LIGHT MICROSCOPIC TECHNIQUES IN BIOLOGY AND MEDICINE Light microscopic techniques in biology and medicine J. JAMES Department of Histology, University of Amsterdam 1976 MARTINUS NIJHOFF MEDICAL DIVISION ISBN-I3: 978-94-010-1416-8 e-ISBN-I3: 978-94-010-1414-4 001: 10.1007/978-94-010-1414-4 No part of this book may be reproduced in any form by print, photoprint, microfilm or any other means, without written permission from the publisher. PREFACE Up to about twenty-five years ago, virtually the entire field of microscopy could be overseen and even practized by any active research worker. The rapid evolution which microscopy in its broadest sense has since undergone and which has contributed greatly to our insight in many fields of biological science and medicine has, however, lead to a progressive specialisation. Both experienced investigators in clinical and biological laboratories and post- graduate students, confronted with a limited number of microscopic tech- niques in their daily research work, have increasing difficulty in keeping (or obtaining) a general idea of the many time-honoured and new possibilities which microscopy has to offer. This book has been written with the aim of presenting general informa- tion on light microscopic techniques, at a level somewhere in between booklets like those provided by microscope manufacturers (which are often too much focussed on the production program of a particular make) and very advanced treatises with a thorough mathematical treatment of all phenomena concerned. The physically oriented texts moreover often do not sufficiently take into account the practical situation in a medical or biolog- ical laboratory; on the other hand, the value of really understanding what one is doing in using a microscopic technique is often underestimated. At- tempt has been made, therefore, to present sufficient background informa- tion necessary for a rational application of the different microscopical tech- niques in their mutual relationship. The text has thus deliberately been given a twofold character, that of a practical guide and of a scientific introduction with references for further reading. Both aspects have a somewhat different emphasis in the two main sections of the book, the first seven chapters dealing with conventional techniques and the second part devoted to ad- vanced techniques of microscopy. In this second part, much attention has been paid to new image-forming principles and quantitative aspects of micro- VI PREFACE scopy. Unlike an earlier version of this book in Dutch published in 1969, electron microscopy has only been dealt with in passing to show certain similarities and dissimilarities between light and electron rays as image- forming agents; several recently published books deal adequately with this rapidly expanding group of techniques. Moreover, the author's experience lies more in the domain of light microscopy and it is a sufficient challenge to deal with this field alone. I am indebted to some colleagues from Holland and abroad for help in reading some parts of the manuscript and/or providing material, tips or references; in particular I want to thank Dr. Goldstein (Sheffield), Dr. Ploem and Dr. de Bruin (Leiden). The photographic work has again been performed by Mr. 1. Peeterse. New drawings and graphs have been made by Mr. J. van Dusschoten (those kept from the older Dutch edition were executed by Mr. E. J. J. Eerkens). Dr. R. D. R. Birtwhistle has been the invaluable adviser in the preparation of the English text, reviewing the entire manuscript, which was read with the proofs by Prof. D. B. Kroon; Dr. P. Mestres (Bochum) helped in translating technical terms into Spanish for the four-lingual technical vocabulary. Miss M. E. Tollenaar performed the laborious task of typing the entire manu- script and bibliography from start to finish. The publisher has met with a great number of reasonable and unreasonable wishes and good co- operation has been maintained during the production of the printed text. Histological Laboratory, J. James University of Amsterdam January 1976 CONTENTS Part I: Conventional techniques of microscopic observation CHAPTER 1 SOME ESSENTIALS OF GEOMETRICAL OPTICS Geometrical optics and wave optics / 3 Resolving power and the eye / 4 The simple microscope / 6 Lens aberrations / 8 Properties oflens combinations / 12 The compound microscope / 15 Suggestions for further reading / 18 CHAPTER 2 FROM OPTICAL PRINCIPLE TO A PRACTICAL INSTRUMENT Some notes about the history of the microscope / 19 The stand and its parts / 26 Stereoscopic vision and the microscope / 32 Literature cited and suggestions for further reading / 38 CHAPTER 3 OBJECTIVES Numerical aperture I 39 Immersion-objectives / 42 The cover glass effect / 45 Types of objective / 49 VIII CONTENTS Qualifications and performance of an objective ! 54 Special objectives ! 56 - mirror- or reflecting objectives ! 57 - objectives for use with invisible light ! 58 - photomicrographic objectives ! 58 Literature cited and suggestions for further reading ! 59 CHAPTER 4 EYEPIECES OR OCULARS Main types of eyepiece ! 60 Exit pupil and the eye ! 64 Eyepiece and field of view ! 66 The meaning of eyepiece magnification ! 69 Special types of eyepiece / 71 Literature cited and suggestions for further reading ! 74 CHAPTER 5 RELATION OF OBJECTIVE AND EYEPIECE Resolving power ! 75 Objective, eyepiece and the eye ! 82 Depth of field ! 86 Literature cited and suggestions for further reading ! 91 CHAPTER 6 CONDENSER AND ILLUMINA TION The function of the condenser ! 92 Critical illumination and Kohler illumination ! 99 Types of condenser / 102 Special types of illumination; incident illumination ! 103 The light source ! 108 Literature cited and suggestions for further reading / 113 CHAPTER 7 SPECIMEN, MICROSCOPE AND OBSERVER; MICROSCOPY IN PRACTICE The microscopic object as an optical model / 114 CONTENTS IX The technique of microscopic observation / 118 - Position of the observer and the placing of the microscope / 118 - General rules for setting up a microscope / 119 - Use of immersion objectives / 121 - Light and illumination in practice / 125 - On the way through the object / 128 - Microscopy for observers wearing spectacles / 132 Maintenance and small technical difficulties / 133 - Care of the stand and the cleaning of optical components / 133 - Frequently occurring minor technical troubles / 137 Literature cited and suggestions for further reading / 140 Part II: Advanced techniques of microscopy CHAPTER 8 SPECIAL TECHNIQUES OF ILLUMINATION Oblique illumination / 143 Dark-field illumination / 145 Fluorescence microscopy / 152 - General principles / 152 - Optical arrangements with a fluorescence microscope / 156 Literature cited and suggestions for further reading / 163 CHAPTER 9 SPECIAL TECHNIQUES OF IMAGE FORMATION Phase contrast microscopy / 165 - Basic principles / 165 - Practical realization of the phase contrast principle / 168 - Some further details about the phase contrast image with different objects / 174 Interference microscopy / 180 - Basic principles; the meaning of a refractive index / 180 - Interferometric measuring systems / 183 - Differential interference contrast / 185 Polarization microscopy / 192 - Basic principles of birefringence / 192 - The polarization microscope / 196 x CONTENTS - Some applications of polarization microscopy in biological research I 202 Literature cited and suggestions for further reading I 205 CHAPTER 10 RECORDING AND REPRODUCTION OF MICROSCOPIC IMAGES Photomicrography I 207 - General principles I 207 - Photomicrographic equipment 209 - The photomicrographic exposure I 213 - Contrasts in the negative I 218 - Colour photomicrography I 229 Some special techniques in photomicrography I 233 - Microflash I 233 - Stereophotomicrography 234 - Holography I 235 - Cinemicrography / 236 Other techniques for registration and reproduction of microscopic images I 239 - Drawing devices 239 - Microprojection 243 - Television-microscopy I 245 Literature cited and suggestions for further reading / 247 CHAPTER 11 MEASUREMENTS WITH THE MICROSCOPE General introduction I 249 Morphometric analysis I 250 - Measurement of length in a focussing plane I 250 - Measurement of distances along the optical axis / 256 - Measurement oflength oblique to the optical axis I 259 Measurement of areas and volumes I 259 - Measurement of areas I 259 - Measurement of volumes; stereology I 264 Automatic and semi-automatic image analysis I 270 Microspectrophotometry and microphotometry I 275 - Microspectrophotometry I 275 CONTENTS XI - Microphotometry / 279 Microspectrofiuorometry and microfiuorometry 283 Literature cited and suggestions for further reading / 285 CHAPTER 12 MICROSCOPY WITH INVISIBLE ELECTROMAGNETIC RADIATION Microscopy and the electromagnetic spectrum / 288 Ultraviolet microscopy / 293 Infrared microscopy / 298 Use of X-rays / 300 - Some fundamental properties of X-rays and their applications / 300 - X-ray microscopy / 304 - Contact microradiography / 304 - Projection-microradiography / 308 Literature cited and suggestions for further reading / 314 Appendix I Table of refractive indices / 316 Appendix II Four-lingual vocabulary of some commonly used technical terms in microscopy / 318 Index / 328