"If, in some cataclysm, all scientific knowledge were to be destroyed, and only one sentence passed on to the next generation of creatures, what statement would contain the most information in the fewest words? I believe it is the atomic hypothesis (or atomic fact; or whatever you wish to call it) that all things are made Seeing atoms of atoms." Mhairi Gass & Andrew Bleloch It is not an unreasonable paraphrase of this famous statement by Richard Feynman (1964) to say that "to understand the properties of all material things we need only to know which atoms are where". Electron microscopy is a technique that in the last decade has been able to image and analyse the atomic structure of a range of materials with atom by atom sensitivity and has led to a greater understanding of materials properties, from nanotubes and nanowires through to grain boundaries in metal alloys and ferritin particles in cells.

The specimen is a 70 nm thick ultra-microtomed section of lung cell from a mouse. The specimen was osmicated and the section poststained with uranyl acetate and lead citrate. The Z-contrast STEM image shows detailed cross sections of a number of cilia consisting of an outer ring of nine microtubial doublets and two central microtubial doublets. The primary function of cilia is to sweep dirt and mucus out of the lung and they are found in the lining of the trachea. The field of view for the image is 3 microns.

70 Issue 18 JUNE 2010 71 Forty years ago, Albert Crewe (Crewe et al. (1970)) showed images of individual atoms acquired from new equipment under development in his laboratory at the University of Chicago, Illinois; the machine was the scanning transmission electron microscope (STEM). Using a novel annular dark field detector (rather than the conventional

bright field imaging method which was popular in Fig. 1. Spherical aberration, a) all the rays that pass through a perfect electron microscopy), Crewe demonstrated, both lens focus at the same point, b) in an imperfect lens the rays that pass furthest from the optic axis are more strongly focused than the rays that pass through the centre of the lens; these aberrations result in a STEM has been a loss of resolution. quadrupole-octupole corrector for the scanning technique that has played electron microscope, Zach and Haider (1995). In an important role in 1997, at the Electron Microscopy and Analysis Group (EMAG) conference in Cambridge, Krivanek materials research and et al. presented results from their prototype has helped solve many spherical aberration electromagnetic quadrupole – octupole corrector for a STEM and in 2002 Batson materials questions over et al. demonstrated sub-angstrom resolution using

the years. such an aberration corrector in a STEM. There Fig. 3. Nickel disilicide. The topmost images are high resolution Z-contrast images of the interface for variant 1 and variant 2; the position of the are now in excess of 100 aberration corrected outer most layers of Ni atoms in the silicide are marked by thin black lines. The bottom images are averaged images of the interface structure and overlaid ball-and-stick display of the deduced model in [-110] (left) and [110] (right) projections. Reprinted Figure with permission from Falke et theoretically and experimentally, that by acquiring STEM machines worldwide, as well as numerous al. ‘Atomic Structure of a (2 x 1) Reconstructed NiSi2/Si(001) Interface’, Copyright (2004), by the American Physical Society. (http://prl.aps.org/ abstract/PRL/v92/i11/e116103). the electrons that had been elastically scattered to aberration-corrected conventional transmission astigmatism that are corrected. The corrector that high angles, individual heavy atoms such as uranium electron microscopes. In 2001 the first dedicated So what is an aberration corrector, what information was used for the first SuperSTEM machine was a and thorium could be imaged on a thin carbon film. AC-STEM facility, SuperSTEM, was opened in can we now achieve that was previously inaccessible Daresbury, UK, with the aim of making aberration and which materials problems can AC-STEM help quadrupole-octupole design by Nion Co. based on a

Ever since, STEM has been a technique that has correction available to academic researchers. solve? derivative of Scherzer’s 1947 design. The corrector played an important role in materials research and was retrofitted into an existing VG HB501 STEM, What is a spherical aberration has helped solve many materials questions over as shown in Figure 2a, improving the resolution to corrector? the years. However, until recently its resolution, as 1Å; the machine was also fitted with an electron Spherical aberration is a defect which afflicts all round with other electron microscopes, has been limited energy loss spectrometer to allow detailed electromagnetic lenses. The effect of this aberration by lens aberrations and thus little improvement in elemental information to be extracted. The second on a round lens is shown in Figure 1, where the rays resolution was achieved during the 30 year period machine to be installed at SuperSTEM was the that pass furthest from the optical axis are more following Crewe’s landmark experiments. Although first dedicated STEM designed for the aberration strongly focused than the rays that pass along the approaches to correct the aberrations had been correction era by Nion Co. and incorporated a fifth optical axis; this results in a range of focal points suggested starting with the fundamental work order aberration corrector and significantly greater and thus the resolution is limited. The addition of by Scherzer in 1936, experimental success was experimental flexibility, Figure 2b. In this article, we a spherical aberration corrector compensates for elusive until the mid 1990s when a combination demonstrate the adaptability of these machines to this effect and brings the focal crossover of the rays of scientific ingenuity, stable electronics, machining a range of materials and provide examples of the to the same point, thus improving the resolution, capabilities and computing power resulted in several variety of information that can be extracted. as illustrated by the “perfect lens” in Figure 1a. successful attempts at aberration correction; e.g. Fig. 2. Aberration corrected scanning transmission electron This is analogous to the way spectacles are added Initial work at SuperSTEM the electromagnetic sextupole corrector for the microscopes at the SuperSTEM facility, a) SuperSTEM 1 is a VG HB501 retro fitted with a Nion 2nd generation aberration corrector, to our eyes to correct aberrations in our vision The germ of the SuperSTEM facility was planted at conventional transmission electron microscope b) SuperSTEM 2 is a Nion UltraSTEM. The electron gun for both though in this case it is usually only defocus and the same 1997 EMAG meeting at which Krivanek by Haider et al. (1995) and the electrostatic machines is beneath the column for stability.

72 Issue 18 JUNE 2010 73 Z-contrast images of the interfaces (variant 1 and Atomic resolution focus” with the aim to infer some 3-dimensional variant 2) where the NiSi2 appears brighter than information from the specimen. An example of the Si due to the heavier atomic species present. Z-contrast images have this is shown in Figure 5 where the locations of The position of the outermost layer of Ni atoms in gold catalyst atoms within a silicon nanowire were the silicide is marked by a thin black line. Averaged revolutionised microscopy investigated, Allen et al. The inclusion of gold within images of the interface structure and an overlaid over recent years. the nanowire would be detrimental to its electrical ball-and-stick display of the deduced model in the properties. A series of Z-contrast images with 1nm [-110] (left) and [110] (right) projection are shown focus steps were acquired of an intrinsic Si nanowire. below. These indicate that variant 1 and variant 2 Figure 5a presents the sum of 7 aligned images from are orthogonal projections of one and the same the series; it shows impurities trapped at a (111)

structure which must then be of a 2 x 1 type. twin defect e.g. d1,d2, and ‘bulk’ impurities e.g. b1,b2. The excess intensity of Au atoms is plotted as a The Z-contrast image is also sensitive to the function of focal depth in Figure 5b indicating that

amount of material being imaged; for small the peak associated with atom b1 lies between the nanoparticles consisting of only one element the defect atom peaks and is therefore located within relationship is linear. This relationship has been used the nanowire as indicated in the schematic diagram,

Fig. 4. Gold 309 nanoparticles. a) – c) Z-contrast images of Au309 to calculate the number of atoms in gold clusters by Figure 5c. clusters on a carbon film demonstrating various outline shapes: Li et al. Z-contrast images of Au309 clusters on a pentagon, square and hexagon. The intensity variation within the clusters clearly demonstrates atomic column resolution. d) Three- carbon film are shown in Figures 4a-c. The intensity Extracting elemental dimensional atom density profile of Au309 was derived from a) and a hard-sphere model for an Ino-decahedral structure is shown with variation within the clusters clearly demonstrates information the electron beam (arrow) parallel to the five-fold axis. Experimental atomic column resolution. A three-dimensional Atomic resolution Z-contrast images have e) and simulated f) intensity line profiles are taken from the central atom column of the cluster to one of the corners (indicated in insets revolutionised microscopy over recent years, with red line) for comparison. Figure first published by Li et al. in Nature, Copyright (2008).

presented his initial results by a paper presented Fig. 6. Automically resolved EELS mapping. Comparison between experiments (tilted images) and simulations of Bi0:5Sr0:5MnO3 by Mick Brown entitled “A Synchrotron in a oriented along the [001] zone axis a) Z-contrast, b) oxygen K edge

c) manganese L2,3 edge. Reprinted Fig. with permission from Bosman microscope”. The facility was funded in 2001 et al. ‘Two-Dimensional Mapping of Chemical Information at Atomic and occupied in 2002 and the first sample system Resolution’, Copyright (2007), by the American Physical Society (http:// prl.aps.org/abstract/PRL/v99/i8/e086102). investigated at the new SuperSTEM facility involved a review of metallic silicides, with a view to both atom density profile of Au309 was derived from

a replacement of TiSi2 as an electrically conducting the first Z-contrast image, Figure 4d, and a hard- interconnect and concerning the growth of small sphere model for an Ino-decahedral structure is scale epitaxial structures for new electronic devices. shown with the electron beam (arrow) parallel The first important results were the elucidation of to the five-fold axis. Experimental (Figure 4e) and a

the structures of NiSi2/Si and CoSi2/Si atomically simulated intensity (Figure 4f) line profiles are taken sharp interfaces. The high angle annular dark field from the central atom column of the cluster to one (HAADF) imaging technique used was similar to of the corners (indicated in insets with a red line) the technique used by Crewe in 1970 and is also for comparison confirming the structure. b c known as atomic number contrast (Z-contrast) - the heavier the element, the brighter it will appear. Fig. 5. Three-dimensional localisation of gold atoms in a silicon Extracting 3-dimensional nanowire. a) A Z-contrast-STEM image of an intrinsic Si nanowire information Furthermore, the images acquired benefit from easy showing impurities trapped at a (111) twin defect (d1,d2) and ‘bulk’ Fig. 7. Intracellular distribution of SWNTs in unstained sections. a) interpretation of atomic positions. The Z-contrast impurities (b1,b2). b) The excess intensity of Au atoms is plotted A consequence of using an aberration corrector is Z-contrast image of SWNTs within a lysosome (2 days exposure). as a function of focal depth. The peak associated with atom b1 Low-loss EELS maps of the b) the plasmon shift (Ep) highlighting lies between the defect atom peaks and is therefore located within that the convergence angle of the electron beam images of the silicide interface are shown in Figure nanotube bundles and c) the Fe M2,3 showing Fe particles in white. d) the nanowire as indicated in the schematic diagram c). Figure first on the specimen is large, resulting in a small depth and e) High-resolution bright-field images of SWNTs. Arrows indicate 3, Falke et al.; the top images are high resolution published by Allen et al. in Nature Nanotechnology, Copyright (2008). the diameter of SWNTs. Figure first published in part by Porter et al. of focus. This can be exploited to image “through in Nature Nanotechnology, Copyright (2007).

74 Issue 18 JUNE 2010 75 Z-contrast image and elemental maps. The first prime example is to understand the pathways Another example of applying Z-contrast imaging to demonstration of atomically resolved elemental and localisation of carbon nanoparticles in cellular a biological problem is demonstrated in Figure 8 EELS mapping was published by Bosman et al. in structures. Nanoparticles such as single walled where Pan et al. applied the technique to determine

2007. Figure 6a shows both experimental and nanotubes (SWNT) and C60 are smaller than the the 3D morphology of a ferritin mineral core.

simulated Z-contrast images of Bi0.5Sr0.5MnO3 typical resolutions used in biological electron Figure 8a shows the Z-contrast image of a number oriented along the (001) zone. In Figure 6b and c, microscopy, further, the low contrast between of ferritin cores. The number of iron atoms per the corresponding oxygen K edge and manganese such small carbon nanoparticles surrounded by the core was determined by correlating the EELS iron

Fig. 8. Reconstruction of a ferritin core. a) An example of one of L2,3 edge maps are shown and compared to the carbonaceous cellular structure makes it almost L2,3 edge and the Z-contrast image. Single particle the Z-contrast images containing multiple ferritin cores, used for theoretical predictions. It can be seen in Figure impossible to image using the traditional bright field reconstruction of a hepatic ferritin core was then the reconstruction. b) Selected views of the initial models and final reconstructions of a core developed from the 750 ferritin cores 6, that from one signal, information from several imaging techniques, particularly when it is combined carried out by classifying 750 Z-contrast imaged each with an iron atom content in the most populated range of between 1100–1850; despite different initial models a cubic-like elements can be obtained. As a result of this paper, with staining, a technique used to highlight the ferritin cores into 9 different projections using the subunit structure with a low density centre is evident in all of the the necessity of selecting the correct experimental cellular structure. A combination of EELS and EMAN program. Despite the three different initial final reconstructions. Reprinted from Pan et al. ‘3D morphology of the human hepatic ferritin mineral core: New evidence for a subunit conditions, such as collection angles for the EELS AC–STEM was used by Porter et al. to locate and models in Figure 8b, the final reconstructions all structure revealed by single particle analysis of HAADF-STEM images’, Copyright (2009), with permission from Elsevier. signal so as not to analyse incorrect information, identify small aggregates of such nanomaterials in indicate a cubic-like subunit structure with a low was highlighted. unstained cellular sections. Figure 7 shows how AC- density centre. however, the image itself is not always sufficient STEM can be used to identify and image individual to fully understand the structure of the material Although electron microscopy is heavily used in SWNTs in macrophage cells. The difference in The final example that demonstrates the wealth of in question. To really understand what the atoms the biological sciences, aberration correction was density and bonding arrangements between the information that can be extracted by combination are, and even obtain some bonding information, it initially a technique applied to hard materials. With graphitic nanoparticles and cell is extracted by aberration corrected imaging with analytical is necessary to obtain spectral information. Using ever increasing interest on the biological effects of mapping the plasmon feature in EELS spectrum information is the verification of monolayer an AC-STEM fitted with an electron energy loss nanotechnology, collaborations between the two images, Figure 7b. This indicates regions where graphene by Gass et al. Figures 9a and b show spectrometer (EELS), it is possible, from the same disciplines are evolving to tackle these issues. A nanoparticle aggregates lie, and smaller features high-resolution bright field and Z-contrast images incident electrons, to simultaneously acquire the of a clean patch of graphene surrounded by a The combination of mono-atomic surface layer; individual contaminant Z-contrast imaging with atoms of higher atomic number can be seen in the Z-contrast image. The inset FFT clearly shows the EELS using AC-STEM, in lattice in the Z-contrast image and by applying a particular, has proven to band pass filter, the atomic structure is apparent, however, it is very difficult to prove the existence be an incredibly successful of mono-layer graphene by imaging alone. As the method for materials plasmon feature in EELS is composed of bulk and surface modes, it thus follows that purely mono- characterisation at the layer graphene with no surface contamination atomic level. will not exhibit the bulk plasmon mode, whilst graphite will exhibit the bulk mode with the

are identified. Extraction of the iron M2,3 edge surface mode being insignificant. Thus, by analysing intensity from the same data set identifies the iron the shape and position of the plasmon feature, catalyst particles used in the growth of the SWNT information on the bulk and surface properties material, Figure 7c, and shows them decorating the can be obtained. An EELS spectrum from a clean SWNT bundle. Higher resolution imaging of the area of graphene is shown as a blue trace in Figure smaller features can then show the existence of an 9c; this is a saw-tooth shape feature with the peak Fig. 9. High-resolution images of mono-layer graphene. a) Bright-field b) Z-contrast images of the monolayer showing a clean patch of graphene surrounded by a mono-atomic surface layer; individual contaminant atoms of higher atomic number can be seen in b). The inset FFT clearly shows individual SWNT lying within the cellular structure, at ~14.5eV confirming the presence of graphene the lattice in the Z-contrast image and the atomic structure is apparent in the filtered inset. c) EELS spectra were acquired from the clean area; and a small bundle of SWNTs end on that were and in agreement with theoretical calculations. a background-subtracted spectrum is shown as a blue trace. For direct comparison spectra acquired for two-layer (red) and five-layer (black) graphene are shown. All spectra are summed over 25 pixels. Figure first published by Gass et al. in Nature Nanotechnology, Copyright (2008). cut during sectioning, Figures 7d and 7e respectively. The thickness of material can then be determined

76 Issue 18 JUNE 2010 77 from both the Z-contrast image and the integrated optics, Nature 418 617-620 Mhairi Gass intensity of the EELS spectrum and, for thin areas, Bosman M, Keast VJ, Garcia-Munoz JL, D’Alfonso AJ, Serco Technical Consultancy Services, Risley, UK the number of graphene layers can be extracted. Findlay SD, and Allen LJ (2007) Two-Dimensional [email protected] Comparable spectra acquired for two-layer (red) Mapping of Chemical Information at Atomic Resolution, After completing her PhD on analytical scanning transmission electron and five-layer (black) graphene from nearby areas Phys. Rev. Lett. 99 086102 microscopy (STEM) at the University of Liverpool, Mhairi moved to highlight the difference in spectral shape. Crewe AV, Wall J and Langmore J (1970) Visibility of Cambridge University as a research associate, where she developed Single Atoms, Science 168 1338-1340 energy filtered tomography to extract elemental information and enable AC-STEM: the future differentiation between carbonaceous materials in 3-dimensions. In 2005 Falke U, Bleloch AL and Falke M (2004) Atomic Structure Mhairi joined the SuperSTEM project where she operated the aberration The emergence of aberration correction in of a (2 x 1) Reconstructed NiSi2/Si(001) Interface, Phys. corrected STEMs for external researchers as well as developing her own transmission electron microscopy has revolutionised Rev. Lett. 92 116103 research in energy loss spectroscopy of graphitic nanostructures and running the day-to-day operations. With a keen interest in promoting science to the way researchers approach materials problems Gass MH, Bangert U, Bleloch AL, Wang P, Nair RR and the general public, Mhairi has been involved in a number of events such as and the wealth of information that can be expected Geim AK (2008) Free-standing graphene at atomic the Royal Society Summer Science Festival and the BA Festival of Science. from these new machines. The combination of resolution Nature Nanotech 3 676 Recently Mhairi moved to industry where she now works as a Materials Z-contrast imaging with EELS using AC-STEM, in Haider M., Braunshausen G and Schwan E (1995) Specialist for Serco. particular, has proven to be an incredibly successful Correction of the spherical-aberration of a 200-KV TEM method for materials characterisation at the atomic by means of a hexapole-corrector Optik 99, 167-179 Andrew Bleloch level. However, the limiting factor now is often the Krivanek O.L., Delby N., Spence A.J., Camps R.A. and SuperSTEM, Daresbury Laboratory, Daresbury, UK www.superstem.org sample preparation technique or damage to the Brown L.M. (1997) Aberration correction in the STEM specimen during analysis due to such small areas Inst. Phys. Conf. Ser. 153(2) 35-40 Andrew Bleloch runs the SuperSTEM aberration corrected electron being heavily analysed. Li ZY, Young NP, Di Vece M, Palomba S, Palmer RE, microscopy facility at Daresbury Laboratories as well as teaching Materials Bleloch AL, Curley BC, Johnston RL, Jiang J and Yuan J Science at the University of Liverpool. He gained his PhD developing new techniques on the VG HB501 STEM in Archie Howie and Mick Brown’s In the decade since the first sub-0.1 nm resolution (2008) Three-dimensional atomic-scale structure of group at the Cavendish Laboratory in Cambridge. More recently he has images were published, a significant fraction of £1 size-selected gold nanoclusters, Nature 451 46-49 pioneered the application of aberration corrected STEM techniques to billion worth of aberration corrected instruments Porter AE, Gass M, Muller K, Skepper J, Midgley PA a wide range of sample systems from the ubiquitous silicon 110 through nanotubes rods and particles to tentative experiments on biological samples. have been sold. Whilst this statement carries and Welland M (2007) Direct imaging of single-walled no scientific weight, it is a testament to the carbon nanotubes in cells Nature Nanotech 2 713-717 demand for these new spectacles for the electron Pan Y, Sader K, Powell J, Bleloch A, Gass M, Trinick J, microscope that hint at a productive future in Warley A, Li A, Brydson R and Brown AE (2009) 3D Robin Keeley materials characterisation whatever the purpose morphology of the human hepatic ferritin mineral It is with sadness that the Society announces the Georgi Markov murder, numerous anti-terrorism of the materials. These instruments are becoming core: New evidence for a subunit structure revealed by death of Dr. Robin Keeley, who passed away in cases, the Libyan Embassy siege and the Jill Dando an essential part of a productive national research single particle analysis of HAADF-STEM images Journal May 2010. He will be greatly missed by his many murder. infrastructure. Structural Biology 166 22-31 friends and colleagues throughout the microscopy Scherzer O (1936) Über einige Fehler von community. Robin was a great mentor and passed on his References Elektronenlinsen, Zeitschrift für Physik 101, 593-603 knowledge with generosity and enthusiasm to Abad JM, Sendroiu IE, Gass M, Bleloch A, Mills AJ and Zach J. and Haider M. (1995) Correction of spherical Robin joined the old Metropolitan Police Forensic several generations of forensic scientists. He was Schiffrin DJ (2007) Synthesis of w-hydroxy hexathiolate- and chromatic aberration in a low-voltage SEM Optik Science Laboratory (MPFSL) in 1971 and was a also a gifted but humble raconteur and when asked protected subnanometric gold clusters JACS 129 12932 99, 112-118 pioneer in the use of SEM in forensic science. about his career he would reply that all he had been Allen JE, Hemesath ER, Perea DE, Lensch-Falk JL, Li ZY, His ground breaking work using SEM-EDX for was a simple "journeyman".

Yin F, Gass MH, Wang P, Bleloch AL, Palmer RE, Lauhon Memoriam In the identification of GSR earned him worldwide LJ (2008) High-resolution detection of Au catalyst atoms recognition as a pioneer in this field. His career at Robin will be remembered for what he was, a in silicon nanowires Nature Nanotech. 3 168-173 both the MPFSL and the FSS spanned four decades pioneering forensic scientist and a generous warm Batson P., Delby N. And Krivanek O.L. (2002) Sub- and he worked on many famous cases, including the hearted man. angstrom resolution using aberration corrected electron

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