Confocal Microscopy
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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 ............................ -
Confocal Microscopy
Confocal microscopy Chapter in Handbook of Comprehensive Biophysics ( in press 2011) Elsevier Brad Amos MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH UK e-mail [email protected] Gail McConnell University of Strathclyde , Centre for Biophotonics 161 Cathedral Street , Glasgow G4 0RE UK [email protected] Tony Wilson Dept. of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK. eMail: [email protected] 1 Introduction A confocal microscope is one in which the illumination is confined to a small volume in the specimen, the detection is confined to the same volume and the image is built up by scanning this volume over the specimen, either by moving the beam of light over the specimen or by displacing the specimen relative to a stationary beam. The chief advantage of this type of microscope is that it gives a greatly enhanced discrimination of depth relative to conventional microscopes. Commercial systems appeared in the 1980s and, despite their high cost, the world market for them is probably between 500 and 1000 instruments per annum, mainly because of their use in biomedical research in conjunction with fluorescent labelling methods. There are many books and review articles on this subject ( e.g. Pawley ( 2006) , Matsumoto( 2002), Wilson (1990) ). The purpose of this chapter is to provide an introduction to optical and engineering aspects that may be o f interest to biomedical users of confocal microscopy. Flying-spot Microscopes A confocal microscope is a special type of ‘flying spot’ microscope. Flying spot systems were developed in the 1950s by combining conventional microscopes with electronics from TV and military equipment. -
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. -
Super-Resolution Imaging by Dielectric Superlenses: Tio2 Metamaterial Superlens Versus Batio3 Superlens
hv photonics Article Super-Resolution Imaging by Dielectric Superlenses: TiO2 Metamaterial Superlens versus BaTiO3 Superlens Rakesh Dhama, Bing Yan, Cristiano Palego and Zengbo Wang * School of Computer Science and Electronic Engineering, Bangor University, Bangor LL57 1UT, UK; [email protected] (R.D.); [email protected] (B.Y.); [email protected] (C.P.) * Correspondence: [email protected] Abstract: All-dielectric superlens made from micro and nano particles has emerged as a simple yet effective solution to label-free, super-resolution imaging. High-index BaTiO3 Glass (BTG) mi- crospheres are among the most widely used dielectric superlenses today but could potentially be replaced by a new class of TiO2 metamaterial (meta-TiO2) superlens made of TiO2 nanoparticles. In this work, we designed and fabricated TiO2 metamaterial superlens in full-sphere shape for the first time, which resembles BTG microsphere in terms of the physical shape, size, and effective refractive index. Super-resolution imaging performances were compared using the same sample, lighting, and imaging settings. The results show that TiO2 meta-superlens performs consistently better over BTG superlens in terms of imaging contrast, clarity, field of view, and resolution, which was further supported by theoretical simulation. This opens new possibilities in developing more powerful, robust, and reliable super-resolution lens and imaging systems. Keywords: super-resolution imaging; dielectric superlens; label-free imaging; titanium dioxide Citation: Dhama, R.; Yan, B.; Palego, 1. Introduction C.; Wang, Z. Super-Resolution The optical microscope is the most common imaging tool known for its simple de- Imaging by Dielectric Superlenses: sign, low cost, and great flexibility. -
Development of Optical Hyperlens for Imaging Below the Diffraction Limit
Development of optical hyperlens for imaging below the diffraction limit Hyesog Lee, Zhaowei Liu, Yi Xiong, Cheng Sun and Xiang Zhang* 5130 Etcheverry Hall, NSF Nanoscale Science and Engineering Center (NSEC), University of California, Berkeley, CA 94720 *Corresponding author: [email protected] http://xlab.me.berkeley.edu Abstract: We report here the design, fabrication and characterization of optical hyperlens that can image sub-diffraction-limited objects in the far field. The hyperlens is based on an artificial anisotropic metamaterial with carefully designed hyperbolic dispersion. We successfully designed and fabricated such a metamaterial hyperlens composed of curved silver/alumina multilayers. Experimental results demonstrate far-field imaging with resolution down to 125nm at 365nm working wavelength which is below the diffraction limit. ©2007 Optical Society of America OCIS codes: (110 0180) Microscopy; (220.4241) Optical design and fabrication: Nanostructure fabrication References and links 1. E. Abbe, Arch. Mikroskop. Anat. 9, 413 (1873) 2. E. Betzig, J. K. Trautman, T. D. Harris, J. S. Weiner and R. L. Kostelak, “Breaking the diffraction barrier – optical microscopy on a nanometric scale,” Science 251, 1468-1470 (1991) 3. S. W. Hell, “Toward Fluorescence nanoscopy,” Nat. Biotechnol. 21, 1347-1355 (2003) 4. M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution,” P. Natl. Acad. Sci. 102, 13081-13086 (2005) 5. J. B. Pendry, “Negative refraction makes a perfect lens,” Phys. Rev. Lett. 85, 3966-3969 (2000) 6. N. Fang, H. Lee, C. Sun and X. Zhang, “Sub-Diffraction-Limited Optical Imaging with a Silver Superlens” Science 308, 534-537 (2005) 7. -
Development of the Optical Microscope
White Paper Development of the Optical Microscope By Peter Banks Ph.D., Scientific Director, Applications Dept., BioTek Instruments, Inc. Products: Cytation 5 Cell Imaging Multi-Mode Reader An Optical Microscope commonly found in schools and universities all over the world. Table of Contents Ptolemy and Light Refraction --------------------------------------------------------------------------------------------- 2 Islamic Polymaths and Optics -------------------------------------------------------------------------------------------- 2 The First Microscope ------------------------------------------------------------------------------------------------------- 2 Hook and Micrographia --------------------------------------------------------------------------------------------------- 2 Van Leeuwenhoek and Animalcules ------------------------------------------------------------------------------------ 5 Abbe Limit -------------------------------------------------------------------------------------------------------------------- 6 Zernicke and Phase Contrast --------------------------------------------------------------------------------------------- 7 Fluorescence Microscopy ------------------------------------------------------------------------------------------------- 7 Confocal Microscopy ------------------------------------------------------------------------------------------------------- 8 BioTek Instruments, Inc. Digital Microscopy ---------------------------------------------------------------------------------------------------------- -
Super-Resolution Microscopy 1
Semrock White Paper Series: Super-resolution Microscopy 1. Introduction Fluorescence microscopy has revolutionized the study of biological samples. Ever since the invention of fluorescence microscopy towards the beginning of the 20th century, significant technological advances have enabled elucidation of biological phenomenon at cellular, sub- cellular and even at molecular levels. However, the latest incarnation of the modern fluorescence microscope has led to a paradigm shift. This wave is about breaking the diffraction limit first proposed in 1873 by Ernst Abbe. The implications of this development are profound. This new technology, called super-resolution microscopy, allows for the visualization of cellular samples with a resolution similar to that of an electron microscope, yet it retains the advantages of an optical fluorescence microscope. This means it is possible to uniquely visualize desired molecular species in a cellular environment, even in three dimensions and now in live cells – all at a scale comparable to the spatial dimensions of the molecules under investigation. This article provides an overview of some of the key recent developments in super-resolution microscopy. 2. The problem So what is wrong with a conventional fluorescence microscope? To understand the answer to this question, consider a Green Fluorescence Protein (GFP) molecule that is about 3 nm in diameter and about 4 nm long. When a single GFP molecule is imaged using a 100X objective the size of the image should be 0.3 to 0.4 microns (100 times the size of the object). Yet the smallest spot that can be seen at the camera is about 25 microns. Which corresponds to an object size of about 250 nm. -
Two-Photon Microscopy for Nanoparticle Imaging in Porous Material
Two-Photon Microscopy for Nanoparticle Imaging in Porous Material Christian Honsaker Dr. Chunqiang Li 2012-2013 ● Nanoparticles ● Microscopes Overview ● Fluorescence ● Confocal vs Two-Photon ● Applications Nanoparticles What’s a nanoparticle? A particle with a typical size between 1 and 100 nanometers (nm). Nanoparticles Examples ● Fullerenes ● Nanotubes ● Silica Powder ● Quantum Dots ● Noble Metals Standard Gold Nanoparticles How are nanoparticles utilized? Nanoparticles Applications ● Agricultural Products ● Automotive Industry ● Ceramics ● Fuel Cell Materials ● Paints Why study nanoparticles? Nanoparticles Concerns ● Environmental ● Health ● Safety https://www.youtube.com/watch?v=Ggy9FuNHGvs Assess potential risks Important to understand how nanoparticles interact with the environment. Nanoparticles Example 1. End up in soil by various means 2. Absorbed by plant roots or reach ground water 3. Enter the food chain and impact the ecosystem How do we observe the transport of nanoparticles? Microscopes Are there different types of microscopes? Scanning Probe Microscope ● Uses fine probe that is scanned over a surface; rather than a beam of light or electrons ● Not restrained by the wavelength of light or electrons ● True 3D maps Electron Microscope ● Powerful magnification ● Applications in various scientific fields ● Destroys sample ○ Ex Vivo Electron Microscope Advantages Types ● Not limited by optical ● Transmission Electron diffraction barrier Microscope ● Scanning Electron ● Wavelength 1000x Microscope shorter than visible light ● -
Assembly and Optimization of a Super-Resolution STORM Microscope for Nanoscopic Imaging of Biological Structures
Dissertation zur Erlangung des Doktorgrades der Fakult¨atf¨urChemie und Pharmazie der Ludwig-Maximilians-Universit¨atM¨unchen Assembly and optimization of a super-resolution STORM microscope for nanoscopic imaging of biological structures Jens Prescher aus M¨unchen, Deutschland 2016 Erkl¨arung Diese Dissertation wurde im Sinne von x7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Don C. Lamb, PhD betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenst¨andigund ohne unerlaubte Hilfe erarbeitet. M¨unchen, den 13. Januar 2016 ............................................ Dissertation eingereicht am 15. Januar 2016 1. Gutachter Prof. Don C. Lamb, PhD 2. Gutachter Prof. Dr. Christoph Br¨auchle M¨undliche Pr¨ufungam 18. Februar 2016 Contents Abstract v 1 Introduction 1 2 Theory of super-resolution fluorescence microscopy 5 2.1 Principles of fluorescence . 5 2.2 Fluorescence microscopy and optical resolution . 8 2.2.1 Principle of fluorescence microscopy . 9 2.2.2 Resolution in optical microscopy . 10 2.2.3 Total Internal Reflection Fluorescence Microscopy . 14 2.3 Super-resolution fluorescence microscopy . 17 2.3.1 Localization-based super-resolution microscopy . 18 2.3.1.1 Photoactivated Localization Microscopy (PALM) . 21 2.3.1.2 Stochastic Optical Reconstruction Microscopy (STORM) . 22 2.3.1.3 Direct Stochastic Optical Reconstruction Microscopy (dSTORM) 23 2.3.2 Determination of resolution in STORM and PALM . 24 2.3.3 Three-dimensional localization-based super-resolution imaging . 25 2.3.4 Overview over other optical super-resolution methods . 27 3 Experimental setup and analysis methods for STORM 29 3.1 Experimental setup . 29 3.1.1 Instrumentation of the STORM setup . -
Basics of Light Microscopy Imaging SPECIAL EDITION of & Imaging Microscopy & RESEARCH • DEVELOPMENT • PRODUCTION
Basics of Light Microscopy Imaging SPECIAL EDITION OF & Imaging Microscopy & RESEARCH • DEVELOPMENT • PRODUCTION www.gitverlag.com GIVE YOUR EYES TIME TO ADJUST TO THE HIGH LIGHT EFFICIENCY. OLYMPUS SZX2. If you’re used to other stereo microscopes, you’d better be prepared before you start working with the new Olympus SZX2 range. The peerless resolution and exceptional light efficiency enable you to see fluorescence with brightness and brilliance like never before, while providing optimum protection for your specimens at the same time. All this makes Olympus SZX2 stereo microscopes the perfect tools for precise fluorescence analysis on fixed and living samples. Importantly, the innovations present in the SZX2 series go far beyond fluorescence. The optimised 3-D effect offers superior conditions for specimen preparation and precise manipulation. The unique ultra-flat LED light base provides bright and even illumination of the entire field of view at a constant colour temperature. Moreover, the ComfortView eyepieces and new trinocular observation tubes make sure your work is as relaxed as it is successful. The future looks bright with Olympus. For more information, contact: Olympus Life and Material Science Europa GmbH Phone: +4940237735426 E-mail: [email protected] www.olympus-europa.com Olympus Microscopy Introduction In this wide-ranging review, Olympus microscopy experts draw on their extensive knowledge and many years of experience to provide readers with a highly useful and rare overview of the combined fields of microscopy and imaging. By discussing both these fields together, this review aims to help clarify and summarise the key points of note in this story. -
How the Optical Microscope Became a Nanoscope
THE NOBEL PRIZE IN CHEMISTRY 2014 POPULAR SCIENCE BACKGROUND How the optical microscope became a nanoscope Eric Betzig, Stefan W. Hell and William E. Moerner are awarded the Nobel Prize in Chemistry 2014 for having bypassed a presumed scientifc limitation stipulating that an optical microscope can never yield a resolution better than 0.2 micrometres. Using the fuorescence of molecules, scientists can now monitor the interplay between individual molecules inside cells; they can observe disease-related proteins aggregate and they can track cell division at the nanolevel. Red blood cells, bacteria, yeast cells and spermatozoids. When scientists in the 17th century for the frst time studied living organisms under an optical microscope, a new world opened up before their eyes. This was the birth of microbiology, and ever since, the optical microscope has been one of the most important tools in the life-sciences toolbox. Other microscopy methods, such as electron microscopy, require preparatory measures that eventually kill the cell. Glowing molecules surpassing a physical limitation For a long time, however, optical microscopy was held back by a physical restriction as to what size of structures are possible to resolve. In 1873, the microscopist Ernst Abbe published an equation demonstrating how microscope resolution is limited by, among other things, the wavelength of the light. For the greater part of the 20th century this led scientists to believe that, in optical microscopes, they would never be able to observe things smaller than roughly half the wavelength of light, i.e., 0.2 micrometres (fgure 1). The contours of some of the cells’ organelles, such as the powerhouse mitochondria, were visible. -
Eric Betzig Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Dr., Ashburn, VA 20147, USA
Single Molecules, Cells, and Super-Resolution Optics Nobel Lecture, December 8, 2014 by Eric Betzig Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Dr., Ashburn, VA 20147, USA. ne of the nicest things about winning the Nobel Prize is hearing from all O of the people in my past and having the time to refect on the important role they’ve each played in getting me to the happy and fulflling life I have now. To all of my friends and colleagues from grade school up to my peers who nomi- nated me for this honor, you have my deepest thanks. I was frst introduced to super-resolution back in 1982 when I went to Cor- nell University for graduate school and met my eventual thesis advisors, Mike Isaacson and Aaron Lewis. Mike had recently developed a means of using elec- tron beams to fabricate holes as small as 30 nanometers (Figure 1A). He and Aaron fgured that if we could shine light on one side of the screen, then the light that initially comes out of the hole from the other side would create a sub- wavelength light source that we could then scan point-by-point over a sample and generate a super-resolution image [1] (Figure 1B). Te ultimate goal was to try to create an optical microscope that could look at living cells with the resolu- tion of an electron microscope. I wanted to become a scientist to do big, impact- ful things, and that certainly ft the bill, so I said, “Please sign me up.” At the time, many people told us that this idea would never work, either because it violated Abbé’s law, or even worse, the Uncertainty Principle.