OPTICAL MICROSCOPY Davidson and Abramowitz OPTICAL MICROSCOPY
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502-13 Magnifiers and Telescopes
13-1 I and Instrumentation Design Optical OPTI-502 © Copyright 2019 John E. Greivenkamp E. John 2019 © Copyright Section 13 Magnifiers and Telescopes 13-2 I and Instrumentation Design Optical OPTI-502 Visual Magnification Greivenkamp E. John 2019 © Copyright All optical systems that are used with the eye are characterized by a visual magnification or a visual magnifying power. While the details of the definitions of this quantity differ from instrument to instrument and for different applications, the underlying principle remains the same: How much bigger does an object appear to be when viewed through the instrument? The size change is measured as the change in angular subtense of the image produced by the instrument compared to the angular subtense of the object. The angular subtense of the object is measured when the object is placed at the optimum viewing condition. 13-3 I and Instrumentation Design Optical OPTI-502 Magnifiers Greivenkamp E. John 2019 © Copyright As an object is brought closer to the eye, the size of the image on the retina increases and the object appears larger. The largest image magnification possible with the unaided eye occurs when the object is placed at the near point of the eye, by convention 250 mm or 10 in from the eye. A magnifier is a single lens that provides an enlarged erect virtual image of a nearby object for visual observation. The object must be placed inside the front focal point of the magnifier. f h uM h F z z s The magnifying power MP is defined as (stop at the eye): Angular size of the image (with lens) MP Angular size of the object at the near point uM MP d NP 250 mm uU 13-4 I and Instrumentation Design Optical OPTI-502 Magnifiers – Magnifying Power Greivenkamp E. -
Dual Beam Detection Technique to Study Magneto-Optical Kerr
DUAL BEAM DETECTION TECHNIQUE TO STUDY MAGNETO-OPTICAL KERR EFFECT By Shankar Chandra Acharya, Msc A thesis submitted to the Graduate Council of Texas State University in partial fulfillment of the requirements for the degree of Master of Science with a Major in Physics May 2019 Committee Members: Wilhelmus J Geerts, Chair Nikoleta Theodoropoulou Alexander Zakhidov COPYRIGHT By Shankar Chandra Acharya 2019 FAIR USE AND AUTHOR’S PERMISSION STATEMENT Fair Use This work is protected by the Copyright Laws of the United States (Public Law 94-553, section 107). Consistent with fair use as defined in the Copyright Laws, brief quotations from this material are allowed with proper acknowledgement. Use of this material for financial gain without the author’s express written permission is not allowed. Duplication Permission As the copyright holder of this work I, Shankar Chandra Acharya, authorize duplication of this work, in whole or in part, for educational or scholarly purposes only. ACKNOWLEDGEMENTS First of all, I would like to than my supervisor Dr. Wilhelmus J Geerts for his constant support and guidance. I feel grateful to have worked under such an inspiring researcher who has given me this opportunity to learn and explore scientific knowledge. I would also like to thank my committee members Dr. Nikoleta Theodoropoulou and Dr. Alexander Zakhidov for their constructive feedback which have contributed to my thesis project. I am grateful for my family and friends for their motivation and encouragement all these years of my studies. My mother and father have supported me during the difficult times and inspired me throughout my research. -
Subwavelength Resolution Fourier Ptychography with Hemispherical Digital Condensers
Subwavelength resolution Fourier ptychography with hemispherical digital condensers AN PAN,1,2 YAN ZHANG,1,2 KAI WEN,1,3 MAOSEN LI,4 MEILING ZHOU,1,2 JUNWEI MIN,1 MING LEI,1 AND BAOLI YAO1,* 1State Key Laboratory of Transient Optics and Photonics, Xi’an Institute of Optics and Precision Mechanics, Chinese Academy of Sciences, Xi’an 710119, China 2University of Chinese Academy of Sciences, Beijing 100049, China 3College of Physics and Information Technology, Shaanxi Normal University, Xi’an 710071, China 4Xidian University, Xi’an 710071, China *[email protected] Abstract: Fourier ptychography (FP) is a promising computational imaging technique that overcomes the physical space-bandwidth product (SBP) limit of a conventional microscope by applying angular diversity illuminations. However, to date, the effective imaging numerical aperture (NA) achievable with a commercial LED board is still limited to the range of 0.3−0.7 with a 4×/0.1NA objective due to the constraint of planar geometry with weak illumination brightness and attenuated signal-to-noise ratio (SNR). Thus the highest achievable half-pitch resolution is usually constrained between 500−1000 nm, which cannot fulfill some needs of high-resolution biomedical imaging applications. Although it is possible to improve the resolution by using a higher magnification objective with larger NA instead of enlarging the illumination NA, the SBP is suppressed to some extent, making the FP technique less appealing, since the reduction of field-of-view (FOV) is much larger than the improvement of resolution in this FP platform. Herein, in this paper, we initially present a subwavelength resolution Fourier ptychography (SRFP) platform with a hemispherical digital condenser to provide high-angle programmable plane-wave illuminations of 0.95NA, attaining a 4×/0.1NA objective with the final effective imaging performance of 1.05NA at a half-pitch resolution of 244 nm with a wavelength of 465 nm across a wide FOV of 14.60 mm2, corresponding to an SBP of 245 megapixels. -
Depth of Focus (DOF)
Erect Image Depth of Focus (DOF) unit: mm Also known as ‘depth of field’, this is the distance (measured in the An image in which the orientations of left, right, top, bottom and direction of the optical axis) between the two planes which define the moving directions are the same as those of a workpiece on the limits of acceptable image sharpness when the microscope is focused workstage. PG on an object. As the numerical aperture (NA) increases, the depth of 46 focus becomes shallower, as shown by the expression below: λ DOF = λ = 0.55µm is often used as the reference wavelength 2·(NA)2 Field number (FN), real field of view, and monitor display magnification unit: mm Example: For an M Plan Apo 100X lens (NA = 0.7) The depth of focus of this objective is The observation range of the sample surface is determined by the diameter of the eyepiece’s field stop. The value of this diameter in 0.55µm = 0.6µm 2 x 0.72 millimeters is called the field number (FN). In contrast, the real field of view is the range on the workpiece surface when actually magnified and observed with the objective lens. Bright-field Illumination and Dark-field Illumination The real field of view can be calculated with the following formula: In brightfield illumination a full cone of light is focused by the objective on the specimen surface. This is the normal mode of viewing with an (1) The range of the workpiece that can be observed with the optical microscope. With darkfield illumination, the inner area of the microscope (diameter) light cone is blocked so that the surface is only illuminated by light FN of eyepiece Real field of view = from an oblique angle. -
De Sénarmont Bias Retardation in DIC Microscopy Stanley Schwartz1, Douglas B
de Sénarmont Bias Retardation in DIC Microscopy Stanley Schwartz1, Douglas B. Murphy2, Kenneth R. Spring3, and Michael W. Davidson4 1Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747. 2Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205. 3National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 6N260, Bethesda, Maryland 20892 4National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 3231 Keywords: microscopy, de Senarmont, Henri Hureau de Sénarmont, Francis Smith, Georges Nomarski, William Hyde Wollaston, Michel- Levy color chart, contrast-enhancement techniques, depth of field, bias, retardation, compensating, plates, DIC, differential interference, contrast, prisms, compensators, shadow-cast, relief, pseudo, three-dimensional, birefringence, optical staining, sectioning, spherical aberrations, wavefronts, shear, fast, slow, axes, axis, linear, circularly, elliptically, polarized light, polarizers, analyzers, orthogonal, path, differences, OPD, gradients, phase, ordinary, extraordinary, maximum extinction, quarter-wavelength, full-wave plates, first-order red, halo, artifacts, interference plane, VEC, video enhanced, VE-DIC, Newtonian, interference colors, Nikon, Eclipse E600, microscopes, buccal, mucosa, epithelial, cheek cells, ctenoid, fish scales, Obelia, hydroids, polyps, coelenterates, murine, rodents, rats, -
Introduction to Light Microscopy
Introduction to light microscopy A CAMDU training course Claire Mitchell, Imaging specialist, L1.01, 08-10-2018 Contents 1.Introduction to light microscopy 2.Different types of microscope 3.Fluorescence techniques 4.Acquiring quantitative microscopy data 1. Introduction to light microscopy 1.1 Light and its properties 1.2 A simple microscope 1.3 The resolution limit 1.1 Light and its properties 1.1.1 What is light? An electromagnetic wave A massless particle AND γ commons.wikimedia.org/wiki/File:EM-Wave.gif www.particlezoo.net 1.1.2 Properties of waves Light waves are transverse waves – they oscillate orthogonally to the direction of propagation Important properties of light: wavelength, frequency, speed, amplitude, phase, polarisation upload.wikimedia.org 1.1.3 The electromagnetic spectrum 퐸푝ℎ표푡표푛 = ℎν 푐 = λν 퐸푝ℎ표푡표푛 = photon energy ℎ = Planck’s constant ν = frequency 푐 = speed of light λ = wavelength pion.cz/en/article/electromagnetic-spectrum 1.1.4 Refraction Light bends when it encounters a change in refractive index e.g. air to glass www.thetastesf.com files.askiitians.com hyperphysics.phy-astr.gsu.edu/hbase/Sound/imgsou/refr.gif 1.1.5 Diffraction Light waves spread out when they encounter an aperture. electron6.phys.utk.edu/light/1/Diffraction.htm The smaller the aperture, the larger the spread of light. 1.1.6 Interference When waves overlap, they add together in a process called interference. peak + peak = 2 x peak constructive trough + trough = 2 x trough peak + trough = 0 destructive www.acs.psu.edu/drussell/demos/superposition/superposition.html 1.2 A simple microscope 1.2.1 Using lenses for refraction 1 1 1 푣 = + 푚 = physicsclassroom.com 푓 푢 푣 푢 cdn.education.com/files/ Light bends as it encounters each air/glass interface of a lens. -
The Microscope Parts And
The Microscope Parts and Use Name:_______________________ Period:______ Historians credit the invention of the compound microscope to the Dutch spectacle maker, Zacharias Janssen, around the year 1590. The compound microscope uses lenses and light to enlarge the image and is also called an optical or light microscope (vs./ an electron microscope). The simplest optical microscope is the magnifying glass and is good to about ten times (10X) magnification. The compound microscope has two systems of lenses for greater magnification, 1) the ocular, or eyepiece lens that one looks into and 2) the objective lens, or the lens closest to the object. Before purchasing or using a microscope, it is important to know the functions of each part. Eyepiece Lens: the lens at the top that you look through. They are usually 10X or 15X power. Tube: Connects the eyepiece to the objective lenses Arm: Supports the tube and connects it to the base. It is used along with the base to carry the microscope Base: The bottom of the microscope, used for support Illuminator: A steady light source (110 volts) used in place of a mirror. Stage: The flat platform where you place your slides. Stage clips hold the slides in place. Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and can be rotated to easily change power. Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X. -
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. -
The-Pathologists-Microscope.Pdf
The Pathologist’s Microscope The Pathologist’s Microscope Rudolf Virchow, the father of Pathology, had available to him wonderful microscopes during the 1850’s to 1880’s, but the one you have now is far better. Your microscope is the most highly perfected of all scientific instruments. These brief notes on alignment, the objective lens, the condenser, and the eyepieces are what you need to know to get the most out of your microscope and to feel comfortable using it. Figure 1 illustrates the important parts of a generic modern light microscope. Figure 1 - Parts of the Microscope UNC Pathology & Lab Med, MSL, July 2013 1 The Pathologist’s Microscope Alignment August Köhler, in 1870, invented the method for aligning the microscope’s optical system that is still used in all modern microscopes. To get the most from your microscope it should be Köhler aligned. Here is how: 1. Focus a specimen slide at 10X. 2. Open the field iris and the condenser iris. 3. Observe the specimen and close the field iris until its shadow appears on the specimen. 4. Use the condenser focus knob to bring the field iris into focus on the specimen. Try for as sharp an image of the iris as you can get. If you can’t focus the field iris, check the condenser for a flip-in lens and find the configuration that lets you see the field iris. You may also have to move the field iris into the field of view (step 5) if it is grossly misaligned. 5.Center the field iris with the condenser centering screws. -
To Take Into Consideration the Propriety Of
his was the subject for discussion amongst the seventeen microscopists who met at Edwin Quekett’s house No 50 Wellclose Square, in the Borough of Stepney, East London on 3rd September 1839. It was resolved that such a society be formed Tand a provisional committee be appointed to carry this resolution into effect. The appointed provisional committee of seven were to be responsible for the formation of our society, they held meetings at their homes and drew up a set of rules. They adopted the name ‘Microscopical Society of London’ and arranged a public meeting on the 20th December 1839 at the rooms of the Horticultural Society, 21 Regent Street. Where a Nathaniel Bagshaw Ward © National Portrait Gallery, London President, Treasurer and Secretary were elected, the provisional committee also selected the size of almost airtight containers. Together with George 3 x 1 inch as a standard for glass slides. Loddiges, he saw the potential benefit of protection from sea air damage allowing the transport of plants Each of the members of the provisional committee between continents. This Ward published in 1834 had their own background which we have briefly and eventually his cases enabled the introduction described on the following pages, as you will see of the tea plant to Assam from China and rubber they are a diverse range of professionals. plants to Malaysia from South America. His glass plant cases allowed the growth of orchids and ferns in the Victorian home and in 1842 he wrote a book on the subject. However glass was subject to a tax making cases expensive so Ward lobbied successfully for its repeal in 1845.