Table Of ContentBIOMEDICAL LIGHT MICROSCOPY
Biomedical Light Microscopy
1 James
Professor of Histology, University ofA msterdam
Faculty of Medicine, Amsterdam, The Netherlands
and
H.l Tanke
Associate Professor of Cell Biology, University of Leiden
Faculty of Medicine, Leiden, The Netherlands
~.
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Springer Science+Business Media, B.V.
Llbrary of Congress Cataloglng-in-Publlcatlon Data
James, J.
Biomedical light microscopy I J. James and H.J. Tanke.
p. em.
Ine 1u des index.
ISBN 978-94-010-5682-3 ISBN 978-94-011-3778-2 (eBook)
DOI 10.1007/978-94-011-3778-2
1. Microseope and microseopy. 1. Tanke, H. J. I!. Title.
[DNLM: 1. Mieroscopy. OH 205.2 J27bl
OH205.2.J36 1991
576' .4--dc20
DNLM/DLC
for Library of Congress 90-5243
ISBN 978-94-010-5682-3
This book is the revised second edition of the version published in 1976
by Martinus Nijhoff Medical Division.
Printed on acid-free paper
All Rights Reserved
© 1991 by Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 1991
Softcover reprint ofthe hardcover 1st edition 1991
No part of the material protected by this copyright notice may be reproduced or utilized in
any form or by any means, electronic or mechanical, including photocopying, recording, or by
any information storage and retrieval system, without written permission from the copyright
owner.
Contents
Preface IX
Chapter 1. Light microscopy as an optical system, the stand and
its parts
1.1 Basic theory 1
1.2 The objective as an optical tool; resolving power 4
1.3 Eyepieces 15
1.4 Objective and eyepiece as an integrated system 18
1.4.1 The interplay between objective and eyepiece 18
1.4.2 Tube length 19
1.4.3 Axial resolving power and depth of field 20
Recommended further reading 23
Chapter 2. The light microscope as a tool for observation and
measurement: illumination and image formation
2.1 Modulation of the illuminating light by the object 25
2.2 The stand and its parts 28
2.3 Illumination and image formation 33
2.3.1 General aspects 33
2.3.2 Types of illumination 36
2.3.3 Special types of illumination: darkground illumination 39
2.3.4 The light source 42
2.3.5 Confocal illumination 46
Recommended further reading 49
Chapter 3. Fluorescence microscopy
3.1 Theoretical background 50
3.1.1 What is fluorescence? 50
3.1.2 Physical properties of fluorescence 50
3.1.3 Spectral properties of fluorochromes 51
3.1.4 Quantum efficiency of fluorochromes 52
3.2 The fluorescence microscope 53
VI Contents
3.2.1 Incident or transmitted light illumination 53
3.2.2 Fluorescence microscopy with transmitted illumination 55
3.2.3 Fluorescence microscopy with incident illumination 55
3.2.4 Components of the fluorescence microscope 56
3.2.5 The two-wavelengths excitation method for fluores-
cence microscopy with incident light 63
Recommended further reading 66
Chapter 4. Special optical techniques of image formation
4.1 Phase-contrast microscopy 67
4.1.1 Basaltheoretical facts 67
4.1.2 Practical realization of the phase-contrast 68
4.1.3 The phase-contrast image with different objects 70
4.2 Interferometry and interference contrast 75
4.2.1 Principles of image formation in interference contrast 75
4.2.2 Differential interference contrast 79
4.3 Modulation-contrast microscopy 83
4.4 Polarization microscopy 85
4.4.1 Anisotropy as an optical phenomenon 85
4.4.2 The polarized light microscope 88
4.5 Reflection microscopy and reflection-contrast microscopy 94
4.6 Acoustic microscopy 97
4.7 Superresolution: modern developments 99
Recommended further reading 101
Chapter 5. Reproduction of microscopic images, microphotography
5.1 Drawing and drawing apparatuses 102
5.2 Microprojection 103
5.3 Television microscopy 105
5.4 Photomicrography 107
5.4.1 Some basic principles 107
5.4.2 Photographic materials 112
5.4.3 Photomicrography in practice 115
5.4.4 Colour photomicrography 119
5.4.5 Photomicrography of fluorescence images 122
5.4.6 Special techniques in microphotography 124
5.4.7 Holographic photomicroscopy 124
5.4.8 Cinemicrography 125
Recommended further reading 126
Chapter 6. Quantitative analysis of microscopic images
6.1 Introduction 127
6.2 Morphometric techniques 128
6.2.1 Estimation of distances perpendicular to the optical
axis 128
Contents Vll
6.2.2 Measurements of distances along the optical axis 131
6.2.3 Measurements of surfaces and volumes: stereo logy 133
6.3 Counting methods 140
6.4 Absorption and fluorescence measurement of cells 142
6.5 Absorption cytophotometry (cytophotometry or microphoto-
metry) 144
6.5.1 Object plane scanners 145
6.5.2 Image plane scanners 148
6.6 Fluorescence cytophotometry (cytofluorometry, microfluor-
ometry) 149
6.6.1 Theoretical background 149
6.6.2 Practical aspects of cytofluorometry 152
6.7 Flow cytometry 152
6.8 Microspectrophotometry 156
Recommended further reading 157
Chapter 7. Automation: image analysis and pattern recognition
7.1 General introduction 159
7.2 Scanning of microscopic objects: special cameras 160
7.3 The digitized image 162
7.3.1 Image processing and image analysis 162
7.3.2 Spatial resolution and grey value resolution 162
7.3.3 Intensity transformations 165
7.3.4 Segmentation of images 166
7.4 Image analysis 168
7.5 Pattern recognition 170
Recommended further reading 170
Chapter 8. Appendix: technical aspects of the microscopical obser-
vation in practice
8.1 Introduction 171
8.2 Setting up a microscope for Kohler illumination 171
8.3 Again: the object 176
8.4 On the way through the object 178
8.5 Maintenance and minor technical problems 180
8.6 Frequently occurring minor defects 182
Recommended further reading 184
Index ofsubjects 185
Preface
New interest in light microscopy of the last few years has not been backed up
by adequate general literature. This book intends to fill the gap between
specialized texts on detailed topics and general introductory booklets, mostly
dealing with the use of the conventional light microscope only. In this short
textbook both new developments in microscopy and basic facts of image
formation will be treated, including often neglected topics such as axial
resolving power, lens construction, photomicrography and correct use of
phase- en interference contrast systems. Theoretical background will be dealt
with as far as necessary for a well-considered application of these techniques
enabling a deliberate choice for the approach of a certain problem.
Over 150 illustrations (photomicrographs and diagrams) complete the
information on microscopy of the nineties in the biomedical field, intended
for scientists, doctors, technicians and research students.
Many drawings have been contributed by the illustrator R. Kreuger; the
photographic work has been executed by J. Peeterse. Secretarial assistance in
preparing the manuscript was given by Ms T. M. S. Pierik. Dr M. J. Pearson
has corrected the English of the final text.
Amsterdam/Leiden, Summer 1990 J. James
H.J. Tanke
IX
Chapter 1
Light microscopy as an optical system, the stand and
its parts
1.1 Basic theory
A microscope is an instrument to produce an enlarged image of objects for
visual observation or for reproduction of that image by video, film, computer
or by other means. In all these cases, the same optical laws apply. These laws
will be dealt with briefly in this first chapter since they provide the foundation
for the construction and the functioning of the microscope. The long and
continuing development of the microscope has been the result of an interplay
between optical and technical problems and practical solutions, and has
known both periods of stagnation and rapid progress.
The term 'microscope' is purely descriptive: the ancient Greek word
mikros means small and skopein to look. Consequently even a magnifying
glass (loupe) is entitled to be called a microscope. In fact the simple micro
scope consisting of a single lens has been an important scientific instrument
in biomedical research. In the hands of the Dutch pioneer Antoni van
Leeuwenhoek (1632-1723) it was superior even to the compound micro
scope of the time, consisting of two lenses. Only when the problems of the
correction of lens aberrations were gradually solved in the second half of the
19th century did it become possible to exploit fully the advantages of the
compound microscope over the simple microscope, i.e., a larger field of view,
more convenient use and the possibility of resolving finer detail. In that same
period also the stands became easier to use.
The term 'microscope' nowadays usually refers to a compound microscope
for visible light in any of its forms as the image forming agent. The electron
microscope (which similar to the light microscope has become a family of
instruments) in which the image is formed by a bundle of accelerated
electrons, will be dealt with only in passing. This book is devoted to light
microscopy in its most important manifestations. The use of other imaging
agents such as X-rays (applied in microradiography), infra-red or ultraviolet
light in microscopy will also not be treated: these are special techniques with
a very limited field of application.
Leaving aside the theoretical aspects of the description of light as a train
1
2 Chapter 1
of moving particles (photons) or as a wave phenomenon moving along
constructible lines (geometrical optics), let us consider what occurs in a
compound microscope. It is a very good didactic model to compare this to a
combination of a dia projector with a magnifying glass. The magnifying glass
(eyepiece) cannot make more visible than is present in the projected image of
the dia projector (the objective). The final magnification is determined by the
product of both magnifications. If an eyepiece is used with too large a
magnification, the final image will be large but hazy, as no new details are
added. On the other hand, a too low magnification of the eyepiece will not
bring out for the eye all details resolved by the objective.
The magnification of a compound microscope is brought about in the first
instance by a real and inverted intermediary image which is formed between
objective and eyepiece (Figure 1.1). The size of this image is determined by
the relation between the object distance and focal length of the objective;
usually the object is slightly beyond the focal point, resulting in the produc
tion of an enlarged image. The relation between the diameter of an object
and its counterpart in the image is called a linear or transverse magnification;
this is engraved on the objective mount referring of course to the special
situation of a focussed microscope. On modern objectives, focal length is not
mentioned.
The intermediary image is observed by the eye using the eyepiece as a
magnifier. The situation is different, however, from that of the real inter
mediary image. Since the latter is positioned just within the focal length of
the eyepiece, the final image of the entire system cannot be projected onto a
screen: it is a virtual and upright image which can be observed via the optical
system of the eye only (Figure 1.2A). As the least strained position for the
eye is that of slight accommodation, the observer in practice focusses in such
a way that the final image seems to come from a distance of 2-3 m.
Theoretically, a positioning of the intermediary image exactly in the focal
plane of the eyepiece would produce a (still virtual) image at infinity. Apart
from the fact that this is technically almost impossible, such an image is far
from ideal for the microscopist, for this would put great strain on the eye.
When the intermediary image is brought still closer to the objective so that it
/',. ......\
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Fig. 1.1 Ray diagram of a compound microscope.
Light microscopy as an optical system 3
A
B
Fig. 1.2 Schematic view of the image formation in a compound microscope set up for obser
vation with an intermediary image inside the focal point of the eyepiece (A) and in a situation
where a real image is projected on a screen (B) with a position of the intermediary image
outside the focal point of the eyepiece, i.e., the projective. FI and F2 focal points of objective
and eyepiece, respectively.
passes beyond the focal distance of the eyepiece, an inverted real image is
again formed which can be projected onto a screen (Figure 1.2B). Such a real
image is used, e.g., in photomicrography. Strictly speaking, the eyepiece then
becomes a projective. If one could bring the eye to the level of the projection
screen no sharp image could be seen: the situation is essentially different
from that with observation.
The magnification obtained with observation cannot be expressed in terms
of linear magnification since the virtual image cannot be measured. This
magnification can be described by an increase of the angle under which the
object is observed with and without the magnifying lens: the angular magnifi
cation. This angular magnification depends on the focal length f of the
eyepiece (the same situation also holds for a hand lens), but also on the
nearest distance for distinct vision of the eye of the observer. This distance is
fixed for convenience at 250 mm (near point or punctum proximum). It is
determined by the ability of the eye to accommodate, which is brought about
by a relaxation of the eye lens to a more spherical shape as a consequence of
muscular action. In young children, the near point is much closer than
250 mm, whereas after the age of 40-45 year, it comes to exceed that value
as a consequence of the reduced elasticity of the eye lens (and hence the
need for reading glasses). For the sake of practical considerations, 250 mm is
kept as the standardized value. Thus, the angular magnification of the
eyepiece (which is not strictly a fixed value) is given somewhat arbitrarily by
the formula:
250
V=-.
f
This is the magnification factor which IS engraved on the eyepiece. In
