Basics of Light Microscopy Imaging SPECIAL EDITION of & Imaging Microscopy & RESEARCH • DEVELOPMENT • PRODUCTION
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Electron Microscopy and the Investigation of New Infectious Diseases
Review Electron microscopy and the investigation of new infectious diseases Alan Curry@) Objectives: To review and assess the role of electron microscopy in the investigation of new infectious diseases. Design: To design a screening strategy to maximize the likelihood of detecting new or emerging pathogens in clinical samples. Results: Electron microscopy remains a useful method of investigating some viral infections (infantile gastroenteritis, virus-induced outbreaks of gastroenteritis and skin lesions) using the negative staining technique. In addition, it remains an essential technique for the investigation of new and emerging parasitic protozoan infections in the immunocompromised patients from resin-embedded tissue biopsies. Electron microscopy can also have a useful role in the investigation of certain bacterial infections. Conclusions: Electron microscopy still has much to contribute to the investigation of new and emerging pathogens, and should be perceived as capable of producing different, but equally relevant, information compared to other investigative techniques. It is the application of a combined investigative approach using several different techniques that will further our understanding of new infectious diseases. Int J Infect Dis 2003; 7: 251-258 INTRODUCTION at individually by a skilled microscopist have con- The electron microscope was developed just before tributed to the decline of electron microscopy. Against World War II in several countries, but particularly in this background, the inevitable question must be Germany.l The dramatic increase in resolution available asked-does electron microscopy still have a useful in comparison with light microscopy promised to role to play in the investigation of emerging or new revolutionize many aspects of cell biology, virology, infectious diseases? bacteriology, mycology and protozoan parasitology. -
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. -
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. -
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. -
Applications of Microscopy in Bacteriology
Microscopy Research, 2016, 4, 1-9 Published Online January 2016 in SciRes. http://www.scirp.org/journal/mr http://dx.doi.org/10.4236/mr.2016.41001 Applications of Microscopy in Bacteriology Mini Mishra1, Pratima Chauhan2* 1Centre of Environmental Studies, Department of Botany, University of Allahabad, Allahabad, India 2Department of Physics, University of Allahabad, Allahabad, India Received 28 September 2015; accepted 2 January 2016; published 5 January 2016 Copyright © 2016 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Abstract Bacteria are smallest primitive, simple, unicellular, prokaryotic and microscopic organisms. But these organisms cannot be studied with naked eyes because of their minute structure. Therefore in search for the information about the structure and composition of bacterial cells, cell biologist used light microscopes with a numerical aperture of 1.4 and using wavelength of 0.4 µm separa- tion. But there are still certain cellular structures that cannot be seen through naked eyes, and for them electron microscope is used. There are certain improved types of light microscope which can be incorporated to improve their resolving power. Hence microscopy is playing a crucial role in the field of bacteriology. Keywords AFM, SEM, TEM, Microscopy, Bacteriology 1. Introduction To get acquainted with the world of bacteria like small organisms, very effective and advanced technique is re- quired. The size of bacteria ranges between 0.5 - 5.0 micrometer in length; the smallest of them are members of mycoplasma which measures 0.3 micrometers [1]. -
The Development of High Performance Liquid
Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 3-23-2010 The evelopmeD nt of High Performance Liquid Chromatography Systems for the Analysis of Improvised Explosives Megan N. Bottegal Florida International University, [email protected] DOI: 10.25148/etd.FI10041603 Follow this and additional works at: https://digitalcommons.fiu.edu/etd Recommended Citation Bottegal, Megan N., "The eD velopment of High Performance Liquid Chromatography Systems for the Analysis of Improvised Explosives" (2010). FIU Electronic Theses and Dissertations. 154. https://digitalcommons.fiu.edu/etd/154 This work is brought to you for free and open access by the University Graduate School at FIU Digital Commons. It has been accepted for inclusion in FIU Electronic Theses and Dissertations by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. FLORIDA INTERNATIONAL UNIVERSITY Miami, Florida THE DEVELOPMENT OF OPTIMIZED HIGH PERFORMANCE LIQUID CHROMATOGRAPHY SYSTEMS FOR THE ANALYSIS OF IMPROVISED EXPLOSIVES A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in CHEMISTRY by Megan Nicole Bottegal 2010 To: Dean Kenneth Furton College of Arts and Sciences This dissertation, written by Megan Nicole Bottegal, and entitled The Development of Optimized High Performance Liquid Chromatography Systems for the Anlysis of Improvised Explosives, having been approved in respect to style and intellectual content, is referred to you for judgment. We have read this dissertation and recommend that it be approved. ____________________________________ Jose Almirall ____________________________________ John Berry ____________________________________ William Hearn ____________________________________ Fenfei Leng ____________________________________ DeEtta Mills ____________________________________ Bruce McCord, Major Professor Date of Defense: March 23, 2010 The dissertation of Megan Nicole Bottegal is approved. -
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 ---------------------------------------------------------------------------------------------------------- -
Examples of High Performance Liquid Chromatography (HPLC) Application in Marine Ecology Studies in the Northern Adriatic
Prejeto / Received: 19.6.2009 Sprejeto / Accepted: 17.12.2009 Examples of high performance liquid chromatography (HPLC) application in marine ecology studies in the northern Adriatic Vesna FLANDER-PUTRLE Marine Biology Station, National Institute of Biology, Fornače 41, SI-6330 Piran; e-mail: [email protected] Abstract. Photosynthetic pigments have proved to be useful biomarkers of the abundance, composition and physiological status of the phytoplankton biomass in the marine environment. Using HPLC pigment analysis, we determined phytoplankton community structure in three different marine environments: in the area of a fish farm, in the area of sewage outlets, and in the mucilaginous aggregates. At the reference site we observed seasonal changes with prevalence of fucoxanthin-containing phytoplankton (i.e. diatoms) during winter/spring and autumn. In the fish farm area the concentration of chlorophyll a degradation products was higher, whereas in the locally enriched environment of sewage outlets we observed only small changes in taxonomic composition and phytoplankton biomass. The impact of season is more expressed than the impact of sewage discharge. With the use of HPLC pigment analysis we determined the development of phytoplankton community in different stages of mucilage aggregates. Phytoplankton biomass was composed primarily of diatoms, and as the aggregates aged, diatoms increased in the relative biomass. Our examples have proven the usefulness and suitability of HPLC pigment analysis in marine ecology studies. Key words: pigments, HPLC, phytoplankton, Adriatic Sea, mucilage, fish farm, sewage outlets Izvleček: PRIMERI UPORABE TEKOČINSKE KROMATOGRAFIJE VISOKE LOČLJIVOSTI (HPLC) PRI ŠTUDIJAH MORSKE EKOLOGIJE SEVERNEGA JADRANA – Fotosintezna barvila so se izkazala kot dobri kazalci abundance, sestave in fiziološkega stanja fitoplanktonske biomase v morskem okolju. -
Microscopy, Optical Spectroscopy, and Macroscopic Techniques Series: Methods in Molecular Biology, Vol
C. Jones, B. Mulloy, A.H. Thomas Microscopy, Optical Spectroscopy, and Macroscopic Techniques Series: Methods in Molecular Biology, Vol. 22 This is the second of three volumes of Methods in Molecular Biology that deal with Physical Methods of Analysis. The first of these, Spectroscopic Methods and Analyses dealt with NMR spec troscopy, mass spectrometry, and metalloprotein techniques, and the third will cover X-ray crystallographic methods. As with the first volume. Microscopy, Optical Spectroscopy, and Macroscopic Techniques is intended to provide a basic understand ing for the biochemist or biologist who needs to collaborate with spe cialists in applying the techniques of modern physical chemistry to biological macromolecules. The methods treated in this book fall into four groups. Part One covers microscopy, which aims to visualize individual molecules or complexes of several molecules. Electron microscopy is the more familiar of these, while scanning tunneling microscopy is a new and rapidly developing tool. Methods for determining the shapes and sizes of molecules in solution are described in Part Two, which includes chapters on X-ray and neutron scattering, 1994, XI, 251 p. light scattering, and ult- centrifugation. Calorimetry, described in Part Three, provides the means to monitor processes involving thermodynamic changes, whether these are A product of Humana Press intramolecular, such as conformational transition, or the interactions between solutes or between a solute and its sol vent. Part Four is concerned with optical and infrared Printed book spectroscopy and describes applications ranging from the measurement of protein concentration by UV absorbance to the analysis of secondary struc ture using circular Hardcover dichroism and Fourier-transform infrared spec troscopy.