Appendix B. Chapter 1 Parts The standard reference for terminology in light microscopy is the RMS Dictionary of Light Microscopy.

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1 Appendix B C. Robert Bagnell, Jr., Ph.D., 2012 Chapter Notes Chapter 0 Introduction The most complete work on light microscopy that I used in compiling these notes is that by Maksymilian Pluta - Advanced Light Microscopy: Volume 1 Principles and Basic Properties, and Volume 2 Specialized Methods. The third volume in the series, Measuring Techniques, I did not use. A more basic text on light microscopy is that by Robert F. Smith Microscopy and Photomicrography, A Working Manual, 2 nd edition. Included in this text is a color illustration containing images made of from an Abbe test plate. Abbe s theory of image formation is beautifully illustrated in this series of images. Many microscopy texts contain some historical information. Here I will only mention Donald L. Padgitt, A short History of the Early American Microscopes, Harold Malies, A Short History of the English Microscope, and American Optical Company, Three American Microscope Builders.. This last is a quaint, out of print, text that retains the flavor of the early days of microscopy in the United States. Chapter 1 Parts The standard reference for terminology in light microscopy is the RMS Dictionary of Light Microscopy. Chapter 2 Alignment An excellent compilation of traditional illumination methods and of Köhler s contribution is contained in the pamphlet titled Köhler Illumination Centenary prepared by the Royal Microscopical Society Included is an English translation of Köhler s original paper. The pamphlet was generally available through Zeiss representatives and from the RMS (37/38 St Clemments, Oxford OX4 1AJ, Phone (0865) , Fax: (0865) Chapter 3 Cleaning Shinya Inoué includes a section on inspecting and cleaning optics in his excellent book: Video Microscopy 2 nd edition, pp The pamphlet How to use a microscope and take a photomicrograph form NIKON Inc. contains a short section on cleaning lenses (pp 32-34). It should be available from your local NIKON representative. Another short section is contained in Optimizing Light Microscopy for Biological and Clinical Laboratories (pp 35-36) by Babara Foster. Chapter 4 Optics, Chapter 5 Illumination Path, UNC-CH Pathology 464, Light Microscopy 1

2 Chapter 6 Image Path, Chapter 7 Lenses Most books on microscopy contain information on optics. Here I will mention only a few of those that I have used. Michael B. Wilson s The Science and Art of Basic Microscopy has some of the best illustrations of geometric optics I have found. Saviel Bradbury s An Introduction to the Optical Microscope, covers a lot of optics in a very small space. A much more detailed book is that by Wolfgang Zieler: The Optical Performance of the Light Microscope Vol 1. A classic work that has been reprinted is The Practical Use of the Microscope by George Herbert Needham. Unfortunately, out of print is Optical Methods in Biology by Elizabeth M. Slayter. Also, unfortunately out of print is Fundamentals of Light Microscopy by Michael Spencer. I highly recommend these two books. They are available in the UNC-CH Health Sciences Library. The Star Test as described in Ch. 7 is adapted from Needham. For those who prefer a mathematical treatment of optics in microscopy, I recommend Light and Electron Microscopy by E.M. Slayter and H. S. Slayter. Chapter 8 Bright Field By far the best descriptions of bright field microscopy methods are contained in the older, out of print texts. Beside those mentioned above an interesting one is The Microscope by Simon Henry Gage, 17 th. While Gage apparently never accepted Abbe s theory of image formation, his book contains a huge quantity of practical advice and many instructive exercises. Contrast Techniques in Light Microscopy by S. Bradbury and P.J. Evennett contains a short chapter on bright field. A good text on histological staining is Theory and Practice of Histotechnology, 2 nd edition by Sheehan and Harpchak. Rheinberg Illumination is briefly described in volume 2 of a short series of books Basics and Beyond Series by Mortimer Abramowitz. A more detailed description is given by Bradbury and Evennett, and in Needham. Chapter 9 Dark Field The Practical Use of the Microscope by George Herbert Needham is the best source I have found for dark field microscopy. More recent publications include those by Robert F. Smith and Bradburry and Evennett. Chapter 10 Phase Contrast Phase Microscopy, Principles and Applications by A. Bennett, H. Osterberg, H. Jupnik, and O. Richards was my main source for this chapter. This out of print text contains a wealth of technical and practical information. Advanced Light Microscopy vol. 2: Specialized Methods by Maksymilian Pluta is the best current source. The physical properties of light involved in phase contrast are well described by Michael Spencer. I also found Fundamentals of Optics 4 th edition by F.A. Jenkins and H.E. White to be of much help in understanding the underlying optical concepts. Chapter 11 DIC UNC-CH Pathology 464 Light Microscopy 2

3 Short descriptions of Normarski Differential Interference Contrast are given in Bradburry and Evennett and in Smith. By far the best treatment is in Pluta vol 2. Jenkins and White have a very good description of polarization, birefringence and the construction of Nicol and Wollaston prisms. The prisms used in DIC are modified Wollaston prisms. Hoffman Modulation Contrast was first described by Robert Hoffman in Barbara Foster gives a description of the technique, and Pluta vol 2 gives more technical details. Chapter 12 Fluorescence Anyone just starting in fluorescence microscopy should read Introduction to Fluorescence Microscopy by J.S.Ploem and H.J. Tanke. This small book covers the fundamentals of fluorescence microscopy and does so very well. A good comparison on episcopic and diascopic fluorescence is in Fluorescence Microscopes by Nikon Inc. Fluorescence Microscopy of Living Cells in Culture part A and B edited by Wang and Taylor covers most of the current techniques in biological fluorescence microscopy. Chapter 13 Confocal Laser Scanning Microscopy Handbook of Biological Confocal Microscopy 3 rd edition ed. James B. Pawley is my primary source for this chapter. For information on video and digital imaging I use Inoué. Minsky has written a fascinating account of his invention of the confocal scanning microscope in the journal Scanning. Chapter 14 Photomicrography John Gustav Delly s book Photography Through the Microscope is one of the best sources on this subject. I have also used Modern PhotoMICROgraphy by Bracegirdle and Bradbury and the Nikon brochure How To Use A Microscope And Take A Photomicrograph. A good reference on CCD chip design is in Inoue and Spring Video Microscopy 2 nd edition. Handbook of Biological Confocal Microscopy 3 rd edition ed. James B. Pawley also has more than you ever wanted to know about CCDs and digital camera technology. Chapter 15 Polarized Light A good, short introduction to polarized light microscopy is in Bradbury and Evennett Contrast Techniques in Light Microscopy chapter 5. A very good, thorough presentation of polarized light in light microscopy is Qualitative Polarized Light Microscopy by Robinson and Bradbury. References Mortimer Abramowitz, Basics and Beyond Series by published by Olympus Corporation. These books are generally available through Olympus dealers. Mortimer Abramowitz is also the principal editor of the extremely good web site for light microscopy learners called Molecular Expressions UNC-CH Pathology 464 Light Microscopy 3

4 American Optical Company, Three American Microscope Builders, American Optical Company Scientific Instrument Division, Out of print. Available in the UNC-CH Health Sciences Library. A. Bennett, H. Jupnik, H. Osterberg, and O. Richards, Phase Microscopy, Principles and Applications, John Wiley & Sons Inc., Out of print. Brian Bracegirdle, Savile Bradbury, Modern PhotoMICROgraphy, RMS Microscopy Handbooks 33, Bios Scientific Publishers, 1995, ISBN Saviel Bradbury, An Introduction to the Optical Microscope, Royal Microscopical Society Microscopy Handbooks 01, Oxford University Press, 1989, ISBN S. Bradbury and P.J. Evennett, Contrast Techniques in Light Microscopy by BIOS Scientific Publishers Ltd, 1996, ISBN John Gustav Delly, Photography Through The Microscope, Eastman Kodak Company, 1988, ISBN X. Babara Foster, Optimizing Light Microscopy for Biological and Clinical Laboratories, published by American Society for Clinical Laboratory Science and Kendall/Hunt Publishing Co, 1997, ISBN Simon Henry Gage, The Microscope 17 th Publishing Co. Out of print. edition published in 1941 by Comstock Robert Hoffman, The modulation Contrast Microscope: Principles and Performance, J. Microscopy, Vol. 110, Pt 3, August 1977, pp Shinya Inoué Video Microscopy 2 nd edition, Plenum Press, NY, 1997, ISBN F.A. Jenkins and H.E. White, Fundmentals of Optics 4 th edition, McGraw-Hill Book Co., 1976, ISBN Köhler Illumination Centenary prepared by the Royal Microscopical Society Reprinted from the Proceedings of the Royal Microscopical Society Volume 28 Part 4, October 1993 (pp i-21); Volume 29 Part 1, January 1994 (pp 22-26), and Zeiss Information No.3, 1993 (pp 27-32). Harold Malies, A Short History of the English Microscope, The Microscope Series, Microscope Publications Ltd., 1981, available from McCrone. UNC-CH Pathology 464 Light Microscopy 4

5 Marvin Minsky, Memoir on Inventing the Confocal Scanning Microscope, Scanning, Vol. 10, 1988, pp George Herbert Needham, The Practical Use of the Microscope originally published by Charles C Thomas Publisher, 1958, is now available as a reprint through McCrone. Nikon Inc. Fluorescence Microscopes Technical Bulletin, Nikon Inc. Instrument Group. This bulletin is available from Nikon representatives. Nikon Inc. How To Use A Microscope And Take A Photomicrograph, Nikon Inc. Instrument Group. This booklet is available from Nikon representatives. Donald L. Padgitt, A short History of the Early American Microscopes, The Microscope Series, Microscope Publications Ltd, 1975, available from McCrone. James B. Pawley, ed. Handbook of Biological Confocal Microscopy 2 nd edition, Springer Press, 2006, ISBN 10: X. J.S.Ploem and H.J. Tanke, Introduction to Fluorescence Microscopy, Microscopy Handbooks 10, 1987, Oxford University Press, ISBN Maksymilian Pluta - Advanced Light Microscopy: Volume 1 Principles and Basic Properties (ISBN ) 1988, and Volume 2 Specialized Methods (ISBN ) Volume 3 is Measuring Techniques.. The ISBN number for the series is These books are published by PWN-Polish Scientific Publishers- Warszawa and are available through Elsevier Science Publishing Co., Inc. PC Robinson, S. Bradbury (1992) Qualitative Polarized Light Microscopy (RMS Handbook no. 9) Oxford University Press, Oxford. (Out of print.) RMS Dictionary of Light Microscopy. This is Microscopy Handbooks # 15 in the Royal Microscopical Society Microscopy Handbooks series. Published by Oxford University Press, 1989, ISBN X. Sheehan and Harpchak, Theory and Practice of Histotechnology, 2 nd edition 1980, Mosby Co., publisher, ISBN Elizabeth M. Slayter,Optical Methods in Biology, Robert E. Krieger Publishing Co, Out of print. E. M. Slayter, and H. S. Slayter, Light and Electron Microscopy, Cambridge University Press, 1992, ISBN Robert F. Smith Microscopy and Photomicrography, A Working Manual, 2 nd edition, CRC Press 1993, ISBN UNC-CH Pathology 464 Light Microscopy 5

6 Michael Spencer, Fundamentals of Light Microscopy Cambridge University Press, 1982, ISBN Out of print. Yu-Li Wang, D. Lansing Taylor ed, Fluorescence Microscopy of Living Cells in Culture, Part A Fluorescent Analogs, Labeling Cells, and Basic Microscopy, Methods in Cell Biology, Vol 29,1989, Academic Press, Inc, ISBN ; Part B, Quantitative Fluorescence Microscopy, Imaging and Spectroscopy, Methods in Cell Biology, Vol 30, Academic Press, Inc, ISBN X. Michael B. Wilson, The Science and Art of Basic Microscopy, produced by the American Society for Medical Technology. Out of print. Wolfgang Zieler: The Optical Performance of the Light Microscope Vol 1, Microscope Publications Ltd., 1972, ISBN This series of books is available through McCrone Accessories & Components. Companies Carolina Biological Supply Company, 2700 Youk Road, Burlington, NC 27215, Ph , Fax The diatoms mentioned in the exercises in these notes are available on a single slide called Deatom Test Plate, catalog number K3-B25D. Chroma Technology Corp. 10 Imtec Lane, PO Box 489, Rockingham, VT also provides filters for fluorescent microscopy. Their web address has extensive information on filter sets. Leica AG, Ch-9435 Heerbrugg, Seitzerland, Ph , Fax , McCrone Accessories & Components, 850 Pasquinelli Drive, Westmont, IL , Ph , Fax , mac@mccrone.com, web: Nikon Inc Dobbin Rd., Suite e, Columbia, MD 21045, Ph Olympus Corp., Precision Instruments Division, 4 Nevada Drive, Lake Success, NY Omega Optical, Omega Optical, Inc Delta Campus Omega Drive Brattleboro, VT Toll free: (866) manufacturers interference filters for fluorescence microscopy. Their web site has a very good tool (Curv-o-matic) for checking UNC-CH Pathology 464 Light Microscopy 6

7 the excitation and emission spectra of just about every known dye plus the Omega filter combination that is good for those dyes. Carl Zeiss, One Zeiss Dr., Thornwood, N.Y , Ph , A very good beginning book on light microscopy - Microscopy from the Very Beginning - is available for download on the Zeiss web site. Software ImageJ is free image acquisition and processing software available from the following web site It was written by Wayne Rasband at NIH and is one of the best image processing programs available. ImageJ runs on any computer equipped with Java (a standard feature of all web capable computers). ImageJ works with 24 bit (real color) images as well as gray scale images. This web site also contains links to many other sites that offer excellent image processing software some of which is either free or shareware. UNC-CH Pathology 464 Light Microscopy 7