD secretin complex (black) and its trypsin-resistant fragment (grey). The Gaussians at 1 MDa and 0.5 MDa, respectively, indicate the presence of 12 monomers and detergent (Chami et al., 2005). (b) Histogram showing the masses measured for the YscC secretin complex (Burghout et al., 2004). The main peak at 1 MDa is compatible with the presence of 13 YscC monomers and detergent. The two higher mass peaks at 2 MDa and 3 MDa arise from two and three associated complexes, respectively. The size and shape of the complexes with masses in the three ranges (insets) allowed the unambiguous interpretation of images recorded from negatively stained microscopy grids. (c) Single-shot STEM images and TEM image averages of negatively stained PulD secretin complexes before (left gallery) and after (right gallery) proteolysis compared with the corresponding averages of images taken in the conventional TEM (Nouwen et al., 2000)). The top views have ringlike projections (top row); note the peripheral densities radiating from the intact complexes. The latter are visible in the STEM image and reveal conformational variability; hence they exhibit only weak contrast in the TEM average composed of 621 projections. After trypsin treatment the radial protrusions are missing in both the STEM single-shot image and the TEM average composed of 276 projections. The side views reveal the channel of single PulD secretin complexes. The radial protrusions are not distinct as they probably collapse during dehydration. (d) STEM images of negatively stained YscC secretin complexes. A ringlike top view (top left) and a rectangular side view of two complexes associated via the flatter ends (bottom left) are compared with the corresponding negative stain TEM averages (right). The association of three complexes is shown on the right; compare inset of (b). Scale bars: (b) 10 nm, (c) and (d) 5 nm. Scanning Transmission Electron Microscopy 375 discussed here to illustrate the versatility of the technique. The first example concerns the Escherichi coli protein folding machinery GroEL- GroES. Discovered in the early 1970s as an essential factor for the morphogenesis of several bacteriophages, GroEL was overexpressed for structural and functional analyses (Hohn et al., 1979). GroEL was also studied by electron microscopy. Its sevenfold symmetry and cylindrical shape were demonstrated by STEM dark-filed images of negatively stained GroEL (Hohn et al., 1979). It was soon recognized that GroEL is a key element in the folding of all E. coli proteins, and that it required the co-factor GroES, which was thought to form the lid on the cylinder to trap the unfolded protein. The Brookhaven STEM revealed that the protein substrate is indeed localized within the central cavity (Braig et al., 1993), an observation confirmed later by cryo-electron microscopy (Chen et al., 1994; Clare et al., 2009). How GroEL and GroES interact in the folding cycles was subject to intense controversy: Was it the GroEL-GroES complex with its characteristic bullet shape or the football-like GroEL-GroES2 aggregate that folded the proteins (see Figures 5a and 6)? Because both the bullet complex and the GroEL tetradecamer can stand on the carbon film with the cylinder axis perpendicular (and often do so) and their top views are essentially indistinguishable, it was not possible to obtain a good measure of their relative abundance compared with the footballs mostly seen in their side view. Yet, the relative population of different complexes was an important piece of information for understanding the structure- function relationship. Hence, mass measurements of such heterogeneous samples were done by STEM (Engel et al., 1995) to reveal the relative abundance of the respective complexes, which can be directly deduced from the mass histograms of top views and side views (see Figures 6a and b). The combination of mass and shape information was key to answer this question. In the following example the single, integral outer membrane proteins of T2 and T3 secretion systems are discussed. They belong to a large superfamily of homologous proteins, the secretins, which are present in the bacterial outer membrane as homo-oligomers. STEM has been used to define the oligomerisation state of two: the T2 secretin complex of Klebsi- ella oxytoca formed by the protein PulD (Chami et al., 2005; Nouwen et al., 1999, 2000) and the corresponding T3 complex of Yersinia enterocolitica formed by the protein YscC (Burghout et al., 2004). In the more extensive study of the K. oxytoca system, STEM mass measurements indicated the presence of 12 monomeric subunits in the intact PulD-PulS complex and in its trypsin-resistant fragment (Nouwen et al., 1999, 2000)(Figure 7a). This stoichiometry was found to be retained when the PulD alone and its trypsin-resistant fragment (Chami et al., 2005) were imaged. In contrast, the T3 secretin complex of Y. enterocolitica was concluded to be a 13-mer from the mass of the complex (Burghout et al., 2004)(Figure 7b). 376 Andreas Engel

Given their mass, the two minor peaks on the latter histogram arise from the aggregation of two and three complexes, respectively. As documented by the insets, sorting the corresponding particles according to mass and inspecting the resulting galleries confirmed this interpretation. The secretin samples were also examined by negative stain microscopy to obtain structural information. As illustrated by Figures 7c and d, the single-shot STEM images revealed details that became visible only on averaging conventional negative stain TEM projections (Nouwen et al., 2000). The top views showed that the PulD-PulS and the YscC secretins form large ring-shaped complexes with a central channel (Figures 7c, d) and revealed peripheral densities around the PulD ring that were lost on trypsin treatment (Figure 7c). Some single complexes of the PulD-PulS complex and the trypsin-resistant PulD fragment sometimes adsorbed side-on, giving a view of the central channel. Because in this case the radial protrusions collapsed on dehydration, single-shot STEM side views did not reveal a significant difference between the uncleaved and cleaved PulD-PulS complexes. However, averages from negatively stained side views recorded by TEM showed distinct differences (Figure 7c). YscC secretin complexes were mainly seen in their top view and adsorbed side- on only when aggregated. In this case, dimers formed by the interaction of the flatter ends and single-shot STEM images revealed an inherent flexibility of the ’’conical’’ region not detected by averaging procedures (Figure 7d). The secretin complex itself is believed to form the translocation pore used in T2 secretion, but the mechanism of the translocation is not known. In K. oxytoca the translocation is possibly facilitated by a short (and as yet undetected) periplasmic pseudopilus formed by the pseudopilin PulG. To obtain structural insights, STEM was employed to characterize the long, flexible pseudopili produced on over-expression of PulG(His)6 in E. coli (Kohler et al., 2004). Indeed, images recorded from negatively stained samples directly revealed their periodic helical structure (Figure 5h and Figure 8a). Straightened filament stretches yielded typical X-shaped diffraction patterns, allowing the pitch of the helix to be determined (Figure 8b). The mass-per-length (Figure 8c) measured for corresponding unstained samples by STEM was key to the following image analysis, restricting the number of helical rules applicable. Accordingly, there were on average 17 PulG subunits in four turns of the left-handed helix, the handedness being established from freeze-dried metal-shadowed pili (Kohler et al., 2004). This information allowed a 3D model of the pseudopilus to be generated based on the X-ray structure determined for a truncated form of PulG. A needle complex that extends into the exterior milieu penetrates the T3 secretin of Y. enterocolitica. In a recent study STEM images clearly revealed a complex at the needle tip (Mueller et al., 2005) (see Scanning Transmission Electron Microscopy 377

~~(a) (b) (d)~~

~~(c)~~

~~20 Number of segments~~

~~0 6 14 22 30 38 Mass-per-length (kDa/nm)~~

FIGURE 8 The PulG(His)6 pseudopilus (Kohler et al., 2004). (a) Negatively stained pseudopili imaged by the STEM; they appear to be rather flexible and to have a helical structure. The inset displays a filament that was straightened computationally. Its width is 8.2 nm. Scale bar 50 nm. (b) Diffraction pattern of a straightened pseudopilus stretch displaying the characteristic X pattern of a helical structure. The strong layer line indicates a pitch of 4.37 nm. Scale bar: 5 nm–1. (c) The mass histogram indicates a mass-per-length of 15.5 kDa/nm. Information from (b) and (c) defines the helical rule of the filament and thus allows a helical reconstruction to be made. (d) Atomic model of the PulG pseudopilus. The left-hand model was based on the average helical selection rule: 17 monomeric subunits in 4 turns, derived from the STEM images. The envelope of the helical reconstruction of the pilus is at 2.5-nm resolution, and the arrangement of the PulG monomers and the interaction of the N-terminal helices (from top to bottom) is shown. Bottom: View up along the pilus axis. (Adapted from Kohler et al., 2004, courtesy of Blackwell Publishing.)

Figure 5g). Combined with further negative stain STEM, mutagenesis and immunolabeling experiments showed this tip to be formed by the protein LcrV (also known as V-antigen). Polyclonal antibodies against LcrV directly visualized by STEM were seen to interact exclusively with the tip complexes, often linking two needles via their variable domains (see Figure 5g). The presence of LcrV at the very tip of the Yersinia needle explains why this protein is essential for pore assembly in the host cell membrane and infection. Averaging about 20 high signal to noise images of the tip complex clearly revealed features faintly visible on the unpro- cessed STEM images; these showed the needle has a central channel that continues into the tip-complex (see Figure 5g [inset]). STEM imaging was also essential to reveal the morphology of tip complexes formed by different chimeric proteins engineered to show how LcrV is oriented in the tip structure, and to provide a first estimate of the number of LcrV proteins building the tip (Broz et al., 2007). 378 Andreas Engel

The last example discussed here concerns the mass of synaptic vesicles. These structures have been characterized by a wide range of biophysical and biochemical techniques. They store neurotransmitters and undergo Ca2þ-dependent exocytosis upon the arrival of an action potential, are subsequently retrieved by clathrin-dependent endocytosis, and are recycled to regenerate exocytosis-competent vesicles. The proteins of synaptic vesicles are presently better understood than those of any other trafficking organelle, and several proteins first discovered in synaptic vesicles are founding members of conserved protein families involved in trafficking (Sudhof, 2004). Nevertheless, even elementary quantitative information is lacking; for example, it is not known how many copies of the essential trafficking proteins are present on each vesicle, how many membrane proteins a vesicle contains, and how these proteins relate to the overall structure of the organelle. Therefore, a purified vesicle fraction from brain was used as the starting point for the description of a prototype synaptic vesicle, resulting in a detailed (albeit averaged) structural model of this trafficking organelle (Takamori et al., 2006). An essential contribution made by STEM is the measurement of the synaptic vesicle mass (Figure 9a), which allowed a wealth of biophysical analyses to be tied together. In addition, STEM images of negatively stained vesicles (Figure 9b) provided further input for assembling a quantitative model of such trafficking vesicles (Figure 9b).

~~7. HIGH-THROUGHPUT VISUAL PROTEOMICS~~

Because it offers the possibility of determining the mass and producing readily interpretable projections of single, negatively stained protein complexes, the STEM appears to be an ideal instrument for visual proteomics. Coined by the Baumeister laboratory, this terminology reflects the promise that the proteome of a single cell is being visualized by using cryo-electron tomography to image individual protein complexes as they exist in the cell (Baumeister, 2005a,b; Nickell et al., 2006). Since eukaryotic cells are in general larger than about 10 to 50 mm, they are too thick to be imaged at the 2- to 3-nm resolution required to identify the complexes present and their subunits. Instead of producing cryo-sections of vitrified cells that are sufficiently thin for tomography at 2- to 3-nm resolution, we propose to use microfluidic circuits to grow and lyse single cells (Breslauer, Lee, and Lee, 2006; Di Carlo et al., 2005; El-Ali, Sorger, and Jensen, 2006) to fractionate their contents (Pamme, 2007; Roman and Kennedy, 2007) and to deposit the fractions by spotting 10 pL drops per square of an electron microscopy grid. In this way, the final sample will be thin enough for high-resolution imaging. Sample deposition devices can be tuned to desalt the sample, to add negative stain, or to vitrify the grid Scanning Transmission Electron Microscopy 379

~~(a) (b)~~

~~20 # of events 10~~

~~0 10 20 30 40 (MDa)~~

~~(c)~~

FIGURE 9 Synaptic vesicles (Takamori et al., 2006). (a) Mass distribution of synaptic vesicles determined by STEM. The histogram displays the mass of 281 vesicles from both untreated and glutaraldehyde-stabilized samples. The average value is 16.2 4.1 MDa excluding the few very high values, and the main peak is at 15.4 3.1 MDa. (b) The uranyl acetate–stained synaptic vesicle imaged by STEM shows the associated V-adenosinetriphosphatase. Scale bar: 20 nm. (c) Molecular model of a synaptic vesicle based on space-filling models of all macromolecules at near-atomic resolution. The view of a vesicle sectioned in the middle is shown. once all grid squares are populated. With current techniques to prepare protein solutions for electron microscopy, which all involve blotting or washing and blotting, only the minute fraction of protein complexes tightly adhering to the grid or remaining in the residual water film in the case of cryo-electron microscopy is available for imaging, and the rest are lost. In contrast, picoliter drop deposition of fractions from the microfluidic system with subsequent drying or vitrification will make the entire proteome available for imaging. Grid squares containing negatively stained or unstained fractions of a single cell will then be read by the STEM and quantitatively analyzed—essentially like the pages of a book. 380 Andreas Engel

The readout will be achieved in either of two ways. First, hierarchical automated particle selection and segmentation algorithms (Coudray et al., 2008) will select all visible particles for mass analysis, and particle projections will be sorted according to their mass (Krzyzanek et al., 2009). Mass- related projections can be classified to obtain mass maps of complexes viewed from different directions and used to calculate the 3D map of the respective complex (Herman and Kalinowski, 2008). This protocol will establish the mass-to-shape relation of complexes in inspected fractions, which will complement the much more accurate mass determination by mass spectrometry. Second, hierarchical automated particle selection will be used to select projections of negatively stained particles, which will be classified based on shapes obtained from mass mapping. Refined classification and pattern recognition protocols will then be used to identify complexes by comparison with lookup tables composed of projections calculated from high-resolution structural models of specific complexes (Frangakis et al., 2002). In this way, variations between single cells submitted to specific perturbations can be efficiently assessed at the level of single complexes. In addition, initial 3D maps of yet uncharacterized, but reproducibly observed complexes can be obtained from projections of negatively stained samples. Ultimately, the visual proteomics chain will be completed by the inspection of vitrified cell fractions, using cryo-electron microscopy. High-resolution 3D maps are likely to be obtained by sorting out particle projections based on all the information established with mass mapping and 3D reconstruction of negatively stained complexes. Combined with mass spectrometry data from the respective fractions these 3D maps will provide a solid foundation for creating atomic-scale models of all complexes identified.

~~8. CONCLUSIONS AND PERSPECTIVES~~

As illustrated by the examples presented here, quantitative biological STEM is a well-developed and invaluable technology for nano-analytics. Because it is possible to associate mass with the shape of individual protein complexes, STEM provides a complementary extension of mass spectrometry to be exploited in the future. STEM’s ability to distinguish between different proteins by means of their mass promises to make this technique of vital importance to many systems biology projects. Considering the enormous progress in instrumentation, it is fair to say that the quality of acquired data now depends to a large extent on the quality of the prepared sample. There is a large misbalance between the effort that has been devoted to instrumentation development and that concentrated on improving sample quality. Although a wide variety of Scanning Transmission Electron Microscopy 381 sample preparation methods have been worked out and implemented since the first electron microscopes were built, most methods currently used were actually first introduced 30 to 50 years ago. The great exception is clearly the vitrification of the specimen introduced by Dubochet and coworkers to keep the sample in a frozen hydrated state (Al-Amoudi et al., 2004; Dubochet et al., 1988). This method has not only been widely used, but has also stimulated a number of other developments, in particular high-pressure freezing. However, vitrification and all other sample preparation techniques used for TEM and STEM, with the exception of block freezing and plastic embedding, waste most of the biological sample by simply blotting or washing it away. Without these wasteful steps the electron microscopic analysis would require sample volumes in the picoliter rather than microliter range. Indeed, even the block-freezing and plastic-embedding techniques are not exempt from wastage since it is rarely, if ever, possible to sample the entire block. Demands placed by systems biology and proteomics to quantitatively and rapidly investigate heterogeneous protein mixtures make future progress on three fronts essential. First, the goal to work with minimal sample loss must be reached; second, it must be possible to examine a large number of different but related samples within a reasonable time frame; and third, image analysis has to be greatly sped up, ideally reaching real time. All three requirements demand automation, and it is here that most progress is to be expected in the near future on both the instrument and sample preparation sides. Automating the microscope for high throughput is taking the lead. The instruments and software required to automate electron tomography are available, and multigrid holders and automated transfer systems have been designed and recently became available for cryo-electron microscopy. Coupled with the corresponding software, such holders promise to make the unattended 24-hour operation of instruments for all types of electron microscopy a realistic prospect within the next few years. Focus must now be placed on the sample preparation, and in particular, on the development of sample preparation robots, which are highly reproducible and preserve the complete contents of cellular fractions in their native state. Microfluidic devices may be a practical solution toward this goal. A prerequisite for all electron microscopy projects aiming at the structural elucidation of single proteins is to have the protein under study available in a highly purified form. In contrast, as implied above, single- cell proteomics requires the examination of multiprotein complexes and heterogeneous protein mixtures. The problem is then how to sort the projection images representing a particular protein species or protein complex from the myriad of particle morphologies found on the grid. One way would be by specifically labeling the species of interest. This would be achieved by adding gold-labeled cognitive proteins (e.g., Fab 382 Andreas Engel fragments, designed ankyrin repeat proteins [DARPins]; Steiner, Forrer, and Pluckthun, 2008) to the fractions produced in the microfluidic system. STEM mass measurement offers an efficient alternative to this approach because it allows the direct correlation of the mass of a given biomolecule and its size and shape. Combining these approaches appears to be feasible and most attractive. Projection sorting based on shape and mass, followed by projection matching to images obtained by negative stain or cryo- electron microscopy from the same sample, allows a first structural model to be derived. In view of this perspective, STEM will be key to the analysis of multiprotein complexes and heterogeneous protein mixtures extracted from single cells.

ACKNOWLEDGMENTS This work was supported by the Swiss National Science Foundation (SNF), the National Center of competence in Research in Structural Biology, SNF grant No. 3100A0-108299, EU grant No. 035995-2, the University of Basel, and the Maurice E. Mu¨ ller Foundation of Switzerland. The author is indebted to Ueli Aebi, Peter Agre, Wolfgang Baumeister, Guy Cornelis, and Tony Pugsley for most fruitful collaborations; to Shirley Mu¨ ller and Patrick Bosshart for numerous stimulating discussions and reading the manuscript; and to Philippe Ringler for beautiful STEM micrographs.

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~~STEM at Cambridge University: Reminiscences and Reflections from the 1950s and 1960s~~

~~K. C. A. Smith~~

Contents 1. STEM as a Diagnostic Tool for SEM 387 2. The Observation of Specimens in Water Vapor by Means of STEM 393 3. High-Voltage STEM Using a Single-Field Condenser—Objective Lens 400 References 405

~~1. STEM AS A DIAGNOSTIC TOOL FOR SEM~~

When I joined Charles Oatley’s group in the Engineering Department as a research student in the autumn of 1952, Dennis McMullan had recently submitted his Ph.D. dissertation. I was thus able to read about the construction of the first operational scanning electron microscope (SEM) since the pioneering work of von Ardenne and Zworykin in the early years of World War II. Dennis describes operating his microscope in the scanning transmission electron microscope (STEM) mode to measure scanning spot size. A standard specimen grid, as used in the TEM, was mounted in the specimen stage of the SEM and the transmitted electrons were detected by an electron multiplier placed below the stage. The video signal at the output of the electron multiplier was displayed on an oscilloscope. Scanning the beam

~~Fitzwilliam College, University of Cambridge, Cambridge CB3 0DG, United Kingdom~~

387 388 K. C. A. Smith across a grid bar produced a square-wave video signal. In chapter 6 of his dissertation (McMullan, 1952), Dennis explained the procedure as follows: (6.1.3) Measurement of Spot Size The electron beam of the microscope was now deflected at 375 cycles per second with the time base described in the last chapter and the time base of the oscilloscope was synchronised to this. The output waveform was observed as the magnification was increased and the sides of the square wave were found to become less and less vertical due to the finite diameter of the scanning spot. Focus could be observed by setting for maximum slope. A typical waveform recorded from the cathode-ray tube is shown in Figure 1. The diameter of the spot is given by the horizontal projection of the sloping part and in this case it is about 500 A.U. with a beam current of À 10 13 A. The calibration had previously been carried out using a 12.5 micron diameter aperture which had been checked optically. The multiple traces in Figure 1 were partly caused by stray mains frequency magnetic fields deflecting the microscope scanning beam. As noted in the previous chapter the magnetic screening surrounding the final lens has only one thickness of mumetal, which is inadequate. Shot noise in the scanning beam is also plainly visible at the top of the pulse. It is also possible by this method to measure the effective diameter of the electron source in the electron gun. The first lens is switched off and the spot size measured. It was found to be 0.2 microns, and from the focal length of the final lens and the distance between lens and source the diameter of the electron source was calculated to be 50 microns.

~~0.1 m~~

~~FIGURE 1 A typical waveform recorded from the cathode-ray tube. STEM at Cambridge University 389~~

Using the same experimental setup, Dennis produced transmission images of latex particles. (6.1.7) Some Results with Transparent Specimens A number of photographs were taken of polystyrene latex particles on a nitrocellulose film and shadowed with gold and iridium. Two micrographs are shown in Figures 2 and 3. The camera used was somewhat inferior to that used later in the work and the scanning lines are hardly visible. The photographs were taken on Ilford H.P.3. 35 mm film using the afterglow of the 5FP7 cathode-ray tube screen. The accelerating voltage was 25 kV and the scanning À beam current about 10 12 A. (6.1.8) Discussion of Results The results with the latex specimens are definitely inferior to these obtained with the instrument operated as a conventional transmission microscope, the main cause being the poor magnetic shielding. Dark field operation was attempted and some pictures observed, but because of lack of time little work was done with it and no noteworthy results have as yet been obtained. Operation with a really thick specimen was tried. A specimen grid was covered with aluminium foil 2.5 m thick. A sharp image of the grid could be obtained when the grid was on the lens side of the foil, but as was expected only a blurred outline was observable with the grid on the other side. The accelerating voltage was 25 kV. This shows that it is possible with the scanning microscope to detect detail in the surfaces of thick specimens, which would be invisible in the conventional microscope, but it must be stressed that the contrast will be very low unless this detail absorbs electrons very strongly. Dennis recalls that these STEM images were obtained in the summer of 1950.1 They are probably the first such images generated since the Lichterfelde STEM of von Ardenne was destroyed in an air raid in 1944 (von Ardenne, 1985).2 Oatley had arranged for Dennis to continue for an additional 2 years, during which he designed and built a new column with better magnetic screening; the new column resulted in much cleaner edge sweep traces and rather better images of latex particles. I used the edge sweep test and latex particle images as diagnostic tools throughout my own research in the development of the SEM, as described in chapter 3 of my dissertation (Smith, 1956):

1 Shortly after Dennis produced these images—less than 2 years after starting work on the microscope—the gain of the electron multiplier proved to be inadequate; it took another 6 months to obtain a satisfactory replacement. He submitted a paper describing his work on the SEM for publication in December 1951, but it was not until June 1953 that this finally appeared in print (McMullan, 1953). 2 The Editor has pointed out that the ’’forgotten’’ French microscope, built in about 1945 at the Ecole Polytechnique, also produced STEM images. (Hawkes and McMullan, 2004). 390 K. C. A. Smith

~~2.5 m~~

~~1.5 m~~

~~FIGURES 2 AND 3Polystyrene latex particles on a nitrocellulose film shadowed with gold and iridium. STEM at Cambridge University 391~~

3.10.3 Measurement of Spot Size The method used for measuring the diameter of the scanning spot is a refinement of McMullan’s. method. To display the waveform McMullan used an oscilloscope, the time basis of which was triggered by the time-base pulses from the generators in the microscope. This method is not entirely satisfactory for accurate measurements, as the current through the scanning and display coils is not linear and, consequently, there will be some non- linearity between the deflection of the scanning spot and the scan on the oscilloscope. In order to avoid this difficulty an old monitor unit was con- verted to magnetic deflection in the x-direction and its coils connected in series with those in the microscope. The magnification on the monitor tube was conveniently made the same as that on the display tube by equalising the scanning amplitudes on the two tubes. For accurate measurement, the opaque edge which is presented to the spot must be microscopically sharp and straight; the edges of standard specimen grid bars were found to be too irregular. A suitable edge was obtained by covering a grid with a Formvar film and then dipping this grid part way into chloroform. It was intended in this way to produce a single straight edge, but instead the film was found to split up into several sections producing a number of suitable edges. To render it opaque the film was heavily coated with gold-palladium by evaporation. The appearance of the waveform displayed on the monitor tube is shown in Figure 4. Shot noise broadens the trace and reduces the accuracy of the measurement. The mean intensity level may be traced either directly from the tube or drawn on to a photograph of the waveform as shown. Figure 4 was produced with a relatively short exposure time and is representative of the appearance of the waveform as displayed on the tube. Longer exposure times give an integrating effect and produce an increased definition of the mean

~~d/ 2~~

~~I 0.63I~~

~~FIGURE 4 Appearance of the waveform displayed on the monitor tube. 392 K. C. A. Smith~~

intensity. For the present spot sizes, the simpler method of tracing directly from the tube was found to give almost identical results. The diameter of the scanning spot has been defined by the points at which the intensity falls to one fifth of the axial intensity, this diameter being represented in Figure 4 by the points which include 93% of the total current in the spot. In practice, because of the small slope of the curve at these points, it is more accurate to measure between the half-diameter points which include 63% of the current. The accuracy of the measurement depends mainly upon the accuracy with which the magnification is known, estimated to be 6% (section 5.6.), and the accuracy with which the measurements on the curve can be made. This latter is estimated to be 10% at present spot size so that the most probable error is about 12%. In 1955 I rebuilt the lower section of the column, incorporating a stigmator, a new final lens, new scan-coil assembly, and new specimen chamber, and made various other improvements. The following micrograph (Figure 5) is representative of the performance of the microscope at this stage of its development. In conclusion, some observations will be mentioned, which were made while carrying out various tests on the microscope. It was noticed that when operating in transmission, the rate of contamination appeared to be particularly high and was sufficient, during the recording of a micrograph

~~0.5~~

FIGURE 5 Micrograph representative of the performance of the SEM in 1955. Latex particles, 0.25 m in diameter on a collodion film shadowed with gold-palladium. STEM at Cambridge University 393 over a 4-minute period, to render the field useless for taking subsequent micrographs. This prevented the use of a through-focal series of the same field to check instrumental performance. The relatively high contamination rate observed when operating in transmission is probably due to the low accelerating voltage used, which results in a greater amount of energy being dissipated in the specimen. This causes the contamination to be more noticeable because of the poor penetrating power of the beam. In this connection it is worth noting that a Formvar supporting film, quite suitable at the voltages normally used for transmission electron microscopy, is useless for the scanning microscope working at 15 kV because of its relatively poor electron transparency.

~~2. THE OBSERVATION OF SPECIMENS IN WATER VAPOR BY MEANS OF STEM~~

In 1955, toward the end of my allotted time as a research student, I commenced what proved to be one of the most difficult experiments I had so far undertaken: the examination of specimens at water vapor pressure, which entailed using the microscope in the transmission mode. I cannot recall whether this was at Oatley’s suggestion or whether I took it upon myself, but certainly Oatley was extremely enthusiastic and followed the work closely. The attempts by various workers to examine biological specimens in the conventional electron microscope by enclosing the specimen in a double-walled cell containing a liquid or air had been followed in the literature, and I suppose it was these reports that prompted us to proceed with the experiments. My first thought was to construct a double-walled liquid cell of the type used by previous workers, despite the difficulties of alignment of the input and output apertures, and the fabrication of a thin liquid film sandwiched between two membranes. But Oatley suggested that it might be more profitable to examine specimens in an atmosphere of water vapor. He also made a further crucial suggestion: the use of a plastic scintillator coupled to a photomultiplier as the detector for transmitted electrons. This transformed the situation, as will be evident from the following description of the work drawn from my dissertation. 9.5 Observation of Specimens in Water Vapour 9.5.1 Introduction One of the outstanding problems in electron microscopy is the observation of biological specimens in a live instead of a dead and desiccated condition (Cosslett, 1951). Attempts have been made to observe, by means of a double-walled cell, specimens immersed in a liquid (Abrams and McBain, 1944) and in an air layer (Sugata, 1954). These attempts have so far been unsuccessful because of 394 K. C. A. Smith

the poor penetration of electrons at normal microscope voltages. Experimen- tal microscopes designed to operate at 500 kV and higher have been constructed to improve the penetration in thick specimens (Coupland, 1954) and these have achieved a limited success, but the application of high voltages to double-walled cells has not been attempted so far. Another difficulty with the liquid cell is to obtain sufficient contrast, as the density of most biological specimens do not differ greatly from the liquid medium in which they are immersed. Even if high voltages are successful in overcoming penetration and scattering problems, and sufficient contrast can be obtained in such cells, it is probable that, although the heating effect is reduced, the ionising effect of a high-voltage beam in a liquid cell would still prove to be lethal to living specimens. Moderate pressures of air and water vapour are sufficient to prevent complete desiccation of the specimen and the difficulties of obtaining adequate penetration and contrast are greatly reduced. In the scanning microscope large energy losses may be tolerated after the electrons have passed through the specimen and the thickness of the cell therefore is not critical. Furthermore, by using a scintillator and photomultiplier as the detector, the necessity of passing the electrons through a second window is avoided. Abrams and McBain used a collodion film to cover the electron window in their cell, and Sugata has found that a 1000 A.U. thick collodion film over a hole 100 microns in diameter will withstand atmospheric pressure. However, as Oatley has pointed out, if the cell is at water vapour pressure (w.v.p 1.3 cm Hg at room temperature) a window of about 50 microns diameter does not require any covering film in a system using a high-speed diffusion pump. Following Oatley’s suggestion I calculated from kinetic theory, assuming a typical pumping speed of 20 L/sec, and a window aperture of 50-m diameter, that the pressure in the microscope specimen chamber would À be about 2 Â 10 4 mm Hg, a reasonable working vacuum. Thus a film is necessary only to support the specimen. 9.5.2 The Water-Vapour Cell Figure 6 shows a cross-section of the cell. The window is a 50 micron standard copper aperture disk which is sealed to the removable top plate of the cell with Tensol cement. The scintillator passes through an O-ring seal so that the distance between the specimen and the face of the cylinder may be adjusted. At w.v.p. the mean free path of an electron is 14 microns, but electrons will lose only a few volts of energy with each ionising collision and the range of a 15 keV electron will therefore be appreciable. The results showed that both the energy and direction of the beam are substantially maintained up to a distance of 4 mm, the limit of the adjustment. Figure 7 shows schematically the arrangement of the apparatus. The cell is fixed in a special holder mounted on the specimen stage. 9.5.3 Experimental Procedure The specimen is mounted on the supporting film after the aperture has been sealed to the top plate. The cell is first pumped down to about 10 cm Hg. with taps A and B open and C closed. C is then opened and is B closed so that the STEM at Cambridge University 395

~~" 3/4 D Aperture~~

~~" 3/8~~

~~Scintillator~~

~~" 1/4 D~~

FIGURE 6 Cross-section of the water-vapour cell. cell slowly attains w.v.p. This procedure speeds up the pumping process and still avoids desiccation of the specimen. When required, the cell may be pumped to high vacuum by closing C and opening B. Use of the cell was found to involve the following experimental difficulties: obtaining a suitable distribution of the specimen over the 50 micron window; splitting of the film during observation, and charging of the scintillator. The effect of this latter was to cause the response of the scintillator to fall as the intensity of bombardment was increased. Thus at low magnifications, with the beam scanning over a large area, the effect was not noticeable, but at sufficiently high magnifications with the beam concentrated over a small area, the charging effect was sufficient to cause complete blackout. Any scattering which had the effect of spreading the beam out, reduced the charging and it was found that with the cell at w.v.p. the effect was less pronounced. However, this improvement only occurred on visual operation; when taking a micrograph, the slower scan caused the charging effect to become troublesome again. Scattering in the specimen also reduced charging of the scintillator and the net result was to cause a semi-reversal of contrast in the picture. Some separate experiments were carried out, with a simplified arrangement, and it was found that coating the scintillator with a thin evaporated silver film and placing it 2 cm below the specimen was sufficient to eliminate charging. Because this was the last project attempted by the author, lack of time prevented the application of this remedy to the cell. 9.5.4 Results Observation at water vapour pressure was first successfully demonstrated using latex particles and polyhedra specimens. The resolution obtained was somewhat inferior to that obtained in normal transmission operation, in which the specimen is placed on top of the film, because of scattering in the film. An attempt was then made to find a specimen which would demonstrate the effects of desiccation. Blood cells were first tried but were found to shrivel in air before they could even be mounted. Bacteria were also tried but it was found impossible to get a suitable distribution over the aperture. They 396 K. C. A. Smith

~~Vacuum~~

~~Flexible tubing Cell~~

~~A Photo- multiplier Specimen B stage movement~~

~~C Head amp.~~

~~Water Mercury reservoir manometer~~

~~FIGURE 7 Schematic arrangement of water-vapour cell experiment.~~

were found to clump together and, as they were used directly from the culture, contained much spurious material. Better results would probably have been obtained if the preparation had been first centrifuged. The best results have been obtained with the fungus spores, Aspergillus nidulans. Figure 8a shows a spore at w.v.p. The semi-reversal of contrast can be seen clearly on the edges of the specimen and on the long sterigmata which branch out from the spore body. Figure 8b shows the spore after about 10 minutes at high vacuum. The appearance has changed slightly but a split developing in the collodion film has caused a small movement which would account for this. There is no evidence of shrinkage. Figure 8c is the same spore taken by means of the usual transmission arrangement with the electron multiplier. The split has caused the spore to move considerably but no shrinkage can be detected. Figure 9 shows another spore on the same film (taken from the same fungus culture), which has a much smoother appearance and is not covered with warts. It is thought that this is a less mature form of the spore than that shown in Figure 8. Figure 9a shows the spore first at high vacuum and Figure 9b after re-admission of water vapour for about 10 minutes. The spore has suffered no change in size, although some change in shape is apparent, which may be accounted for by movement during the exposure. However, Figure 9c, which was taken with the usual transmission arrangement at the same time as Figure 8c, shows a marked reduction in size. This result was found also for another smooth spore on the same film. Thus for both the spores shown in the micrographs, there is no difference between high vacuum and water vapour pressure conditions for the short period of 10 minutes between exposures. But the micrographs of Figure 8c and Figure 9c were taken after the spores had been let down to air and then STEM at Cambridge University 397

~~(a)~~

~~(b)~~

~~(c)~~

FIGURE 8 Fungus spore, Aspergillus nidulans. (a) A spore at w.v.p. (b) The spore after about 10 minutes at high vacuum. (c) The same spore taken by means of the usual transmission arrangement with the electron multiplier. The split has caused the spore to move considerably but no shrinkage can be detected. 398 K. C. A. Smith

~~(a)~~

~~(b)~~

~~(c)~~

FIGURE 9 Another spore on the same film as in Figure 8. (a) The spore at high vacuum and (b) after readmission of water vapour for about 10 minutes. The spore has no change in size, although some change in shape is apparent. (c) Image obtained with the usual transmission arrangement at the same time as Figure 8c shows a marked reduction in size. STEM at Cambridge University 399

returned to high vacuum again for an appreciable time. In this case the rough spore showed no shrinkage and the smooth spore a shrinkage of 20%. 9.5.5 Conclusions Although it has not yet been possible to demonstrate differences between water vapour and high vacuum conditions, it is to be expected that a more complete investigation will produce some interesting results. The operation of the cell at water vapour pressure has been proved; indeed, it was found possible to work at a total pressure of about 4 cm Hg before the vacuum in the microscope became inadequate. The present diffusion pump is very slow and with a higher pumping speed it should be possible to work at a still higher pressure. Alternatively, for the same pressure, a larger area of window could be used which would reduce the difficulties of mounting the specimen. The poor quality of the micrographs obtained so far is due firstly to charge effects in the scintillator (which could be eliminated by providing a longer path through the vapour to spread the beam, and by coating the scintillator with silver), and secondly, to the low accelerating voltage (15 kV). Normal transmission voltages of 50–100 kV would bring about an improvement in penetration and resolution, and in addition, would permit a thicker film to be used, which in turn would allow higher working pressures and/or an increase in window area. It was unfortunate that these experiments had to be conducted in such a hurried fashion and terminated abruptly owing to lack of time. The fact that it was unnecessary to cover the input aperture of the cell pointed to the next obvious step, which would have been to mount the specimen on a standard electron microscope grid placed a millimeter or so below the aperture, well within the water vapor atmosphere. Apertures placed in front of the scintillator would have allowed light- and dark-field operation to be investigated and opened up the possibility of a range of experiments. But this was not to be; the microscope had to be handed over to the next research student, Tom Everhart, who had wisely decided to study ‘‘voltage contrast’’ modes and semiconductor applications of the SEM. Fortunately, he persevered with the plastic scintillator-photomultiplier detector, which led him to the development of what has become known as the Everhart–Thornley detector. Although the water vapor cell experiments were terminated inconclu- sively, and consequently were never reported in the literature, looking back on the work after the space of over half a century, I regard it in a rather more positive light. We had, in fact, begun to look into some important aspects of what has become the environmental SEM: differential pumping, ionization of gas and vapor in the detector, and its response to varying pressure. Who knows where the work would have led had it been continued? 400 K. C. A. Smith

~~3. HIGH-VOLTAGE STEM USING A SINGLE-FIELD CONDENSER—OBJECTIVE LENS~~

In 1960 I was invited by Ellis Cosslett to join his electron microscope group at the Cavendish Laboratory in Cambridge.3 Cosslett left everyone in the group very much to their own devices, and in the absence of any clear directive I decided to follow Bill Nixon’s work on the point projection X-ray microscope. However, toward the end of my first year in the group, Cosslett announced that he had obtained a large grant from the Paul Instrument Fund of the Royal Society to build a high-voltage microscope, and I learned that he would be appointing an assistant to work on the project. Having spent all my time in the field working on SEM, my knowledge of conventional transmission microscopy was fairly minimal and, initially, he did not offer me a position on the project, nevertheless I decided to draw up a tentative design, which I submitted to him, with the result that I got the job. In 1962 or early ’63, after I had been working on the high-voltage project for about a year, as I was shuffling through the great pile of reprints and articles on electron microscopy that every week came flood- ing into Cosslett’s office, I came across a paper by Riecke (1962) describing the single-field condenser-objective (SFCO) lens for transmission microscopy. It immediately struck me that here was a way of combining scanning transmission microscopy and conventional microscopy within the same lens, using the prefield to produce a fine electron spot. At the time, the Cavendish microscope was still in the design stage, and consequently, this germ of an idea had to be relegated to the future. In fact, it was not until some time, I think in 1966, that I mentioned the idea at a local meeting of microscopists in Cambridge (of which no record exists), and it was not until 1968 that a formal presentation of the SFCO scanning mode was made at a meeting of the Institute of Physics, Electron Microscopy and Analysis Group, held at Churchill College, Cambridge. The paper was presented jointly with Kevin Considine, who had been my assistant during the construction of the Cavendish microscope, but who was now studying for a Ph.D., investigating the Ichinokawa electron energy analyzer. The abstract of this paper reads as follows: Scanning Transmission Microscopy at High Voltages K.C.A. Smith (Department of Engineering, University of Cambridge) K. Considine (Cavendish Laboratory, Surface Physics Department, University of Cambridge)

~~3 I filled the slot left vacant by Bill Nixon’s move to the Engineering Department. STEM at Cambridge University 401~~

The Cavendish High Voltage Microscope has been operated in a scanning mode at voltages up to 500 kV using the objective lens as a single-field condenser-objective with the specimen placed approximately in the mid- plane of the lens, With this arrangement the illuminating spot may be reduced to the order of 100 A.U. Deflection coils placed above the objective lens allow the specimen to be scanned, and the transmitted beam is detected by a scintillator-photo multiplier combination. In principle there is no restriction to the solid angle and energy range over which the transmitted electrons may be collected, thus the method should allow the examination of thicker specimens. This advantage, however, is offset by the decrease in contrast which results when the objective aperture is removed. Preliminary studies are being carried out comparing micrographs obtained by the scanning and normal modes of observation on the same area of the specimen, both in light and dark field. This abstract had been submitted to the organizers of the meeting at least a couple of months before the event, and at that time no results had yet been obtained with the method. But on June 15, according to an entry in my lab book, we managed to assemble all the requisite electronics (mainly borrowed from the Engineering Department) and on June 25 a series of pictures of a thick aluminum specimen, in both normal and scanning modes, was obtained (the 500 kV mentioned in the abstract was overoptimistic). The paper was presented July 10, 1968—a close call! (Alas, over the intervening years, all traces of negatives and prints obtained in this experiment have vanished.) A little later that same year, Kevin presented an almost identical paper at the Rome Conference (Smith and Considine, 1968). It was about this time that I learned that the electron microscope division of the Philips Laboratories at Eindhoven had been working on the same idea and had, indeed, decided to incorporate an SFCO scanning facility in their latest TEM (Ong, 1968). It was pleasing to have the validity of my idea confirmed, but disappointing to learn that others were so far in advance. As will be evident from the above abstract, I had by this time moved from the Cavendish back to the Engineering Department. My commit- ments there ruled out any further substantial involvement with the Cavendish microscope. Kevin’s time on the microscope was severely limited, and he had to fit his analyzer work in without interfering with the needs of many other users. We did one or two more runs of the apparatus looking at a biological specimen, but then decided reluctantly that no further work on the STEM project was possible. Toward the end of 1968, Kevin left the Cavendish to take up a post with Tektronix in the United States. From there he wrote his Ph.D. dissertation and submitted in 1969. His dissertation contains an appendix describing the SFCO scanning mode of operation, which is the only coherent account of our work. 402 K. C. A. Smith

High Voltage Scanning Transmission Microscopy (Extract from Ph.D. dissertation, K. T. Considine, 1969) A.1 Introduction In the high voltage microscope, the major interest, so far, has been in the examination of thick specimens. Cosslett (1967) has shown that the maximum usable specimen thickness is limited ultimately by image visibility: as the specimen thickness is increased a greater proportion of the electron beam is scattered outside the objective aperture until a point is reached when the detail in the image falls below the threshold of visibility. The objective aperture can only be increased at the expense of the image resolution which, for thick specimens, will be determined by the chromatic aberration of the objective lens. In scanning transmission electron microscopy, there is, in principle, no restriction on the solid angle over which the scattered beam may be collected.

~~Source~~

~~Accelerator~~

~~Condenser 1~~

~~Condenser 2 Scan Field limiting coils aperture S.F.C.O. Specimen plane objective~~

~~Scintillator Photo- multiplier~~

~~FIGURE 10 Arrangement of the Cavendish microscope for operation in the 300-kV scanning mode. STEM at Cambridge University 403~~

It might be expected, therefore, that this method would allow the examination of thicker specimens. There are certain disadvantages in the scanning method (e.g., small field of view, lack of contrast etc.) but the advantages include reduced specimen bombardment, single line scanning across the image to obtain direct intensity contours, and (of more relevance to the work described previously) the possibility of combining the technique with energy analysis to produce energy-filtered images. If the scanning technique can be incorporated in a conventional transmission microscope, then the advantages of both methods are available. A.2 Scanning Operation of the Cavendish Microscope The Cavendish microscope has been operated in the scanning mode at 300 kV, using the arrangement shown in Figure 10. The condenser lenses are employed to focus a fine electron probe onto the specimen; the first condenser is operated at high excitation to obtain as small a probe size as possible. The specimen is placed at the centre of the objective lens which is operated in the single-field-condenser-objective mode (Ruska, 1964, 1965; Riecke and Ruska, 1966). This allows the probe size to be further reduced by the focusing action of the first half of the magnetic field in the objective lens (see Figure 10). The objective lens has not been modified for this mode of operation and this has limited the maximum accelerating voltage that can be employed to approximately 300 kV.

~~5 m~~

~~Bright field~~

~~5 m~~

~~Dark field~~

~~FIGURE 11 Bright-field image (top) and dark-field image of a specimen of fish muscle fiber. 404 K. C. A. Smith~~

The probe is scanned on the specimen, in a raster, by means of coils placed above the field limiting aperture. The scattered electrons are detected by a scintillator-photomultiplier combination which, for convenience, is placed in the position normally occupied by the selected-area diffraction aperture. The scintillator can be withdrawn from the beam to allow conventional imaging. Thus, it is possible to examine the same area of the specimen by both methods. In the preliminary work so far carried out, the objective aperture has been employed to provide contrast in the scanning image. The geometry of the arrangement is such that dark field scanning images can be obtained by displacing the objective aperture sideways and collecting only the scattered electrons. One of the advantages in having both scanning and conventional microscopy incorporated in the one instrument is that one can form dark field scanning images of crystalline specimens using only selected diffracted beams. Conventional microscopy is employed to produce the diffraction pattern from the specimen and to place the objective aperture so that only a chosen diffracted beam passes through it. The ‘‘selected’’ dark field scanning image can then be obtained. Scanning images (bright and dark field) have been obtained of both metal and biological specimens. Good contrast has been obtained using large objective apertures. Figure 11 shows a bright and a dark field image of a specimen of fish muscle fibre. The objective aperture used for these images was 400 microns. The conventional transmission images of this specimen were particularly lacking in contrast and the visibility was poor. The specimen thickness was not known accurately but was believed to be of the order of one micron. The recording times for these images were of the order of 10 seconds. The image resolution (i.e., the probe size) is typically 1000 A.U. or less but no serious attempt has yet been made to improve on this figure. It is expected that a probe size of the order of 100 A.U. should be attainable, containing sufficient current to produce scanning images of good signal-to-noise ratio. One method of reducing the probe size is to operate with the second condenser lens off and the first condenser lens at high excitation. This would reduce the beam current available but operation at high voltages eases the problems of signal detection considerably. A.3 Application to Energy Analysis The ability to form a scanning image of the specimen, as described above, allows the elemental area of the specimen, chosen for analysis, to be selected accurately (cf. section 7.3.1). The scanning image is examined and the probe ‘‘stopped’’ over the image point selected for analysis. The detector is then withdrawn from the beam which is allowed to pass through to the analyser (suitable deflection coils, located below the specimen, are required to direct the beam into the analyser assembly). For this operation the magnification of the microscope is set so that the image of the probe is equal to the size of the input aperture of the analyser. The energy analyser may be incorporated with the scanning mode of operation to provide an alternative means of contrast-formation in the image (i.e., energy-filtered imaging). The analyser (set for any particular energy-loss, see section 5.6.1), would be the detector of the signal. Additional STEM at Cambridge University 405

scan coils would be required below the specimen to compensate for the deflection of the beam as it is scanned across the specimen. The apparatus for investigating this mode of operation has recently been assembled but there has been insufficient time available for any results to be obtained. A.4 Summary The preliminary investigation into the application of scanning techniques to the high voltage microscope, though only qualitative, has produced encouraging results.The method appears to be worth developing and this should include modification of the objective lens for SFCO operation at high voltages.

Kevin’s departure for the United States and my own heavy commit- ments in the Engineering Department brought to an end our experiments with STEM. At Tektronix, Kevin rose rapidly to become director of research (later a vice president of the corporation). In that capacity, and with his scanning background, he seriously considered the possibility of persuading the company to manufacture either a SEM or possibly a STEM; he proposed to use a new type of field emission gun and took out a patent. However, the company eventually decided against the idea. Perhaps this was just as well in view of the difficulties experienced by several established manufacturers of transmission electron microscopes who ventured into scanning (Agar, 1996). In 1988 Kevin left Tektronix to set up a consulting firm, Cambridge Consultants. Sadly, he died prema- turely from a heart condition in 1991 at the age of 53. Postscript In about 1976, Alex Strojnik came on sabbatical leave from Arizona State University to work in Cosslett’s group. At the suggestion of Cosslett, he decided to take up the challenge of improving the performance of the microscope as a STEM instrument. Working with the assistance of Tim Sparrow, he made numerous modifications and improvements with the result that, in 1977, a paper was published in the Journal of Physics (Strojnik and Sparrow, 1977), the abstract of which is given below. After 15 years, my germ of an idea had been realized.

~~REFERENCES~~

Abrams, I. M., and McBain, J. W. (1944). A closed cell for electron microscopy. J. Appl. Phys. 15, 607–609. Agar, A. W. (1996). The story of the European commercial electron microscopes. Adv. Imaging Electron Phys. 96, 415–584. Considine, K. T. (1969). An energy analyser for high-voltage electron microscopy. PhD dissertation, University of Cambridge. Cosslett, V. E. (1951). Practical Electron Microscopy. London: Butterworths Scientific Publications. 406 K. C. A. Smith

Cosslett, V. E. (1967). Electron penetration in high voltage electron microscopy. Optik 25, 383–388. Coupland, J. H. (1954). A new experimental high-voltage electron microscope. Proc. Third Int. Conf. on Electron Microscopy, pp. 159–161. Royal Microscopical Society, London. Hawkes, P. W., and McMullan, D. (2004). A forgotten French scanning electron microscope and a forgotten text on electron optics. Proc. Roy. Microscopical Soc. 39(4), 285–290. McMullan, D. (1952). Investigations relating to the design of electron microscopes. PhD dissertation, University of Cambridge. McMullan, D. (1953). An improved scanning electron microscope for opaque specimens. Proc. IEE 100(Pt II), 245–259. Ong, P. S. (1968). A combined conventional and scanning electron microscope. Proc. 5th Int. Conf. of X-ray Optics and Microanalysis, Tu¨bingen, pp. 84–85. Springer, Berlin. Riecke, W. D. (1962). Ein Kondensorsystem fur Eine Starke Objektivlinse. pp. KK-4–KK-5. Proc. Fifth International Congress for Electron Microscopy, Philadelphia. Riecke, W. D., and Ruska, E. (1966). A 100 kV transmission electron microscope with single- field condenser objective. Proc. 6th Int. Cong. Electron Microsc. pp. 19–20, Kyoto. Ruska, E. (1964). Travaux re´cents destine´sa` ame´liorer le pouvoir se´parateur expe´rimental du microscope e´lectronique. J. Microscopie 3, 357–372. Ruska, E. (1965). U¨ ber die Auflo¨sungsgrenzen des Durchstrahlungs-Elektronenmikroskops. Optik 22, 319–348. Smith, K. C. A. (1956). The scanning electron microscope and its fields of application. PhD dissertation, University of Cambridge. Smith, K. C. A., and Considine, K. T. (1968). Scanning transmission microscopy at high voltages. Proc. 4th Eur, Reg. Conf. Electron Microsc., Rome, 1968, p. 73. Strojnik, A., and Sparrow, T. G. (1977). An improved scanning system for a high-voltage electron microscope. J. Phys. E: Sci. Instrum. 10, 502–504. Sugata, E., Nishitana, Y., Kaneda, S., Tatcishi, M., and Yokoya, H. (1954). Fundamental researches for observing specimens in gas layers. Proc. Third Int. Conf. on Electron Micros- copy, pp. 452–459. Royal Microscopical Society, London. von Ardenne, M. (1985). On the history of scanning electron microscopy, of the electron microprobe, and of early contributions to transmission electron microscopy. In ‘‘The Beginnings of Electron Microscopy,’’ (P. W. Hawkes ed.), Academic Press, Orlando. Advances in Electronics and Electron Physics Supplement 16, 1–21. Contents of Volumes 151–158

~~VOLUME 1511~~

C. Bontus and T. Ko¨hler, Reconstruction algorithms for computed tomography L. Busin, N. Vandenbroucke, and L. Macaire, Color spaces and image segmentation G. R. Easley and F. Colonna, Generalized discrete Radon transforms and applications to image processing T. Radlicˇka, Lie agebraic methods in charged particle optics V. Randle, Recent developments in electron backscatter diffraction

~~VOLUME 152~~

N. S. T. Hirata, Stack filters: from definition to design algorithms S. A. Khan, The Foldy–Wouthuysen transformation technique in optics S. Morfu, P. Marquie´, B. Nofie´le´, and D. Ginhac, Nonlinear systems for image processing T. Nitta, Complex-valued neural network and complex-valued backpropaga- tion learning algorithm J. Bobin, J.-L. Starck, Y. Moudden, and M. J. Fadili, Blind source separation: the sparsity revoloution R. L. Withers, ‘‘Disorder’’: structured diffuse scattering and local crystal chemistry

~~VOLUME 153~~

Aberration-corrected Electron Microscopy H. Rose, History of direct aberration correction M. Haider, H. Mu¨ller, and S. Uhlemann, Present and future hexapole aberration correctors for high-resolution electron microscopy

~~1 Lists of the contents of volumes 100–149 are to be found in volume 150; the entire series can be searched on ScienceDirect.com~~

~~407 408 Contents of Volumes 151–158~~

O. L. Krivanek, N. Dellby, R. J. Kyse, M. F. Murfitt, C. S. Own, and Z. S. Szilagyi, Advances in aberration-corrected scanning transmission electron microscopy and electron energy-loss spectroscopy P. E. Batson, First results using the Nion third-order scanning transmission electron microscope corrector A. L. Bleloch, Scanning transmission electron microscopy and electron energy loss spectroscopy: mapping materials atom by atom F. Houdellier, M. Hy¨tch, F. Hu¨e, and E. Snoeck, Aberration correction with the SACTEM-Toulouse: from imaging to diffraction B. Kabius and H. Rose, Novel aberration correction concepts A. I. Kirkland, P. D. Nellist, L.-Y. Chang, and S. J. Haigh, Aberration-corrected imaging in conventional transmission electron microscopy and scanning transmission electron microscopy S. J. Pennycook, M. F. Chisholm, A. R. Lupini, M. Varela, K. van Benthem, A. Y. Borisevich, M. P. Oxley, W. Luo, and S. T. Pantelides, Materials applications of aberration-corrected scanning transmission electron microscopy N. Tanaka, Spherical aberration-corrected transmission electron microscopy for nanomaterials K. Urban, L. Houben, C.-L. Jia, M. Lentzen, S.-B. Mi, A. Thust, and K. Tillmann, Atomic-resolution aberration-corrected transmission electron microscopy Y. Zhu and J. Wall, Aberration-corrected electron microscopes at Brookhaven National Laboratory

~~VOLUME 154~~

~~H. F. Harmuth and B. Meffert, Dirac’s difference equation and the physics of finite differences~~

~~VOLUME 155~~

~~D. Greenfield and M. Monastyrskiy, Selected problems of computational charged particle optics~~

~~VOLUME 156~~

V. Argyriou and M. Petrou, Photometric stereo: an overview F. Brackx, N. de Schepper, and F. Sommen, The Fourier transform in Clifford analysis N. de Jonge, Carbon nanotube electron sources for electron microscopes E. Recami and M. Zamboni-Rached, Localized waves: a review Contents of Volumes 151–158 409

~~VOLUME 157~~

~~M. I. Yavor, Optics of charged particle analyzers~~

~~VOLUME 158~~

P. Dombi, Surface plasmon-enhanced photoemission and electron acceleration with ultrashort laser pulses B. J. Ford, Did physics matter to the pioneers of microscopy? J. Gilles, Image decomposition: Theory, numerical schemes, and performance evaluation S. Svensson, The reverse fuzzy distance transform and its use when studying the shape of macromolecules from cryo-electron tomographic data M. van Droogenbroeck, Anchors of morphological operators and algebraic openings D. Yang, S. Kumar, and H. Wang, Temporal filtering technique using time lenses for optical transmission systems Index

A T4 tail fibers, 111 heavy metal cluster labeling Aberration-corrected CFE-STEM monofunctional undecagold atomic-level characterization instrument, preparation, 107–108 170–171 nanogold clusters, 108 HD-2700C uranium single atom imaging, 106 (Ca CoO ) (CoO ), EELS column- 2 3 0.62 2 instrument design parameter by-column mapping, 178, 180–181 annular detectors, 104 EELS stability, 176–177 computer systems, 105–106 Pd/Pt core shell catalyst, EELS ion-pumped freeze dryer, 104–105 spectrum mapping, 178–179 soundproof room location, 103 single uranium atom, 177–178 STEM optics, 102 high-angle ADF-STEM image, M/L amyloid fibril measurement, 115 information transfer, 173–174 low-kilovolt capability, 174–176 C theoretical consideration of advantages, 172–173 Castaing–Henry filter, 328, 340 AEI STEM-1 Chromatic aberrations advantage, 191 coefficient of, 23 cold field emission gun, 189 electron probe size, 6, 11 double focusing magnetic sector, 190 mirror corrector, 26 source size, 189 Cold-field electron (CFE) cathode 200-kV Analytical CFE-TEM, HF-2000 applications of, 64 acceleration voltage, 152 column optics Aharonov-Bohm effect, 151 aberration contribution, 85 Argonne accelerator, 8 beam size vs. current, 84 chromatic and spherical aberration B coefficients, 83 column magnification, 82, 86 Beryllium cartridges, 269 long-term stability test, 93 Brookhaven National Laboratory (BNL) source parameters, 83 early user/collaborator projects working distance and aberrations, 83 chaperone and heat shock protein, 114 current stability DNAÀprotein interaction, 110 adsorbate-induced noise, 88 fibrinogen image study, 109 equilibrium coverage, 94 filamentous virus projects, 114 Faraday cup, 90 invertebrate protein complex, 114 gas flux, 89 microtubule motor complex high-frequency fluctuations, 87–88 dynein, 112 life cycle regions, 91 M/L of intermediate filament, 110–111 normalized angular intensity vs. time, native and reconstituted nucleosome 89, 91 study, 108–109 work function fluctuations, 87 needle complexes, 113 end-of-life mechanisms prion protein filament analysis, 111 carbon contamination, 95–96 ribosome projects, 112 catastrophic vacuum arc, 94

~~411 412 Index~~

Cold-field electron (CFE) cathode (cont.) HB5, 242 field emission patterns, 97 single-electron counting, 243 surface tension-based radius-dulling tobacco mosaic virus, 244 model, 95 DNA labeling, 21–22 typical emitter shapes, 96 DNAÀprotein interaction study, 110 energy distribution, tungsten analytical vs. numerical calculation, E 70–71 Electron energy-loss spectroscopy (EELS), angular current density (I’), 73–74 288, 327 b factor, 68 atomic column imaging, 168 current density equation, 68–69 atomic resolution analysis, 148 emission characteristics, 77 boron nitride analytical results, 150 emission parameters, 74 column-by-column mapping, 178–181 energy-broadening Boersch effect, detectors, 301–302 76–78 elemental mapping, 169 FowlerÀNordheim (FN) equipped with STEM, 149 equation, 67 HB603 system, 303 FWHM vs. I’, 78 serial system, 305 FWHM(N) vs. T, 72–73 single uranium atom, 177–178 normalized retarding potential spectrum mapping, 178 current, 75 stability, 176–177 SEM photos, 77 Electron gun, 226, 233–234 Sommerfeld free-electron model, 67 Electron holography, 150–151 surface potential diagram, 69 epoch making instrument, 152 TED vs. I’, 76 field emission gun (FEG), 152 tunneling coefficient, 69 high-angle takeoff geometry, 154 key source characteristics, 64 recording media magnetization and source optics thickness, 151 emission parameters, 81 Electron optics, 27–28 mutual Coulomb interactions, 80 gun optics, 297 source reduced brightness, 79, 81 column optics, 298 work function ELMISKOP ST100F STEM emission pattern, tungsten advantages emitter, 65 bright-field imaging, interpretation, 198 and flicker noise values, 66 ease of operation, 200–201 Column optics low radiation damage, imaging, 198 aberration contribution, 85 optimum analysis possibilities, 199–200 beam size vs. current, 84 signal processing and electronic chromatic and spherical aberration contrast enhancement, 199 coefficients, 83 thick specimens, imaging conditions, 199 column magnification, 82, 86 construction long-term stability test, 93 CRT, 197 source parameters, 83 high-voltage and field emission working distance and system, 197 aberrations, 83 microscope column and ray path, Cryo-transfer system, 270–271 196–197 D objective lens, focusing control, 196–197 recording system, 197 Dark-field detector (DFD) scanning system, 196 biological specimens, 244 signal detection and processing design, 242 system, 197 DNA strands, 245 vacuum control, 196 Index 413

major optical components, 201, 203 system/pumping, 294–295 Energy analyzer Mk1 HD-2700 Cs-corrected STEM, 170 energy-loss spectrum, 250 High-excitation objective hysteresis, 249 lens, 266–268, 276 micrometer-driven slit mechanism, 249 High-voltage STEM project, Toulouse schematic diagram, 242, 249 building Energy analyzer Mk2 diagram proposal, funding, 329 aberration correction, 264 high-resolution holography outline drawing, 264–265 installation, 332 parallel EELS (PEELS) system, 265–266 MEBATH building, 332 solid angle of acceptance, 264 tank schematic, 334 yttrium-aluminum-garnet column (YAG), 264–265 accelerating tube, 342–346 Energy-broadening Boersch effect, 76–78 Castaing–Henry filter, 340 Everhart–Thornley detector, 245, 399 CFEG source, 336 F lenses, 346–348 schematic diagram, 337 Faraday cup, 90, 91 source image position, 340 Fermi–Dirac function, 68 spectrometers, 348–349 120-kV FE ultrahigh vacuum (UHV), spherical aberration, 341 nanotubes tetrode gun and 3D structural and elemental analysis, parameters, 338, 342 169–170 triode gun calculations, 341 image drifts, 166–168 electron energy-loss spectroscopy manganite (La1.2Sr1.8Mn2O7), 168 (EELS), 327 postspecimen lenses, 165 field emission experiments, 328 b-SiAlONphosphor, europium, 166–168 generator Field emission SEM, 19–20 dielectric gas under pressure, 334 Filamentous virus projects, 114 locked tanks, 335 FowlerÀNordheim (FN) equation, 67 nominal current and voltage, 333 top of accelerator, 335 G irradiation experiment, 326 1.6 MeV accelerating voltage, 326 Gold lattice fringes, 255–256 Graphite lattice fringes, 230 plastic deformation, BCC material, 327 H signal recording, 351–352 suspension of the platform and HB501 microscope, 349–350 development, 271–272 Hitachi cold-field emission scanning HB501UX and high-resolution transmission electron microscopes imaging, 273 (CFE-STEM) HB601, 278, 280 aberration-corrected HB603 300-KV stem instruments atomic-level characterization column electron optics, 298 instrument, 170–171 detectors, 301–302 HD-2700C, 176–181 gun electron optics, 297 high-angle ADF-STEM image, HT tank-gun and accelerator, 296 information transfer, 173–174 objective lens, analytical and high- low-kilovolt capability, 174–176 resolution pole pieces, 298–299 theoretical consideration of projector system, 301 advantages, 172–173 software architecture, 302–304 200-kV analytical CFE-TEM, HF-2000 specimen stage, 300–301 acceleration voltage, 152 414 Index

Hitachi cold-field emission scanning electron energy-loss spectrometer, transmission electron microscopes 147–149 (CFE-STEM) (cont.) schematic diagram, basic design, Aharonov-Bohm effect, 151 141–142 electron holography, 150–151 single-atom observation, 144–147 epoch making instrument, 152 specifications, 141 field emission gun (FEG), 152 high-angle takeoff geometry, 154 I recording media magnetization and Image forming systems thickness, 151 analytical microscopy, requirements, cutting-edge applications, HD series 215–217 diffraction, 161–163 imaging process, 204–205 energy-dispersive x-ray lens systems, CTEM and STEM, 210–214 systems, 161 magnetic lenses, image forming HD-2300 SEG and auto functions, properties 164–165 image rotation, 206 development ray trajectories, 206–207 HCRL, 128–130 schematic cross section, 205 Naka Works, 131–132 theoretical resolution limit, lens 300-kV FE-TEM, HF-3000, 152–153 system, 207 120-KV FE UHV, nanotubes weak lens focal length, 206 3D structural and elemental analysis, strong lenses, 208–210 169–170 Invertebrate hemoglobin work image drifts, 166–168 ‘bracelet’ protein, 34–35 manganite (La Sr Mn O ), 168 1.2 1.8 2 7 dissociation-reassociation process, 34 postspecimen lenses, 165 3D reconstruction, 34 b-SiAlONphosphor, europium, Lumbricus hemoglobin, 33–34 166–168 protein field emission gun development crystal structure and sequence emitter characteristics, 127–128 alignment, 37 metal work function, 127 subunit stoichiometry, 35 schematic diagram, 128 Ion-pumped freeze drying system, 104–105 field emission microscopes, trigger CFE gun (CFEG), 125–126 K nanometer-order analysis and imaging, 126 Klebsiella oxytoca, 375 field emission stability critical dimension (CD)-SEM, 135, 137 L emission current fluctuations, 134, 136 general behavior, field emission Lehigh design, 310 current, 132, 134 Lens aberration corrections, 23–27 gun B, experimental apparatus, 134, 136 aperture calculation, 9 off-axis aberrations, 7 improvements, 132 relative emission fluctuation, 134–135 spherical and chromatic work function, absorbing gas, 134 aberrations, 6, 23 third-order aberrations, 7, 10 first analytical model, 139–140 gate viewer, 154–161 zero aberration system, 11 Lumbricus hemoglobin, 33–34 HD-2700 Cs-corrected, 170 holography studies, 137–138 M multistage acceleration CFEG, 138–139 50kV STEM Mass measurements bright-field images, 142–143 biological samples, 371–373 Index 415

GroEL-GroES, 375 source size, 189 LcrV, Yersinia needle, 377 BNL (see Brookhaven National principle, 366, 368 Laboratory (BNL)) PulD and YscC secretins, 374–376 construction, 16–17 PulG(His)6 pseudopilus, 376–377 development and atomic images, 18–19 statistical error, 369–371 diagnostic tool, SEM stoichiometry, 375 contamination rate, 393 synaptic vesicles, 378–379 dark field operation, 389 T2 and T3 secretion systems, 375–376 microscope performance, 392 MEBATH project. See High-voltage STEM polystyrene latex particles, project, Toulouse nitrocellulose film, 389–390 Microdiffraction coils, 246–248 spot size measurement, 388, 391–392 MIDAS project DNA labeling, 21–22 gun lens, 289–290 ELMISKOP ST100F objective lens, 290–291 advantages, 198–201 performance, 291–292 construction, 195–197 side-entry stage, 291 major optical components, 201, 203 system, 289 micro area diffraction, 203 energy loss and radiation damage, N 20–21 field emission SEM, 19–20 Non-dispersive x-ray detector, 257–259 hemoglobin work O ‘bracelet’ protein, 34–35 dissociation-reassociation process, 34 Oak Ridge design 3D reconstruction, 34 detector system, 309 Lumbricus hemoglobin, 33–34 HB603U, 309 protein crystal structure and sequence objective lens pole pieces, 307 alignment, 37 optical transfer function, 308 protein subunit stoichiometry, 35 Si(110) image, 308 high-throughput visual proteomics Omega filter cryo-electron microscopy, 379 calculation, 331 cryo-electron tomography, 378, 380 installation, 331 hierarchical automated particle schematic diagram, 349 selection, 380 Q microfluidic circuits, 378 picoliter drop deposition, 379 Quadrupole-octupole correctors sample deposition devices, 378 applied to 30-kV STEM, 8 segmentation algorithms, 380 spherical aberration correction, 7 high voltage project, Toulouse vacuum flange mounting, 3 accelerating tube, 342–346 building diagram proposal, R funding, 329 electron energy-loss spectroscopy Ribosome projects, 112 (EELS), 327 S field emission experiments, 328 generator, 334–335 Scanning transmission electron microscope high-resolution holography (STEM) installation, 332 AEI STEM-1 irradiation experiment, 326 advantage, 191 lenses, 346–347 cold field emission gun, 189 MEBATH building, 332 double focusing magnetic sector, 190 1.6 MeV accelerating voltage, 326 416 Index

Scanning transmission electron microscope Siemens ST 100 F (STEM) (cont.) adenovirus image, 192–193 plastic deformation, BCC column and control panel, 192–193 material, 327 CT150, 194 signal recording, 351–352 single-field condenser-objective lens source, 336–342 Cavendish microscope, 401–404 spectrometer, 347–348 disadvantages and advantages, 402 suspension of the platform and energy analysis, 404–405 microscope, 349–350 objective aperture, 402 tank schematic, 334 scanning mode of operation, 400–401 image formation source development, 17–18 detectors, 360–361 specimen observation, water vapor electron-sample interactions, Aspergillus nidulans, 396–397 359–360 desiccation, 395 imaging modes, 363 double-walled liquid cell, 393 optical system, 360 Everhart–Thornley detector, 399 single-electron counting, 361–363 experimental procedure, 394–395 image forming systems schematic arrangement, cell analytical microscopy, requirements, experiment, 394, 396 215–217 transmission mode, 393 imaging process, 204–205 voltage contrast modes, 399 lens systems, CTEM and STEM, 210–214 water-vapor cell, cross-section, 394–395 magnetic lenses, image forming 50kV STEM properties, 205–208 atom image contrast and electron strong lenses, 208–210 probe diameter, relationship, image processing, 30 144–145 lens aberration corrections, 23–27 bright-field images, 142–143 aperture calculation, 9 carbon film, dark-field images, 146–147 off-axis aberrations, 7 electron energy-loss spectrometer, spherical and chromatic aberrations, 147–149 6, 23 probe size and electron gun brightness, third-order aberrations, 7, 10 relationship, 145 zero aberration system, 11 scattered electrons, intensity angular mass measurements distribution, 144 biological samples, 371–373 schematic diagram, basic design, GroEL-GroES, 375 141–142 LcrV, Yersinia needle, 377 schematic diagrams, detecting principle, 366, 368 conditions, 146 PulD and YscC secretins, 374–376 specifications, 141 PulG(His)6 pseudopilus, 376–377 specimen contamination, 145–146 statistical error, 369–371 thorium atoms, dark-field image, stoichiometry, 375 147–148 synaptic vesicles, 378–379 theoretical electron optics, 27–28 T2 and T3 secretion systems, 375–376 thin section imaging negatively stained samples, 365, 367 advantages, 365 nucleosome, 22–23 plastic embedding and sectioning, 363 optimization and super-high-resolution, septate junction, 365–366 28–29 three-dimensional reconstruction, 31–32 quadrupole-octupole correctors, 3 Scherzer’s theorem, 6, 23, 27 schematic mirror sysstem, 5 Secondary electron detector, 245 secondary electron production, 21 Sextupole corrector sextupole corrector, 4, 10–11, 25 advantages, 11 Index 417

construction, 200-kV STEM, 25 V partially assembled, 4 Siemens ST 100 F Vacuum generator 100-kV STEM adenovirus image, 192–193 beam-blanking plates, 256–257 column and control panel, 192–193 design considerations CT150, 194 analytical facilities location, 226–227 Single-atom observation, 50kV STEM electron gun, 226 atom image contrast and electron probe HB200, 224 diameter, relationship, 144–145 objective lens, 225 carbon film, dark-field powerful analytical tool, 224 images, 146–147 resultant assembly, 227 preliminary irradiation, 146 specimen stage, 225–226 probe size and electron gun brightness, energy analyzer Mk2, 264–266 relationship, 145 first STEMs and HB5 development scattered electrons, intensity angular airlock and manipulator, 240–241 distribution, 144 basic imaging and diffraction modes, schematic diagrams, detecting optical column, 234–236 conditions, 146 basic structure, 231 specimen contamination, 145–146 column development, 238 thorium atoms, dark-field dark-field detector (DFD), 241–245 image, 147–148 differential pumping aperture, 231 Single-field condenser-objective lens, diffraction pattern observation screen, high-voltage STEM 248–249 Cavendish microscope, 401–404 electron gun, 233–234 disadvantages and advantages, 402 energy analyzer Mk1, 249–250 energy analysis, 404–405 extraction electrode, 233 objective aperture, 402 first optical column design, 236–238 scanning mode of operation, 400–401 graphite lattice fringes, 230 Sommerfeld free-electron model, 67 isolation valve housing, 231 Source optics microdiffraction coils, 246–248 emission parameters, 81 microscope control console, mutual Coulomb interactions, 80 electronics, 251–254 source reduced brightness, 79, 81 modus operandi, 230 Spherical aberrations packing note, 227–228 coefficient of, 23 performance specification, 230 electron probe size, 6 secondary electron detector, 245 fifth-order aperture aberration, 29 spot knocking, 230 mirror corrector, 26 stage development, QEC instrument, Spot knocking, 230 238–240 Stage motor drives, 261–262 vacuum controls, electronics, 254 STEM. See Scanning transmission electron gun lens, 266 microscope HB501 Surface tension-based radius-dulling development, 271–272 model, 95 HB501UX and high-resolution imaging, 273 T HB601, 278, 280 high-excitation objective lens, 266–268 Thermionic electron current density, 127 new airlock, 262–264 T4 viral tail fiber study, 111 non-dispersive x-ray detector, 257–258 U resolution first gold lattice fringes, 255–256 Ultra-high vacuum (UHV) MIT HB5, 254–255 engineering, 16 special and variant instruments 418 Index

Vacuum generator 100-kV STEM (cont.) objective lens pole pieces, 307 HB501A, 276–278 optical transfer function, 308 post-specimen lens (PSL) Si(110) image, 308 system, 276 source brightness, cold-field stage motor drives, 261–262 emission, 316 stages and cartridges testing, 311–316 basic cartridges, 268–269 X-ray microanalysis beryllium cartridges, 269 Cu alloy specimen, 318–319 cold stage, 269–270 output count rate (OCR), 318 cryo-transfer system, 270–271 P/B ratio, 318–319 virtual objective aperture, 259–261 Virtual objective aperture, 259–261 windowless x-ray detectors, 258–259 Visual proteomics 300-kV Vacuum generator STEM cryo-electron microscopy, 379 ADF resolution, 316–317 cryo-electron tomography, 378, 380 energy spread, cold-field emission, 316 hierarchical automated particle HB603 300-KV stem instruments selection, 380 column electron optics, 298 microfluidic circuits, 378 detectors, 301–302 picoliter drop deposition, 379 gun electron optics, 297 sample deposition devices, 378 HT tank-gun and accelerator, 296 segmentation algorithms, 380 objective lens, analytical and high- resolution pole pieces, 298–299 W projector system, 301 Water vapor, specimen observation software architecture, 302–304 Aspergillus nidulans, 396–397 specimen stage, 300–301 desiccation, 395 system/pumping, 294–295 double-walled liquid cell, 393 Lehigh design, 310 Everhart–Thornley detector, 399 MIDAS project experimental procedure, 394–395 gun lens, 289–290 schematic arrangement, cell experiment, objective lens, 290–291 394, 396 performance, 291–292 transmission mode, 393 side-entry stage, 291 voltage contrast modes, 399 system, 289 water-vapor cell, MIT and Argonne National Lab (ANL) cross-section, 394–395 designs Wentzel-Kramers-Brillouin (WKB) Argonne preparation chamber, approximation, 69 306–307 Windowless x-ray cylindrical mirror analyzer, 306 detectors, 258–260 MIT HB603 column, 305 virtual objective aperture, 306 Y Oak Ridge design detector system and HB603U, 309 Yersinia enterocolitica, 375–376