Impact of Nanotechnology on New Instrumentation
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Second Harmonic Imaging Microscopy
170 Microsc Microanal 9(Suppl 2), 2003 DOI: 10.1017/S143192760344066X Copyright 2003 Microscopy Society of America Second Harmonic Imaging Microscopy Leslie M. Loew,* Andrew C. Millard,* Paul J. Campagnola,* William A. Mohler,* and Aaron Lewis‡ * Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, CT 06030-1507 USA ‡ Division of Applied Physics, Hebrew University of Jerusalem, Jerusalem 91904, Israel Second Harmonic Generation (SHG) has been developed in our laboratories as a high- resolution non-linear optical imaging microscopy (“SHIM”) for cellular membranes and intact tissues. SHG is a non-linear process that produces a frequency doubling of the intense laser field impinging on a material with a high second order susceptibility. It shares many of the advantageous features for microscopy of another more established non-linear optical technique: two-photon excited fluorescence (TPEF). Both are capable of optical sectioning to produce 3D images of thick specimens and both result in less photodamage to living tissue than confocal microscopy. SHG is complementary to TPEF in that it uses a different contrast mechanism and is most easily detected in the transmitted light optical path. It also does not arise via photon emission from molecular excited states, as do both 1- and 2-photon excited fluorescence. SHG of intrinsic highly ordered biological structures such as collagen has been known for some time but only recently has the full potential of high resolution 3D SHIM been demonstrated on live cells and tissues. For example, Figure 1 shows SHIM from microtubules in a living organism, C. elegans. The images were obtained from a transgenic nematode that expresses a ß-tubulin-green fluorescent protein fusion and Figure 1 also shows the TPEF image from this molecule for comparison. -
Optical Sectioning in Fluorescence Microscopy by Confocal and 2
Optical Sectioning in Fluorescence achieved with this methodology, Calcium sparks are microscopic calcium release Downloaded from events inside living muscle cells and their properties are giving new insight into Microscopy by Confocal and how excitation leads to contraction (Cannell et al., 1995; Lopez-Lopez et al,, 2-Photon Molecular Excitation 1995; Gomez et al., 1997). Although the wide field microscope had been applied to calcium imaging since about 1985, calcium sparks had not been observed Techniques previously. This is probably because the presence of fluorescence from outside https://www.cambridge.org/core M.B. Cannell & C.Soeller the focal plane results in a marked loss of in-plane contrast for wide field St. George's Hospital Medical School, microscopy. (Note also that fluo-3 was used as the calcium indicator in these Cranmer Terrace, London SW17 ORE experiments as it has low fluorescence in the absence of calcium which also improves image contrast.) The calcium spark illustrates the high sensitivity of Confocal Microscopy current confocal optical methods - the calcium spark finally occupies about 10 fl Fluorescence microscopy has proved to be an invaluable tool for 14 4 (10" l) and represents calcium binding to only -10 indicator molecules, Until biomedical science since it is possible to visualise small quantities of labeled recently, the laser scanning confocal microscope has been the only instrument materials (such as intracellular ions and proteins) in both fixed and living that could measure fluorescence with a spatial resolution of about 0.4 x 0.4 x 0.8 cells, However, the conventional wide field fluorescence microscope suffers . -
Two-Photon Excitation Fluorescence Microscopy
P1: FhN/ftt P2: FhN July 10, 2000 11:18 Annual Reviews AR106-15 Annu. Rev. Biomed. Eng. 2000. 02:399–429 Copyright c 2000 by Annual Reviews. All rights reserved TWO-PHOTON EXCITATION FLUORESCENCE MICROSCOPY PeterT.C.So1,ChenY.Dong1, Barry R. Masters2, and Keith M. Berland3 1Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; e-mail: [email protected] 2Department of Ophthalmology, University of Bern, Bern, Switzerland 3Department of Physics, Emory University, Atlanta, Georgia 30322 Key Words multiphoton, fluorescence spectroscopy, single molecule, functional imaging, tissue imaging ■ Abstract Two-photon fluorescence microscopy is one of the most important re- cent inventions in biological imaging. This technology enables noninvasive study of biological specimens in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. It also produces higher-contrast im- ages and is a novel method to trigger localized photochemical reactions. Two-photon microscopy continues to find an increasing number of applications in biology and medicine. CONTENTS INTRODUCTION ................................................ 400 HISTORICAL REVIEW OF TWO-PHOTON MICROSCOPY TECHNOLOGY ...401 BASIC PRINCIPLES OF TWO-PHOTON MICROSCOPY ..................402 Physical Basis for Two-Photon Excitation ............................ -
Innovations of Wide-Field Optical-Sectioning
Innovations of wide-field optical-sectioning fluorescence microscopy: toward high-speed volumetric bio-imaging with simplicity Thesis by Jiun-Yann Yu In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2014 (Defended March 25, 2014) ii c 2014 Jiun-Yann Yu All Rights Reserved iii Acknowledgements Firstly, I would like to thank my thesis advisor, Professor Chin-Lin Guo, for all of his kind advice and generous financial support during these five years. I would also like to thank all of the faculties in my thesis committee: Professor Geoffrey A. Blake, Professor Scott E. Fraser, and Professor Changhuei Yang, for their guidance on my way towards becoming a scientist. I would like to specifically thank Professor Blake, and his graduate student, Dr. Daniel B. Holland, for their endless kindness, enthusiasms and encouragements with our collaborations, without which there would be no more than 10 pages left in this thesis. Dr. Thai Truong of Prof. Fraser's group and Marco A. Allodi of Professor Blake's group are also sincerely acknowledged for contributing to this collaboration. All of the members of Professor Guo's group at Caltech are gratefully acknowledged. I would like to thank our former postdoctoral scholar Dr. Yenyu Chen for generously teaching me all the engineering skills I need, and passing to me his pursuit of wide-field optical-sectioning microscopy. I also thank Dr. Mingxing Ouyang for introducing me the basic concepts of cell biology and showing me the basic techniques of cell-biology experiments. I would like to pay my gratefulness to our administrative assistant, Lilian Porter, not only for her help on administrative procedures, but also for her advice and encouragement on my academic career in the future. -
Imaging with Second-Harmonic Generation Nanoparticles
1 Imaging with Second-Harmonic Generation Nanoparticles Thesis by Chia-Lung Hsieh In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2011 (Defended March 16, 2011) ii © 2011 Chia-Lung Hsieh All Rights Reserved iii Publications contained within this thesis: 1. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, "Three-dimensional harmonic holographic microcopy using nanoparticles as probes for cell imaging," Opt. Express 17, 2880–2891 (2009). 2. C. L. Hsieh, R. Grange, Y. Pu, and D. Psaltis, "Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes," Biomaterials 31, 2272–2277 (2010). 3. C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, "Second harmonic generation from nanocrystals under linearly and circularly polarized excitations," Opt. Express 18, 11917–11932 (2010). 4. C. L. Hsieh, Y. Pu, R. Grange, and D. Psaltis, "Digital phase conjugation of second harmonic radiation emitted by nanoparticles in turbid media," Opt. Express 18, 12283–12290 (2010). 5. C. L. Hsieh, Y. Pu, R. Grange, G. Laporte, and D. Psaltis, "Imaging through turbid layers by scanning the phase conjugated second harmonic radiation from a nanoparticle," Opt. Express 18, 20723–20731 (2010). iv Acknowledgements During my five-year Ph.D. studies, I have thought a lot about science and life, but I have never thought of the moment of writing the acknowledgements of my thesis. At this moment, after finishing writing six chapters of my thesis, I realize the acknowledgment is probably one of the most difficult parts for me to complete. -
NNT '09 Program
NNT '09 Program 9-Nov Wednesday, November 11 12:00 Exhibit set-up, Room J2/J3, San Jose Convention Center 12:00 Registration 17:00 Welcome Reception and Equipment Exhibit, Room J2/J3, San Jose Convention Center Thursday, November 12, Room J1/J4 , San Jose Convention Center Plenary Session - Session Chairs: S. Chou (Princeton) & L. Montelius (Lund) 8:15 Welcome: Stephen Chou and Christie Marrian 8:30 Plenary NNT is Losing the Propaganda War Fabian Pease Stanford University 9:00 Invited 1 Template Infrastructure for Nanoimprint Lithography Nobuhito Toyama Dai Nippon Printing 9:20 Invited 2 NaPANIL: Consolidation of Nanoimprinting for Production Jouni Ahopelto VTT Microsystems and Nanoelectronics 9:40 Invited 3 Shrink-Induced Nanostructures Michelle Khine University of California, Irvine 10:00 Break Magnetics/Biology/Solar - Session Chairs: G. Willson (U. Texas) & H. Schift (PSI) 10:30 Invited 4 The Nano-imprinting Process towards Patterned Media Manufacturing Tsai-Wei Wu HGST 10:50 c1 Large Scale Fabrication of Nanoimprinted Magnetic Nanoparticles with Self-Assembled Templates Wei Hu Stanford University 11:05 c2 Photocatalytic Nanolithography: An Emergent Patterning Technique Relevant to Biotechnology Jane P Bearinger LLNL 11:20 c3 Fabrication of 3D Cell Containers with Integrated Topography by Combined Microscale Arne Schleunitz Paul Scherrer Institut Thermoforming and Thermal Nanoimprint 11:35 c4 Nanoimprinting of Subphthalocyanines for Photovoltaic Applications Xiaogan Liang Lawrence Berkeley National Laboratory 11:50 c5 Nanopatterned anode for organic solar cell by nanoimprint Dae-Geun Choi Korea Institute of Machinery & Materials 12:05 Lunch Electronics/Optoelectronics - Session Chairs: J. Randall (Zyvex) and S. Matsui (Hyogo) 13:30 Invited 5 Nanoimprint lithography for organic thin film transistors Barbara Stadlober Joanneum Research 13:50 c6 Fabrication of organic TFT arrays on an A4-sized flexible sheet using microcontact printing Hiroshi Fujita Dai Nippon Printing Co., Ltd. -
Etude Des Techniques De Super-Résolution Latérale En Nanoscopie Et Développement D’Un Système Interférométrique Nano-3D Audrey Leong-Hoï
Etude des techniques de super-résolution latérale en nanoscopie et développement d’un système interférométrique nano-3D Audrey Leong-Hoï To cite this version: Audrey Leong-Hoï. Etude des techniques de super-résolution latérale en nanoscopie et développement d’un système interférométrique nano-3D. Micro et nanotechnologies/Microélectronique. Université de Strasbourg, 2016. Français. NNT : 2016STRAD048. tel-02003485 HAL Id: tel-02003485 https://tel.archives-ouvertes.fr/tel-02003485 Submitted on 1 Feb 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE MATHEMATIQUES, SCIENCES DE L'INFORMATION ET DE L'INGENIEUR (MSII) – ED 269 LABORATOIRE DES SCIENCES DE L'INGENIEUR, DE L'INFORMATIQUE ET DE L'IMAGERIE (ICUBE UMR 7357) THÈSE présentée par : Audrey LEONG-HOI soutenue le : 2 DÉCEMBRE 2016 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline / Spécialité : Electronique, microélectronique, photonique Étude des techniques de super-résolution latérale en nanoscopie et développement d'un système interférométrique nano-3D THÈSE dirigée par : Dr. MONTGOMERY Paul Directeur de recherche, CNRS, ICube (Strasbourg) Pr. SERIO Bruno Professeur des Universités, Université Paris Ouest, LEME (Paris) RAPPORTEURS : Dr. GORECKI Christophe Directeur de recherche, CNRS, FEMTO-ST (Besançon) Pr. -
All Optical Histology of Brain Tissue: Serial Ablation and Multiphoton Imaging with Femtosecond Laser Pulses
All optical histology of brain tissue: Serial ablation and multiphoton imaging with femtosecond laser pulses Philbert S. Tsai, Beth Friedman, Varda Lev-Ram*, Qing Xiong*, Roger Y. Tsien* and David Kleinfeld Departments of Physics and *Pharmacology University of California, San Diego La Jolla, CA 92093 Agustin I. Ifarraguerri and Beverly D. Thompson Science Applications International Corporation Arlington, VA 22203 Jeff A. Squier Department of Physics Colorado School of Mines Golden, CO 80401 Ph:303-384-2385, FAX:303-273-3919, E-mail:[email protected] Abstract: We demonstrate the first use of femtosecond laser pulses for serial histology. Successive iterations of multiphoton imaging and ablation provide diffraction-limited volumetric data that is used to reconstruct the architectonics of labeled cells or microvasculature. ”2002 Optical Society of America OCIS codes: (000.0000) General 1. Introduction Current techniques in histology involve the manual slicing of frozen or embedded tissue, which is both labor intensive and may affect tissue morphology [1]. Advances in molecular labeling and the introduction of transgenic animals have brought about a need for high throughput analysis of architectonics and patterns of gene expression. Figure 1. The iterative process by which tissue is imaged and cut. A sample (left) containing two fluorescently labeled structures is imaged by two-photon microscopy to collect optical sections through the ablated surface until scattering of the incident light reduces the signal-to-noise ratio below a useful value; typically ~ 150 mm in fixed tissue. Labeled features in the stack of optical sections are digitally reconstructed (right). The top of the now-imaged region of the tissue is cut away with femtosecond pulses to expose a new surface. -
Multilayer Three-Dimensional Super Resolution Imaging of Thick Biological Samples
Multilayer three-dimensional super resolution imaging of thick biological samples Alipasha Vaziri1, Jianyong Tang, Hari Shroff, and Charles V. Shank1 Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA 20147 Contributed by Charles V. Shank, October 23, 2008 (sent for review September 30, 2008) Recent advances in optical microscopy have enabled biological versely proportional to pulse width squared and is called tem- imaging beyond the diffraction limit at nanometer resolution. A poral focusing (16, 17). Temporal focusing is experimentally general feature of most of the techniques based on photoactivated achieved by first broadening the pulse using a dispersive optical localization microscopy (PALM) or stochastic optical reconstruction element such as a grating. The illuminated spot on the grating is microscopy (STORM) has been the use of thin biological samples in then imaged onto the specimen plane using a telescope. This combination with total internal reflection, thus limiting the imag- results in a pulse broadened everywhere in the sample except at ing depth to a fraction of an optical wavelength. However, to study the image plane, where the dispersion is compensated and the whole cells or organelles that are typically up to 15 m deep into pulse reaches its minimum width (Fig. 1). Compared with an the cell, the extension of these methods to a three-dimensional epi-fluorescence technique, this minimum width results in a (3D) super resolution technique is required. Here, we report an depth of field that is orders of magnitude smaller (see supporting advance in optical microscopy that enables imaging of protein information (SI) Fig. S1 and SI Materials and Methods). -
Introduction to Confocal Laser Scanning Microscopy (LEICA)
Introduction to Confocal Laser Scanning Microscopy (LEICA) This presentation has been put together as a common effort of Urs Ziegler, Anne Greet Bittermann, Mathias Hoechli. Many pages are copied from Internet web pages or from presentations given by Leica, Zeiss and other companies. Please browse the internet to learn interactively all about optics. For questions & registration please contact www.zmb.unizh.ch . Confocal Laser Scanning Microscopy xy yz 100 µm xz 100 µm xy yz xz thick specimens at different depth 3D reconstruction Types of confocal microscopes { { { point confocal slit confocal spinning disc confocal (Nipkov) Best resolution and out-of-focus suppression as well as highest multispectral flexibility is achieved only by the classical single point confocal system ! Fundamental Set-up of Fluorescence Microscopes: confocal vs. widefield Confocal Widefield Fluorescence Fluorescence Microscopy Microscopy Photomultiplier LASER detector Detector pinhole aperture CCD Dichroic mirror Fluorescence Light Source Light source Okular pinhole aperture Fluorescence Filter Cube Objectives Sample Plane Z Focus Confocal laser scanning microscope - set up: The system is composed of a a regular florescence microscope and the confocal part, including scan head, laser optics, computer. Comparison: Widefield - Confocal Y X Higher z-resolution and reduced out-of-focus-blur make confocal pictures crisper and clearer. Only a small volume can be visualized by confocal microscopes at once. Bigger volumes need time consuming sampling and image reassembling. -
Label-Free Multiphoton Microscopy: Much More Than Fancy Images
International Journal of Molecular Sciences Review Label-Free Multiphoton Microscopy: Much More than Fancy Images Giulia Borile 1,2,*,†, Deborah Sandrin 2,3,†, Andrea Filippi 2, Kurt I. Anderson 4 and Filippo Romanato 1,2,3 1 Laboratory of Optics and Bioimaging, Institute of Pediatric Research Città della Speranza, 35127 Padua, Italy; fi[email protected] 2 Department of Physics and Astronomy “G. Galilei”, University of Padua, 35131 Padua, Italy; [email protected] (D.S.); andrea.fi[email protected] (A.F.) 3 L.I.F.E.L.A.B. Program, Consorzio per la Ricerca Sanitaria (CORIS), Veneto Region, 35128 Padua, Italy 4 Crick Advanced Light Microscopy Facility (CALM), The Francis Crick Institute, London NW1 1AT, UK; [email protected] * Correspondence: [email protected] † These authors contributed equally. Abstract: Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. -
How Single-Molecule Localization Microscopy Expandedour
International Journal of Molecular Sciences Review How Single-Molecule Localization Microscopy Expanded Our Mechanistic Understanding of RNA Polymerase II Transcription Peter Hoboth 1,2 , OndˇrejŠebesta 2 and Pavel Hozák 1,3,* 1 Department of Biology of the Cell Nucleus, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeˇnská 1083, 142 20 Prague, Czech Republic; [email protected] 2 Faculty of Science, Charles University, Albertov 6, 128 00 Prague, Czech Republic; [email protected] 3 Microscopy Centre, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeˇnská 1083, 142 20 Prague, Czech Republic * Correspondence: [email protected] Abstract: Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression. Citation: Hoboth, P.; Šebesta, O.; Hozák, P. How Single-Molecule Keywords: cell nucleus; gene expression; transcription foci; transcription factors; super-resolution Localization Microscopy Expanded microscopy; structured illumination; stimulated emission depletion; stochastic optical reconstruc- Our Mechanistic Understanding of tion; photoactivation RNA Polymerase II Transcription.