light microscopic techniques in biology and medicine

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 postgraduate students, confronted with a limited number of microscopic techniques 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 information 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 biological laboratory; on the other hand, the value of really understanding what one is doing in using a microscopic technique is often underestimated. Attempt has been made, therefore, to present sufficient background information necessary for a rational application of the different microscopical techniques 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 advanced 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 imageforming 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 manuscript and bibliography from start to finish. The publisher has met with a great number of reasonable and unreasonable wishes and good cooperation has been maintained during the production of the printed text.

Histological Laboratory, University of Amsterdam

J. James 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

- 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

XI

- 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

PART I

CONVENTIONAL TECHNIQUES OF MICROSCOPIC

OBSERVATION

CHAPTER I

SOME ESSENTIALS OF GEOMETRICAL OPTICS

GEOMETRICAL OPTICS AND WAVE OPTICS

Light is a form of electromagnetic radiation and as such a wave phenomenon. The wavelength is so small, however, that light rays can be considered often as rays propagating linearly. What happens when e.g. a band of light passes a lens, can be illustrated quite adequately with geometric constructions. When phenomena are studied near the level of the wavelength of light, however, geometrical optics fail to explain what happens and light should be treated purely as a wave phenomenon. This is the case when light is used in microscopy; objects with a diameter of about 0.0005 mm - such as bacteria and parts of animal cells, e.g. mitochondria - are in the same size range as the wavelength of the light with which they are to be studied.

In view of these small - and even much smaller - dimensions of object size and wavelength, which would enforce the use of increasingly smaller fractions of millimeters, the following measures have been introduced in microscopy:

micrometer, abbreviation [Lm (formerly micron or [L) = 10-6 m

nanometer, abbreviation nm = 0.001 [Lm = 10-9 m.

0.001 mm

These measures form a part of the internationally adopted SI (Systeme International) system in which the meter has become the basic unit of length (with this system, which also holds for weight, the prefix milli- means 10-3,

micro- 10-6 and nano- 10-9). The term millimicron (m[L) thus has become obsolete, although it is still occasionally used. Another unit of length which does not fit into the SI system is the Angstrom-unit, abbreviation Au or A (called after a Swedish physicist from the nineteenth century) which is 0.1 nm or 10-10 m. It is still often employed in electron microscopy and for the expression of wavelengths, although a tendency now exists to replace this unit which dates from before the international metric system by an universal expression in [Lm and nm.

4 SOME ESSENTIALS OF GEOMETRICAL OPTICS

The term microscopy is associated in first instance with the use of visible light, with a wavelength from about 380 nm to 760 nm viewed by the eye as violet or red, respectively. In microscopy other types of radiation from the electromagnetic spectrum than light are also used, such as infrared light with a longer wavelength than that of visible light and ultraviolet light with a shorter one. Infrared and ultraviolet light do not differ essentially in their physical properties from visible light. This is no longer the case with X-rays or rontgen-rays with wavelengths of a few Angstrom-units, or fractions thereof (chapter 12). With bundles of accelerated electrons (comparable in their physical behaviour with electromagnetic waves) the wavelength is likewise a small fraction of an A. In contrast to the situation with X-rays, electron waves can be influenced in their course by electromagnetic fields, which has made possible the development of electron microscopy. Both in X-ray microscopy and in electron microscopy, mainly geometric phenomena have to be taken into account in explaining the formation of the image. Objects which come even approximately within the size range of these short wavelengths cannot be observed with X-rays or electron microscopy, but wave optics can be applied here for sUbmicroscopic analysis (X-ray diffraction, electron diffraction).

All image forming agents used in microscopy have in common physically that they are electromagnetic radiations, or behave as such. They have been shown to propagate with equal velocity in the same matter. Because in a homogeneous medium the propagation velocity of an electromagnetic wave equals the product of wavelength and vibration frequency, the latter quantity is inversely proportional to the wavelength.

RESOLVING POWER AND THE EYE

With a normally built, so-called emmetropic eye an object at infinity (in practice: far removed from the eye) forms a sharp image on the retina (fig. 1.IA). When the object is approximated to the eye, the image remains sharp up to a distance of about 250 mm. This is brought about by the fact that the refractive power of the eye lens is continuously enhanced hy what is called accommodation. The closest distance to the eye at which a sharp image can be formed is called the near point (punctum proximum); although large individual variations exist, it has been standardized at 250 mm for optical calculations. The observation of objects at a distance of much less than 250 mm is as a rule possible with younger persons; the accommodation (brought about by an increase of the curvature of the eye lens when the

RESOLVING POWER AND THE EYE 5

A

c

Fig. 1.1. A Observation of a distant object. B Observation of the same object, moved towards the eye up to the near point. The accommodation is maximal, the angle of vision ()( as large as possible. C Observation of the same object with a simple microscope (Joupe). The angle of observation has been enlarged to ~, the image is far removed ('in the infinite'), due to the positioning of the object in or near the focal plane of the lens; accommodation is slight or absent, the eye lens has a flattened form.

circular tension exerted on it is relieved by muscular contraction) then puts a sensible strain on the eye.

In moving an object towards the eye, the image on the retina will grow: one takes a 'closer look' (fig. l.lA and B). This enlargement of the image is obviously limited by the nearest point to the eye at which distinct vision is still possible as mentioned before (P in fig. l.lB). This distance increases with age, from about 7 cm at ten to about 20 cm as forty is approached, after which it increases slowly. Apart from age, variations in this distance can also be due to other causes, e.g. myopy in which it may be considerably shorter.

Except by the mutual distance of the photoreceptors in the retina, which can be considered as fairly constant, the ability of the eye to recognize two closely related points as discrete entities is determined by the angle at which the light rays from both points enter the eye. This angle will be the greater when the near point is closer to the eye. When the smallest distance which can be separated is 8 mm, the resolving power is defined as 1/8.


Normal visual acuity is sufficient to distinguish points which subtend an angle of one minute of arc, which corresponds with a distance between retinal images of 4-5 [Lm. As the diameter of the retinal receptors is just less than 3 [Lm, it can be concluded that two adjacent points of light can be distinguished if their images fall on two separate photo receptors with an intermediate, unstimulated retinal receptor. If it is assumed that the near point at which the largest sharp image is obtained is at the standard 250 mm, this would correspond with a minimal distance of about 0.07 mm. This can be reached under the most ideal circumstances, however; this minimal distance is usually reckoned at 0.1-0.2 mm.

It is obvious that if the near point is closer to the eye than the standardized 250 mm, the image in the retina will increase correspondingly, enhancing the resolving power. As explained before, this is possible with younger persons and in the case of myopy. These two factors are independent from one another so that they can have a cumulative effect; a myopic teen-ager sometimes can accommodate until 50 mm, enabling the separation of distances of 0.020 to 0.015 mmI . On the other hand, the difference in refractive power of the eye in the two states of complete relaxation and maximal accommodation decreases with age; hence the near point recedes, so that between 40 and 50 years it comes to exceed 250 mm. This involves a corresponding decrease in resolving power of the eye, without the quality of the retina being involved. As a rule, this phenomenon can be adequately compensated by reading glasses.

Apart from the formation of the geometric image of the object discussed so far, contrast and brightness of the image play a role. It can be easily demonstrated that two closely applied dots of ink on brown paper will fuse more easily for the eye than two similar dots on white paper; illumination with a candle or a strong light bulb will also make a great difference. These are influences on the resolving power which affect the quality and not the size of the retinal image. In contrast to the effects related to the accommodation, these phenomena occur also in observing objects at larger distances.

THE SIMPLE MICROSCOPE

Assuming that brightness and contrast are optimal, it is obvious that the resolving power of the eye can be increased only when the angle of vision,

1. Probably this forms an explanation for the extremely fine detail discernible only with a loupe in some miniatures from the early middle ages. It is beyond question that these small paintings were made in a period when magnifying glasses were not yet in use.

THE SIMPLE MICROSCOPE 7

under which the object is observed, is increased beyond one minute of arc. Instruments for modifying the course of the light rays to this end are called microscopes. (The name in itself has no pretentions: the greek mikros means small, and skopein = to look). Distinction is made between simple microscopes, which consist of a single lens, and compound microscopes, formed by two separate lenses, which cooperate in a well-defined way. For different reasons which will be discussed later on, it makes sense to build up each of those lenses from a combination of lenses (compound lens).

The simple microscope, which may be called a magnifying glass, consists of a positive lens which is placed between the object and the eye. In this situation, the observer more or less unconsciously puts the object in, or near to, the focal point of the lens. This puts less strain on the observer, as the light rays from the object thus reach the eye with a virtually parallel course, so that accommodation is absent or slight (Fig. 1.1 C). This is of itself an advantage, apart from the magnification. The image is in the infinite (in practice: at a certain distance) and is in the same position as the object. This is called an upright, virtual image formed by the magnifying glass; it cannot be projected on a screen.

From fig. 1.1 can be derived that when the object is in the near point in the situation of l.lB, the magnification A, when using the magnifying glass in fig. l.IC, will amount to ~/(l. When (l and ~ are small angles, one may substitute the tangents of the angles for the angles, so that:

A = L = tan ~ = 250 (l tan (l f

in which f stands for the focal length of the lens used. The magnification attained is thus inversely proportional to the focal length.

The refracting power of a lens can thus be expressed in mm focal length, or alternatively in dioptres. The power in dioptres is the reciprocal of the focal length in meters. This classic optical measure which is seldom applied in microscopy is highly arbitrary. It is only important to note that a magnifying glass with a power of 15 dioptres has by no means a magnification of 15 x, but (when used as a loupe) 250/66, consequently slightly under 4 x. The magnifying power of a lens expressed in this way, in which the apparent size of an object seen through a lens is related to its angular size without that instrument, is called the angular magnification. This should be distinguished clearly from the linear or transverse magnification, which just means the ratio of image size to object size, in the situation of a real image at a given image distance (e.g. in the intermediary image of the compound microscope, seep. 15).


If one takes another look at fig. 1. 1 C, one could argue that accommodation could be brought into play here again; if the object is approached to the lens, the image will be brought from the 'infinite' to a distance closer to the eye. By approaching his eye as close as possible to the lens, a young observer could theoretically enhance the value of A by bringing the image in his near point, close to the eye. The gain is rather slight, however, and must be paid for by the strain of maximal accommodation; with more powerful lenses the difference becomes nearly nil. As the loupe is seldom used nowadays for magnifications higher than 10-15 x, the problem of the resolving power of the simple microscope no longer plays a role of any importance. As will be explained in the next chapter, the simple microscope has been applied in the past (until about 1830) for magnifications up to several hundreds of times.

Finally, in using a loupe, it is advisable to place the eye as near to the lens as possible, as the entire field of view of the loupe cannot otherwise be utilized, due to the fact that the pupillar border in the eye comes to limit the rays entering the eye (cf. fig. 1.1 C); this occurs especially with lenses of a higher refractive power. This can be demonstrated simply by holding a lens at focal distance against a page of a book and moving the eye subsequently from the lens. At a given moment an ever increasing 'keyhole-effect' occurs and finally only a few letters can be overseen. As will be explained in chapter 4 and 5, these problems with the simple microscope also playa role with the eyepiece of the compound microscope.

LENS ABERRA nONS

So far, it has been assumed that light can be considered as homogeneous beams of light which behave exactly according to the rules of geometrical optics. Even apart from wave optics this is a rather crude picture, however, which can be applied only for a global consideration. It appears that a lens, even with the most pure spherical form of its surfaces, does not produce a perfect image of the object. Of these lens aberrations (which would better be called image errors) a number are known, which can be divided into two groups:

1. Image errors which occur with light of a single wavelength: monochromatic aberrations;

2. Aberrations in the image which are caused by differences in refraction of light of different wavelength: chromatic aberrations.

LENS ABERRATIONS 9

A variety of monochromatic aberrations exists; for practical microscopy the most important are spherical aberration and curvature offield.

Spherical aberration or aperture error which is invariably present in each simple lens, is due to the fact that rays which pass through the outer portion of a lens appear to have a different focal point than rays which pass through the lens near the axis (fig. 1.2). This phenomenon occurs with refraction at

Fig. 1.2. Spherical aberration. When the object is a luminous point emlttmg monochromatic hght, a bnght circle with darker border will be seen on a screen held in the object space at A, at plane B a bright ring with a darker centre will be seen. AP angular aperture of the lens.

spherical surfaces, but also with reflection. As the phenomenon increases with the distance from the optical axis, it is clear that with increasing angular aperture of the lens (AP in fig. 1.2) the phenomenon will become more pronounced. With curvature of field, the image of a flat plane perpendicular to the optical axis becomes a curved surface. This error increases also with the lens aperture, but it should be corrected independently from the spherical aberration. The same is the case for other monochromatic errors such as coma, astigmatism and distorsion which have a lesser significance for practical light microscopy (but not for technical optics !).

Chromatic aberration arises from the fact that the refractive index of the lens material is not the same for different wavelengths; the focal point will be nearer to the lens with shorter wavelengths. On each point of the lens surface, so-called dispersion takes place of the emanating light rays (D in fig. 1.3). As a result of this, the image of a point that sends out 'white' light, is a spectrum along the optical axis and not a single white image point. The points for the violet range of the spectrum are the nearest to the lens and those for the red side of the spectrum the farthest away; those for green


Fig.I.3. Chromatic aberration. As a consequence of different refraction of rays of various wavelength coming from a luminous point emitting mixed light, dispersion occurs for which the distance D is a measure. On the optical axis a spectrum of image points will be formed with points for violet (V) and red (R) at the extremities.

light would fall about half-way. At the image points for light of shorter wavelength (violet-blue) near the lens, a screen would show an unsharp ring of orange-red light, whereas vice versa a red image-point further from the lens (near R in fig. 1.3) would be surrounded by blue l • The degree of dispersion (i.e. the length of the distance V-R) is dependent on the physical nature of the lens material with a given focal distance.

The kind of chromatic defect considered so far is that occuring with a point source on the axis of an optical system, giving rise to an infinite number of separate axial images; this defect is often referred to as longitudinal chromatic aberration. Similarly, with images of the optical axis, light of each wavelength will form a series of images of an object which all will have a different size. This phenomenon is called lateral chromatic aberration or chromatic difference of magnification. Thus longitudinal chromatic aberration causes the images to be at different places along the optical axis, while lateral chromatic aberration causes the images to have different sizes. Both types of chromatic aberration seriously impair the image quality.

The phenomenon of dispersion is related to the fact that the propagation velocity of an electromagnetic radiation in a medium is a function of the vibration frequency - and therefore the wavelength - of that radiation. When light, or another electromagnetic radiation, is reflected by a mirror, no change in medium takes place and no dispersion occurs. Consequently, an

1. The use of monochromatic light to avoid this lens error seems simple enough; it has been tried by different investigators in the previous century, but has not found more general application in microscopy, partly because it is impossible to use staining techniques to enhance the contrasts in the image.

LENS ABERRATIONS 11

image formed by a concave spherical mirror is free from chromatic aberration (but not from other lens aberrations !). This principle is applied in mirror objectives (chapter 3).

In table I some data are collected concerning the dispersion properties of a few materials used in the construction of lenses compared with water. v is the dispersion index of Abbe, which can be calculated from the refractive indices at certain fixed wavelengths. The smaller this index, the larger the dispersive power of a material and the larger the angle D in fig. 1.3. The value of n is given at 20° C, as usual (a normalized refraction index nD 20

refers to the refractive index at 20° C at the yellow sodium D-line of the spectrum). It is to be noted that the dispersion does not vary with the refractive index, as thought by Newton; this would make the correction of chromatic aberration impossible.

TABLE 1. DISPERSION PROPERTIES OF SOME MATERIALS USED IN THE MANUFACTURE OF LENSES.

n for red n for green n for violet light light light

(A = 656 nrn) (A = 546nrn) (A = 435 nrn) v

crown glass 1.507 1.511 1.520 61.9 flint glass 1.615 1.624 1.642 36.3 fluorspar (CaF,) 1.432 1.435 1.439 95.2 quartz 1.542 1.546 1.554 69.9 water 1.331 1.334 1.340 55.6

When an image of an object is formed by a lens, both chromatic aberration and spherical aberration (along with other monochromatic errors) will distort the image in a varying degree. As in microscopy lenses are often used with a large aperture which suffer greatly from these errors, it is prerequisite that the lens aberrations should be suppressed as much as possible. This can be overcome technically because lens errors, as physical phenomena, are subject to laws that can be formulated; they can thus, in given circumstances, be predicted. With a combination of lenses manufactured from different types of glass and with a given curvature, the chromatic aberration for e.g. two colours can be suppressed by compensating the differences in dispersion. In fig. 1.4 such a situation is illustrated in a so-called achromatic doublet.

Such a system should be further corrected for spherical aberration, without jeopardizing the suppression of the chromatic aberration already


Fig. 1.4. Correction of chromatic aberration for two colours by the combination of a positive and a negative lens of materials with different dispersion; both image points come to coincide, but it entails an increase in the focal distance of the refractive complex.

achieved. This is possible e.g. by adding to the system a second doublet with an opposite effect on the spherical aberration.

All these problems hardly arise with the simple microscope, but they are of utmost importance in the compound microscope, as this instrument is exploited to the most extreme limits of feasability in forming an image with light. It is very clear that with the large lens apertures involved, the degree of lens correction will influence to a large extent the effective resolving power that can be reached. In the past, this was a great obstacle in the evolution of the compound microscope (chapter 2).

PROPERTIES OF LENS COMBINATIONS

To allow correction for image errors, the essential units forming the image with a compound microscope, the ob;ective, the eyepiece (or ocular), and the condenser for illumination, consist as a rule of a combination of single or compound lenses.1 Before the forming of the image in the compound microscope can be dealt with, the ray tracing within a compound system should be very briefly treated.

As is known from elementary optics, the following rules hold true for the ray tracing of an image by a thin lens (fig. 1.5): a. a light ray, entering the lens parallel to the optical axis, passes through the

second focal point F2 at the image side. In accordance with the rule of the reversibility of the light path, a ray entering through the object side focal point FI will leave the lens parallel to the optic axis.

1. With regard to the eyepiece this is not only a matter oflens errors, as will be explained in chapter 4.

PROPERTIES OF LENS COMBINATIONS 13

H o .. .. •

Fig. 1.5. Image-forming by a thin lens: 0 object distance, i image distance, H principal plane, PI and p. focal points, f focal distance. The supposition is that the same refractive index exists in object- and image space.

b. a light ray passing through the optic centre of the lens, is not refracted at all.

c. the relations between object distance 0 and image distance i is given in the well-known formula

111 -+-=o i f

in which f is the focal length. Strictly spoken this only holds true when there is air on both sides of the lens, otherwise the formula would be

~+~2=~=~, o 1 fl f2

in which n1 en n2 and f1 and f2 are the refractive index and focal distance in object space and image space, respectively.

d. the total magnification M follows from the conformity of the two pairs of triangles which touch in the focal points on both sides:

f o-f M=-=-.

i-f f

Again, this only holds when the lens is in air; otherwise the second and

third term become ~ and o-f2 . O-fl f2

When an image is formed by a thick lens or a combination of lenses separated by a given distance, the course of the rays is to be traced in a different way as in the case of a thin lens (fig. 1.6). It appears now that the figure seems to be pulled apart in two halves; the so-called principal plane H in


o

Fig. 1.6. Image-forming by a thick lens complex: H, and H. principal planes, other symbols as in fig. 1.5.

which the refractive power of the thin lens of fig. 1.5 could be assumed to be concentrated, is replaced now by two principal planes Hl and H2 which are images of each other with a linear magnification of + 1. If one could approach both principal planes to each other, exactly the situation of fig. 1.5 would arise.

A thin lens thus can be considered as a combination of two refractive surfaces in which both principal planes coincide; for simple lenses with a certain thickness, this is no longer the case.

For ray tracing outside ofa lens complex, the combination can be considered as a single lens. Certain special circumstances exist, however, in a compound system with regard to bundle limitation for paraxial rays reaching the combination. Here it cannot be assumed, that a light ray striking the first refracting surface and not being reflected can take part in the formation of the image. With a complex system as shown in fig. 1.7, the following facts can be established.

If the light bundle passing through such a combination is limited not only by the diameter of the lenses but also by other factors such as stops (D1,

D 2, Da), the situation can become complicated. The essential question is

0,

I I I 0;

Fig. 1.7. Entrance pupil (02) and exit pupil (02') of a complex lens system with different stops !imitating the bundle; further explanation in the text.

PROPERTIES OF LENS COMBINATIONS 15

how great an angle of the apex of the cone emanating from the object point A can be, so that all rays from the bundle participate in the formation of the image. This question may be answered by constructing an image in the object space of all the stops and other bundle limitations and discover subsequently which aperture is the most narrow as seen from A. In fig. 1.7 this is clearly the image 0'2 from stop O2, The aperture at 0'2 thus indicates the limitation of the light cone accepted by the system and is called the entrance pupil of the lens combination. The exit pupil in the image space (02" in fig. 1.7) is conjugate to the entrance pupil. It is easy to see that O 2'

and O 2'' will be images of each other. In complex lens combinations such as objectives the entrance pupil is usually made to coincide more or less with the mount of the front lens; light-rays which would reach the front lens at the periphery of the entrance pupil cannot in any case take part in the formation of the image.

THE COMPOUND MICROSCOPE

After all that has been said so far about image formation in general, the following facts can be stated about the formation of an image in the compound microscope with its coordinative action of objective and eyepiece (see fig. 1.8). 1. The objective, usually of short focal length, forms a magnified image of

the object at a certain distance. This image, the intermediary image, is inverted and real: it can be demonstrated on a screen or with a photographic plate. As the image distance is much greater than the object distance, the object should be placed somewhat outside the focal point of the objective.

2. The intermediary image, serving as an object in its turn, is observed by the eye using the eyepiece as a magnifier. As the intermediary image is near to the focal point of the eyepiece, the final image is formed at some distance from the eye. The place of the intermediary image is, of course, dependent on the object distance. As the least strained position for the eye is that of slight accommodation, the object is focussed so that the intermediary image falls just within the focal distance of the eyepiece. Consequently the final image, at some distance from the eye, is virtual and, since a magnifier used under these conditions does not invert the image again, inverted like the intermediary image (fig. 1.8). It should be noted that this virtual image cannot be shown on a screen or with a photographic plate; the image used in photomicrography is another one (cf. chapter 10).


f~~"""",::;::;~ __ I -'-_ I - ..... -_

I --~~--_ l - ..... --:---_ I " I ~~~~~

I ___ 1 ___ _

,

FOB Foe

Fig. 1.8. Ray diagram of a compound microscope; the object is just outside of the first focal plane of the objective (FOB); the intermediary image is just within the focal plane of the eyepiece (FOC); compare with fig. 1.1.

3. The magnification is clearly the product of the magnification of objective and eyepiece, Mo x Me. From the formula d) on page 13 it follows that the magnification of the objective is equal to the distance Ll between focal plane and object plane of the objective, divided by the focal distance of the objective. The angular magnification of the eyepiece is, according

250 to the formula on page 7, - Consequently, the total magnification

f of the compound microscope is:

Ll 250 Magnification objective x Magnification eyepiece = x --.

fobj. foe.

The value for Ll or optical tube length virtually equals the distance between the focal points of objective and eyepiece, when the image plane of the objective falls just within the focal length of the eyepiece. It is clear that this factor may change considerably; the focal lengths of ordinary objectives usually vary between 2 and 50 mm and the position of the intermediary image is kept at a fixed plane within the microscope tube. The mechanical tube length is merely the length of the metallic tube in which the eyepiece is inserted at one end, with the objective at the other end. It has been standardized at 160 mm for virtually all makes, although a few manufacturers construct microscopes with a mechanical tube length of 170 mm.

With all modern microscopes the nominal magnifications of objectives and eyepieces are engraved on the mounts; to calculate the final magnification with a given combination one simply multiplies one number by the other. For measurements it is important to know that a 10 x objective may have a Lljf of 9.7, or perhaps 10.2: the magnification engraved on the mount can never be completely relied upon, partly because the value for Ll may

THE COMPOUND MICROSCOPE 17

vary somewhat. In some cases a multiplication factor of 1.25 or 1.50 has to be taken into account for the final magnification, when the optical tube length is altered by a binocular unit or other intermediary optical device.

Finally a few words about the measuring of light intensities or photometry in its connection with microscopy; some confusion exists here with regard to notions and unities which have changed continuously. The most recently accepted international system is the following (13th general conference on weights and measures, Paris 1967). The unity of luminous intensity, defined as luminous flux proceeding from a point source per unit solid angle is the candela (contraction cd). An extended light source and/or a light-reflecting surface will give off light in all directions. The luminous power leaving the surface per unit solid angle is called the luminance; it is expressed in candela per square meter, or Stilb (contraction Sb). When one looks at a surface which emits or reflects light it is the luminance which, for a given state of adaptation, determines the impression of brightness of the surface. Brightness as a subjective phenomenon should be clearly distinguished from luminance as a physically determined amount of luminous flux emitted; until recently the term brightness has been used both for the subjective and the objective phenomenon.

When a luminous flux is incident upon a surface, it is said to be illuminated and the flux received per unit area is called the illuminance or lighting intensity; it is measured in units of lumen/m2, or lux. When an image is formed from an illuminated or light emitting object, the luminance of the image will be the same as that of the object, as the angle of the bundle is subject to a change which corresponds with the alteration in the surface: consequently, the total luminous flux remains theoretically the same. The illumination changes, however, proportionally to the alteration in the surface area of the object with regard to the image. When in a given situation an image has a linear magnification of q, the lighting intensity (on which e.g. the exposure time in photomicrography depends) will diminish by a factor q2. Moreover, the illuminance of the image changes proportionally with the size of the light cone accepted by the image forming system and consequently with the second power of the effective angular aperture used (AP in fig. 1.2). With regard to the formation of the intermediary image by the compound microscope, this has the following consequence. Although the aperture augments with increasing magnification of an objective, resulting in a certain compensation, this gain falls far short of the decrease in illuminance, so that a special illumination apparatus becomes necessary to maintain a workable intensity of illumination up to higher magnifications.


Moreover, this apparatus has the even more important task to provide a cone of light with a sufficient top angle for exploiting the full power of the objective with significant consequences, as will be dealt with in chapter 6.

So far it has been taken for granted that in forming the image, the total luminous energy remains unaltered; in any optical system, however, losses do occur to a varying degree. These can be caused by unclean lenses resulting in light absorption, but a certain percentage of the light energy is lost by reflection which occurs at virtually any refracting surface. If light is falling onto a clean interface between media of refractive index n1 and n2, a propor-

2

tion of the light equalling ( n1 - n2) is lost by reflection. At an air-glass n1 + n2

boundary this loss amounts to something like 3-8%, depending on the type of glass. With higher magnifications, especially with highly corrected objectives in which the light has to pass a large number of surfaces, the total loss by reflection may amount to over 50%. Not only is this light lost for the formation of the image, but it also diminishes the contrast in the image through generation of stray light within the system. Many lens surfaces are therefore coated with a very thin film of a material having a refractive index intermediate between that of air and glass. When a certain relation exists between the thickness of this layer and the average wavelength of light falling on this coated boundary, the reflection of oblique rays can be considerably suppressed. The reflection at a glass boundary treated in this way (which can be recognized by a purple hue when held oblique to incident light) can be limited to about 1%.

SUGGESTIONS FOR FURTHER READING

A. C. S. van Heel and C. H. F. Venzel: What is light? Weidenfeld and Nicolson, London 1968.

R. S. Longhurst: Geometrical and physical optics, 3rd ed. Longmans, London 1965. W. R. McCluney: Radiometry and photometry. Am. J. Physics 36 (1968) 977-979. J. R. Meyer-Arendt: Introduction to classical and modern optics. Prentice-Hall, Englewood

Cliffs 1972. H. G. Zimmer: Geometrical optics. Springer Verlag, New York-Heidelberg-Berlin 1970.

CHAPTER 2

FROM OPTICAL PRINCIPLE TO A PRACTICAL INSTRUMENT

SOME NOTES ABOUT THE HISTORY OF THE MICROSCOPE

While lenses, used as magnifying glasses or primitive spectacles, were known by the end of the thirteenth century, the use of apparatus consisting of a combination of two lenses (astronomic telescopes and compound microscopes) is of much more recent origin. In the beginning of the seventeenth century Gallileo made his famous discoveries of ceiestial bodies with a telescope, consisting of a convex and a concave lens placed at a certain distance apart. The oldest-known descriptions and illustrations! of a compound microscope come from Holland; opinions differ as to who may be considered its inventor, if anyone.

Important scientific investigators in the second half of the seventeenth century who were engaged in systematic application of this microscope for biological and medical purposes were Marcello Malpighi in Italy and Robert Hooke, curator of experiments at the Royal Society in London. In 1665 Hooke published an illustrated book 'Micrographia'. In this curious collection of observations of such divergent objects as the leaves of the stinging nettle, the anatomy of a louse, the eyes of insects and the functioning of an alcohol thermometer, also the first description is given of 'cellulae' in different botanical tissues, amongst which cork. This first evidence for the existence of cellular structures in living organisms - regarded as a curiosity by his contemporaries - was given by Hooke on the basis of observations made with the only microscope in the possession of the Royal Society in this period, of which a description together with a picture was given in the preface of the 'Micrographia'. It is known that these instruments cost about three pounds (a large sum in those days) and that they had a magnification of 30-40 x; a few have still been preserved. At both ends of the often ingeniously decorated tube a simple objective lens and a simple eyepiece were mounted. The light from a candle or spirit flame could be concentrated

1. A drawing has been found in a diary of Isaack Beeckman of Middelburg from the year 1625; the instrument depicted probably was manufactured by the spectacle maker Zacharias Jansen at Middelburg.

20 FROM OPTICAL PRINCIPLE TO A PRACTICAL INSTRUMENT

Fig.2.1. Robert Hooke's compound microscope, with illumination apparatus and a transverse section of an eyepiece (marked fig. 4); drawing printed in the Micrographia (1665).

on the object by means of a glass sphere filled with water (fig. 2.1); after inclining the tube forwards so that it adopted a horizontal position, observation with transmitted light was possible.

Even if it is taken into account that the focussing (with the thread of a screw) was somewhat crude, it can be said that Hooke's instrument as a whole showed a certain degree of technical perfection. In comparison, the microscope shown in fig. 2.2 dating from the same period makes a very primitive and small impression. Yet, spectacular discoveries have been made with this type of instrument by Anthoni van Leeuwenhoek, citizen of the city of Delft in Holland and 'amateur' research-worker. It should be noted in passing that the gap between professional and amateur scientist was really not great in that period. It may be of some interest in this connection

SOME NOTES ABOUT THE HISTORY OF THE MICROSCOPE 21

Fig. 2.2. Left image: microscope of van Leeuwenhoek, approximately natural size; right image: use of van Leeuwenhoek's microscope focussed onto the border of a snip of paper fixed to the object-pin.

that van Leeuwenhoek owned a linen drapers shop; in this trade rather strong lenses were used for control of the quality of the linen, whereby it might have occurred to him to start his work on microscopy. In a period of fifty years, from 1671 until his death at the age of 91 in 1723, this unique personality made his observations on the most divergent matters, searching down to an order of dimensions beyond reach for any of his contemporaries. In an early stage he was able to improve some of the observations of Hooke and even check them with measurements. In a later stage of his investigations, he gave descriptions of bacteria, spermatozoa and blood cells which could not be confirmed and further extended until the nineteenth century. The microscope from fig. 2.2 consisted, like the many others which he made with his own hands, of two rather coarsely manufactured plates of copper or silver, which were clinched together. Between two openings in those plates he fixed the small hand-ground lens; the instrument in fig. 2.2 has a focal distance of just under a millimeter. The object was attached to a pin on which it could be focussed with two screws; the instrument was held upright and brought against the light close to the eye.

Van Cittert (1934) has examined a few of these microscopes. Those of the type depicted in fig. 2.2 appeared to have an angular magnification of 240-280 x ; although the surfaces were scratched, they appeared to be able to


separate a grating structure of 1/700 mm. In the period they were made, they were certainly capable of separating distances of less than a micrometer. No other microscope from the seventeenth or eighteenth century was able to achieve this and it was only after 1825 that the compound microscope reached this level of performance.

It has often been discussed what the reason might be for this curious advance of van Leeuwenhoek, and why most of his observations could not be confirmed by other microscopists for more than a century. To a large extent the reason is a purely optical one. In the case of a compound microscope such as used by Hooke and others, the errors of the simple lenses of objective and eyepiece were multiplied through their use in series; with magnifications of over 30-40 X the image quickly became distorted. The simple microscope only suffers once from lens errors, so that it can be used at a much higher angular aperture and shorter focal length. The 'secret of van Leeuwenhoek' consisted thus essentially in the simplicity of his system. The price he had to pay for this very considerable gain in resolving power, however, was the uncomfortable way in which such a short-focussed lens must be used; to trap the very oblique rays leaving the lens, the eye had to be pressed against the plate (fig. 2.2). Few people besides van Leeuwenhoek could tolerate this for a longer period and many of his visitors complained about the strain this method of observation puts on the eye, resulting in headaches (this latter contention was confirmed by the author almost three centuries later, in preparing fig. 2.2). Apart from his perseverance, van Leeuwenhoek might well have been gifted with an exceptional visual acuity. The simple microscope, used together with the compound microscope throughout the eighteenth century, gradually fell into obscurity for use at higher magnifications when the major problems of the compound microscope - correction of the objective, the development of an adequate illumination and a practical stand - gradually found a solution in the course of the nineteenth century. The advantages of the compound microscope, such as a more comfortable method of observation, a greater effective field of view and a real intermediary image which made measurements possible, could only then be fully exploited.

In the eighteenth century, this development was far off. As for the stand, an important development was the new microscope stand made by Cuff in 1744 which had a free stage and a good fine adjustment (fig. 2.3). The uncorrected lenses, however, gave a highly distorted picture, even at comparatively low magnification (fig. 2.4). Certainly Cuff's stand meant a considerable improvement over the earlier clumsy and heavy tripod stands without improvement in the optical system, however, these instruments


Fig. 2.3. Microscope stand of Cuff, about 1750 (photograph made of an instrument from the collection of the National Museum of the History of Science in Leiden).

were doomed to serve as playthings of wealthy amateurs, who confronted their fantasies with these unclear images. A trade card of the early eighteenth century of Edward Scarlett, optician to His Majesty the King (printed, by the way, in English, French and Dutch) offers 'the greatest variety of single and double microscopes', along with reading glasses and 'magick lanternes' (Sherwood Taylor, 1957). Official science turned away from this pastime and most of the fundamental investigations in biology and medici-

24 FRO M OPTICAL PRINCIPLE TO A PRACTICAL INSTRUMENT

A

8

Fig. 2.4. Striated scales and hairs in the wing of a mosquito, about 300 x. A Photograph made with an uncorrected objective of the Cuff microscope of fig. 2.3.; the striations are not resolved; B the same object, photographed with an achromatic objective of Hartnack from about 1865; the striations are clearly resolved now.

Details of photographs made by P. van der Star, National Museum of the History of Science in Leiden, with historical microscopes.

ne in the eighteenth century were performed with the simple microscope, which then had the reputation of being more reliable. The French anatomist Bichat, who founded histology about 1800, even worked without a microscope on principle, as he held the view that this instrument always enabled one to observe what one wished to see.

The struggle with the lens errors showed its first preliminary sign of victory at the end of the eighteenth century in the combination - by haphazard trying out - of positive and negative lenses of different kinds of glass. The first succesfull more or less achromatic microscopes of low magnification were constructed near 1800; among the first was one manufactured in 1791 by a Dutch cavalry officer with optics as a hobby, Fran~ois Beeld-


snijder. In the first quarter of the nineteenth century low-power achromatic objectives became commercially available. Only after 1850 the purely empirical manufacture of complex lenses was gradually replaced by calculation of lens combinations. Although the results were at first still far removed from present day developments, the advantages of these primitive complexes over uncorrected lenses (also with regard to spherical aberration) were very striking (fig. 2.4). The simple microscope was definitely left behind.

As a consequence of the loss in magnification when e.g. a convex lens of crown glass is combined with a concave flint glass lens to form an achromatic doublet (fig. 1.4), only low-power corrected lenses could be constructed at first. The solution for the problem of correction of high-power objectives was given by Amici in 1830 when he proposed to make use of combinations of achromatic complexes, avoiding the application of lenses with very short focal length, the correction of which proved to be unsurmountable. The perfecting of the objective in the second half of the nineteenth century, especially the high level reached by Carl Zeiss and Ernst Abbe after their association in 1866, already belongs to another era of development, in which the limit of resolving power with visible light microscopy was gradually reached near the end of the nineteenth century. Abbe constructed in 1878 the first oil-immersion system with a numerical aperture larger than 1.0 and presented in 1883 an apochromatic objective with a correction of chromatic errors for three colours, in which no less than seven different types of glass had been applied. This was made possible by a specialized glass factory, founded by Otto Schott in the vicinity of the Zeiss works at Jena. Unlike those of earlier periods, these new developments immediately found their way to the consumers. Different discoveries waiting, as it were, for an improvement in resolving power, were made in this period between 1880 and 1890 by means of those new lenses in combination with the newly developed condensers. Among them can be mentioned the precise analysis of the cell division (Flemming, 1882), the discovery of the myofilaments in the contractile fibrils of the muscle cell (Kolliker, 1887) and Koch's discovery ofthe tubercle bacillus (1882).

Different further refinements of the optical and mechanical parts have taken place till far into the twentieth century. A maximal correction for chromatic aberration and curvature of field in a single objective has been realized in commercially manufactured objectives only after 1950 (planapochromats). In calculating these lens combinations, use is made of matrix algebra and computers. These refinements, how important they may be for the present day, cannot be compared, however, with the great leap forward in the period 1810-1865 (fig. 2.4).


Eyepieces generally are not as complicated in their construction as most objectives; not only are the demands not as high because of the lower magnifications at stake, eyepieces moreover have a different function than objectives. In many cases a simple system consisting of two lenses is employed, in which the lower lens is a field lens for enlarging the field of view. The principle of this ocular was given as early as 1690 by Christiaan Huygens. The evolution of the eyepiece did not stop here, of course, but the developments have been far less spectacular than with the objectives. The Huygenseyepiece has been kept in use till to-day for more simple work.

THE STAND AND ITS PARTS

Without following the historical events any further, the present-day microscope and its parts with their appropriate terminology will be reviewed briefly in the following section.

Apart from the larger types of universal research microscopes and microscopes made for special purposes, two types of microscope stands are used nowadays; 1. stands with upright (straight) tubes, or hinged stands; 2. stands with oblique (inclined) tubes, or fixed stands. The stand with an upright tube, an example of which is given in fig. 2.5, can be considered to be derived from the Cuff stand (fig. 2.3). In order to have a more comfortable attitude in looking down the tube, it is possible to incline the upper part of the stand forward about a hinge joint near the foot (M in fig. 2.5). Until about 1940, this hinged type was the universal type of microscope stand; it is still used and manufactured to-day for routine work. As the stage inclines when the upper part of the stand is bent about the axis, in the case of wet preparations movement can arise in the object in using this type of stand. This does not occur in the case of a fixed stand in which the optical axis of the tube is inclined towards the observer via a prism. This type of stand has come more and more into general use since about 1950. It is shown in a schematic drawing of a larger type of binocular microscope with built-in illumination in fig. 2.6. In the case of a more simple type of microscope of this design, a mirror-fork is fixed to the base of the stand. The fixed inclination of the upper part of the tube - monocular or binocular - towards the observer is mostly 45 0

• With all the advantages of a stand in which the tube is inclined and the stage horizontal, this fixed inclination of the upper part of the tube sometimes has certain drawbacks compared to the hinged stand of fig. 2.5, a.o. in finding the correct sitting

THE STAND AND ITS PARTS 27

Fig. 2.5. Bench microscope stand of the hinged type. An eyepiece in the draw tube (B) with scale division, sliding vertically inside the body tube.

C coarse adjustment, D fine adjustment, E revolving nosepiece with objectives F, G and H. J specimen stage with detachable specimen holder, movable by means of a vertical (K)

and a horizontal (L) sledge, with their controls. M hinge joint, enabling a variable inclination of tube and stage, N limb, 0 substage condenser holder with condenser and P filter holder, Q mirror (flat side) over which the iris diaphragm of the condenser is shown. R condenser focussing adjustment, S foot of the stand.

position for microscopic observation (see Chapter 7). Microscopes with straight or inclined tubes have no fundamental differences; in the following description of the stand and its parts both types will not be described separately, therefore, except where necessary.

Essential to any complete microscope stand are the following parts: a. A metallic tube, in which the eyepiece can be inserted at one side, whereas

at the other side objectives can be screwed in. The objectives are mounted as a rule in a so-called revolving or rotating nosepiece; this is a rotating disc with 3, 4 or 5 holes into which objectives can be screwed ready for


alternating use (fig. 2.5E). As a rule a system for centering individual objectives towards the optical axis is not present.

b. A mechanical system for varying the distance between the object and the tube with objective and eyepiece. This can be achieved with a tube which can be moved in the arm of the stand (fig. 2.5), or with a tube fixed to the arm, while the stage can be moved up and down. This latter system which is applied in virtually all fixed stands (fig. 2.6) has the advantage

~ 13

Fig. 2.6. Schematic vIew of the optic elements of a modern microscope stand with inclined binocular tube and built-in illumination. 1 low-voltage incandescent lamp, 2 collectorsystem, 3 fixed mirror, 4 field diaphragm, 5 filter holder, 6 aperture diaphragm, 7 condenser, 8 specimen, 9 objective, 10 inclining prism, 11 beam-splitting prism complex, 12 eyepieces, 13 co-axial controls of mechanical stage, 14 co-axial coarse and fine adjustment, 15 condenser focussing adjustment.

that a camera or other heavy apparatus can be attached to the tube. With a tube which moves with rack-and-pinion as with the stand of fig.


2.5, it would too easily descend with the extra weight of such an apparatus. The movement of the tube with regard to the stage has a coarse

adjustment (macrometer) and a fine adjustment (micrometer). These systems, which are independent, are usually served by two separate knobs, but can be mounted on a common axis (so-called co-axial adjustment, fig. 2.6, 2.7). In some cases a single adjustment knob serves both,

Fig. 2.7. Universal research stand with built-in illumination (arranged for transmitted illumination).

still independent, mechanical systems in which a light movement operates the fine adjustment some resistance being felt when the coarse adjustment comes into play. Low-power stereoscopic microscopes often have a coarse adjustment only (fig. 2.8). As a rule, the fine focussing control is graduated, enabling one to read the displacement ofthe objective towards the specimen (either by movement of the tube or of the stage, as already stated). This can be applied to measuring thickness with the microscope (chapter 11).

c. With the conventional microscope using transmitted light, the object stage is fixed under the tube, connected with the latter by the limb or arm of the stand (N in fig. 2.5). The stage has an opening, through which the light from the illumination apparatus can enter the object. Different mechanical systems exist for moving the object on the stage in two dimensions perpendicular to the optical axis. The most simple


Fig. 2.8. Large stand of an universal research- and photomicroscope, including a groundglass projection head for displaying the image.

device is moving the slide on the stage manually, while the object is held down with a pair of spring clips. This gives rise to difficulties with higher magnifications, of course; not only the object, but also all movements of the object are magnified by the optical system of the microscope1 . The following aids exist to perform these movements more easily.

With so-called mechanical stages, the specimen can be moved in a controlled and systematic manner. In the larger type of research micro-

1. In carrying out certain delicate manipulations under the microscope, the movements of the hand sometimes have to be reduced with a so-called micromanipulator to accomplish excursions with a needle or pipette small enough (down to a few fLm) to be performed under higher magnification.


scope (fig. 2.6), the stage itself can be moved in two directions with the object attached to the stage top by a special clip (built-in mechanical stage). A simpler device is the so-called detachable mechanical stage, in which the stage proper does not move and the object can be moved in a specimen holder in two coordinates by two control knobs (fig. 2.5). With the mechanical stage of modern design, as applied in the researchtype of stands, the controls are often coaxial (fig. 2.8) so that in moving the mechanical stage in different directions it is not necessary to switch from the horizontal to the vertical and vice versa. As a rule, mechanical stages are fitted with scales and verniers, so that a given spot on a slide can be located to 0.1 mm (see chapter 7 for applications of this principle). With certain stands the stage is circular and can be rotated around the optical axis of the microscope; if this is combined with a centering device the object can easily be turned in any desired direction e.g. for photomicrography or in polarization microscopy. In some older types of stand, the stage can be rotated and centered, in which the centering device can be used as a mechanical stage with limited range of action.

A rather new development for the moving of objects under the microscope is the sliding stage, which consists essentially of two flat discs with a central opening of which the upper one can be moved smoothly in all directions over the lower one which is fixed to the stand. This can be very useful for the study of objects which have to be searched in all directions at a magnification for which manual effecting of the movement is too coarse and a mechanical stage not sufficiently free.

d. Under the stage is located the so-called illumination apparatus which consists of a lens complex, the substage condenser with an iris diaphragm, under which a swing-out filter holder and a mirror are arranged (fig. 2.5). The mirror usually has a flat and a concave surface and is capable of being turned in any direction on its two axes. The mirror-fork is generally attached along with the substage to an extension of the limb below the stage. With built-in illumination the mirror is located somewhere in the foot of the stand (fig. 2.6). As will be explained in chapter 6, the concave mirror is hardly ever used, but it is still invariably supplied with any new microscope. The substage condenser holder - which may vary as to form, size and method of attachment to the stand - can be moved up and down along the optical axis in all good microscopes. In the research types of microscope, the substage can be aligned moreover with the objective in use with a centering adjustment. The vertical movement of the substage condenser is generally of rack-and-pinion construction similar to the coarse adjustment of the tube and in some cases even provided with a


fine adjustment. More simple students' stands sometimes have a worm -and nut action for focussing the condenser; this is in most cases unsuitable for any type of serious work, as the centering is totally unreliable and incapable of adjustment.

With the complete illumination apparatus of Abbe, the condenser fitting is swung out when the condenser has reached its lowest point. Moreover, it is possible with this type of illumination apparatus - seldom found with modern microscopes - to shift the position of the substage condenser iris diaphragm separately, to centre it or give some degree of oblique illumination.

e. The stand, with its tube, arm, stage and substage, stands with a pillar on a foot or base which supports the whole on the microscope table. The foot which can be horseshoe-shaped or (in older stands) constructed as a tripod, is generally heavy in a stand of the hinged type (fig. 2.5) as it must also function as a counterweight. The movement of the hinge joint in this type of stand is generally such that with extreme forward inclination, the tube comes into an entire horizontal position; the stand should still remain perfectly stable when so placed. For ordinary microscopy this is of no importance, but this position is sometimes used without the mirror for simple photomicrography or microprojection in an optical bench situation.

In fixed stands, the foot can be much lighter and is often rounded in many modern microscopes. With built-in illumination the whole base becomes heavier. With the larger types of research stand, which should have great stability for the attachment of different accessories, not much attention is paid to weight and dimensions. These large universal microscopes are generally used in a fixed position in a room and not taken from place to place. With the largest types of universal microscopes, transport is anyhow virtually out of the question (fig. 2.8). On the other hand, for excursions and other employment in which easy transportation is of primary importance, specially light and small stands are manufactured. With these travelling microscopes, ease of transport and convenience in use seldom form a happy marriage.

STEREOSCOPIC VISION AND THE MICROSCOPE

With the classical microscope type, as shown in fig. 2.5, the microscopic observation is made with one eye only. Although this does not necessarily entail great difficulties in practice (chapter 7), the use of both eyes for ob-

STEREOSCOPIC VISION AND THE MICROSCOPE 33

servation naturally occasions less strain. Already in the course of the nineteenth century, constructors of microscopes had considered the fact that nature has endowed us with two eyes and therefore binocular microscopic observation should be a natural aim. Moreover, stereoscopic vision could seem to be within reach as well with the development of some kind of binocular microscope. A great deal of experimentation since the tum of the century has resulted today in the following situation

The construction of microscopes with a stereoscopic image is possible without great difficulties. In fact two separately functioning microscopes are involved with their optical axes at a small angle (generally 12°_14°) to each other. With a stereoscopic microscope of the so-called Greenough type two definitely separate objectives, tubes and eyepieces are present (fig. 2.9).

Fig. 2.9. Stereo-microscope of the classical Greenough type, focussed onto the head of a match to show the free-working distance. The re-inverting prisms are localized in the drumlike extensions half-way along the tube.

For the stereoscopic effect as such, two separate tubes or even two separate objectives are not necessary, however, provided the objective is of sufficient diameter to admit the two beams at a mutually acute angle. This can be easily verified by using a large magnifying glass with both eyes. Most modem stereoscopic microscopes are of the single-objective type (fig. 2.11).

The question arises then whether microscopes for observation with both eyes (see fig. 2.6, 2.7 and 2.8) can be used for stereoscopic observation. This is not generally so, for the following reasons. In the first place, with somewhat higher magnifications (say 150-200 x) the stereoscopic effect is lost by


Fig. 2.10. Scheme of the course of the rays in an image-dividing prism complex according to Jentzsch; both eyes receive light from left- and right-hand side of the image.

the reduction in depth of focus of the image, which is, as will be explained in detail in chapter 5, inversely proportional with the magnification and even the square of the aperture of the objective. The image thus quickly becomes a thin optical section of the object; as a consequence differences in depth (which are essential for stereoscopic vision) can no longer be discerned. In the second place, it is technically very difficult to divide the light beam from the objective over both eyepieces (fig. 2.6), keeping the beams from both halves of the objective totally separated, without an appreciable loss in the effective aperture of the objective. With most modern microscopes the division of the light in the binocular eyepiece is arranged in quite another way, therefore, as it makes no sense to accept such a considerable loss in resolving power for a largely fictive goal. With the so-called Jentzsch prisms often applied with binocular microscopes with a single objective, the primary beam splitting is made by a semi-reflecting plate in the manner shown in fig. 2.10. Both eyes consequently are met by rays from right and left half of the objective; the stereoscopic effect is totally lost,1 but both eyes receive light from the entire aperture of the objective. The full resolving power can thus be used, moreover the light loss is minimal. It is evident that the lighting intensity of the image per eyepiece will be halved and some light will be lost by reflection from the extra glass surfaces across the light path. The use of a binocular tube, which should be considered primarily as a means of working more comfortably but not of seeing more, calls therefore for a more powerful illumination of the object. In using the time-honoured high voltage 40-60

1. By means of polarization filters the stereoscopic effect can be regained (Huber, 1963); this is, however, of little significance.


Watt bulb as a light source, the lighting intensity of the image will certainly be too low when using e.g. a 100 X oil immersion objective. With any type of a binocular head, the separation of the eyepieces should be adjustable in order to accommodate the varying interpupillary distances of different observers. Moreover, at least one eyepiece should have adjustable focussing, in order to compensate for differences in refraction between the eyes. It is clear, therefore, that a binocular tube head is a rather complicated - and therefore costly - part of equipment. With all the prisms and other devices it has a relatively considerable weight, moreover, mounting a binocular eyepiece on a simple stand as shown in fig. 2.5 would lead to forcing down the tube, as the rack-and-pinion movement of the tube could not hold this weight. Stands with binocular eyepieces are mostly of the type shown in fig. 2.6, in which the tube is firmly attached to the stand and the stage can be adjusted instead of the tube.

A binocular eyepiece is of advantage when the microscopic observation must be conducted for lengthy periods as it then lessens eye strain. On the other hand, it is not in any case the preferred method of microscopic observation. In this connection, both optical factors (such as an image of extreme low brightness that can occur in fluorescence microscopy) and more subjective human factors may be of importance. With the latter, a monocular tube is to be preferred where a microscope is used alternatively by different persons for quick observation. The absurd (but quite common!) situation that only one eyepiece of a binocular head is used, merely to avoid all this adjusting and extra focussing, will otherwise be the result.

As explained previously, the use of a microscope with a stereoscopic image only makes sense with lower magnifications. The field of application of these stereoscopic microscopes is quite different, therefore, to that of the conventional microscope. In working with a stereoscopic microscope, incident illumination is used in the majority of cases, often with a spot light attached to the stand, or with built-in illumination parallel to the optical axis.

Although objectives in these stereoscopic microscopes can be changed -in older stands of the Greenough-type often with a sledge-type of objective changer (fig. 2.9) - a resolving nosepiece is for technical reasons seldom applied. Variation in the total magnifications is achieved by substituting eyepieces, or in modern stands with a step-wise magnification changer or a zoom-system (chapter 4).


Fig. 2.11. Large free-standing stereomicroscope as used for micro-surgical interventions with built-in magnification changer. The image has been focussed on the upper free border of a match-box, so that the long working distance can be seen.

In any form of the compound microscope the image is inverted with respect to the object (chapter 1); with conventional mono-objective microscopy this is hardly felt as a hindrance. In preparative work, however, it can be less convenient when e.g. a needle brought towards the specimen from lowerleft appears in the image as coming from upper-right. To meet this difficulty, all stereo-microscopes are provided with a set of reinverting prisms. For ordinary microscopy reinverting the image makes no sense, and would entail unnecessary light losses and deterioration of image quality. A similar relation exists here as between the high-power astronomic telescope with inverted image and the well-known prism binoculars with upright image.

As can be seen from the drawing of fig. 2.12, it is not possible to reach a total inversion of an image with a single prism. A complex of 4 reflecting surfaces in a given spatial relation to each other is mostly applied for reinverting an image; the complete set of two crossed separate prisms, as shown in fig. 2.12, is called a Porro prism set. It is found with most older types of stereoscopic microscope, where these prisms are mounted in clearly visible boxes somewhere half-way along the tube (fig. 2.9). In more recent models, other types of prism, so-called Thomp'Son or Z-prisms are used, which occupy less space.


s

Fig. 2.12. Course of a light bundle in a set of reinverting prisms according to Porro; the position of the image can be derived from the figure 5.

A stereoscopic microscope for preparation work should not only have a reinverted image, but also a sufficient distance between object and objective to enable a certain amount of free movement. Generally, this so-called free working distance will amount to something like 8-12 cm, but in some cases even this is insufficient. For certain surgical interventions, such as on the internal ear, deep in the petrosal bone of the skull or other preparative work necessitating delicate manoeuvering in the object space, special stereomicroscopes have been developed with a very large free working distance. The operation microscope of fig. 2.11 has a working distance of no less than 20 cm. In order to leave both of the surgeon's hands free, such stands are sometimes provided with pedals, so that they can be controlled (inclusive focussing and change of magnification) entirely with the feet. These very large special stands are often provided with extra image splitting prisms, so that observation by two persons and/or the mounting of a camera is possible.

Other types of specialized stereomicroscopes will be mentioned briefly. The slit lamp microscope is an instrument used in ophthalmology in which a narrow band of light is directed through the transparent part of the eye and the 'optical section' studied with a stereomicroscope. This technique is used for localizing foreign bodies, especially in the cornea. In gynecology a colposcope is used which is essentially a microscope for low-power incident illumination, used to examine certain parts of the internal genital organs. It is mainly applied for the early detection of tumours in this region. A socalled capillary microscope used in yet another medical field, dermatology,


is again a (stereo )microscope for incident illumination to study the vascular patterns just beneath the epidermis, which can be easily observed after certain treatment. In biology and technical sciences a series of other stereoscopic microscopes are used for special purposes and with stands adapted to these ends of which there is no point in treating them here, as they are all based on general principles already discussed.

LITERATURE CITED AND SUGGESTIONS FOR FURTHER READING

J. R. Benford and H. E. Rosenburger: Microscopes, in: Applied optics and engineering, Vol. IV, ed. R. Kingslake. Academic Press, New York-London 1967.

H. Beyer: Handbuch der Mikroskopie. VEB-VerJag Technik, Berlin 1973. S. Bradbury: The evolution o/the microscope. Pergamon Press, New York 1967. P. H. van Cittert: Descriptive catalogue 0/ the collection 0/ microscopes in charge 0/ the

Utrecht University Museum. Noordhoff, Groningen 1934. M. Espinasse: Robert Hooke. Heinemann, London 1956. H. Haselmann: Das Mikroskop, Werkzeug und Object der Wissenschaft. Z. wiss. Mikr. 67

(1966) 244-256. P. Huber: Ein gewohnliches binokuliires Mikroskop wird Stereomikroskop. Mikroskopie

18 (1963) 231-234. A. Hughes: A history 0/ cytology. Abelard-Schuman, London-New York 1959. L. C. Martin and W. T. Welfort: The light microscope in: Physical techniques in biological

research, 2nd ed., Vol. I, part A, ed. G. Oster. Academic Press, New York-London 1971. M. Rooseboom: Microscopium. Leiden: National Museum for the history of science, 1956. F. Sherwood Taylor: An illustrated history o/science. Heinemann, London 1957.

CHAPTER 3

OBJECTIVES

NUMERICAL APERTURE

All modern microscope objectives consist of a number of lenses, separate or in combinations of two or three. The properties and the relative position of the different components of such an objective are the result of meticulous calculation for correction of aberrations of the individual lenses necessitating rather advanced mathematical techniques. Nowadays, lens designs are generally made by computer; with appropriate programming a computer can calculate not only the optical properties (curvature, thickness, types of glass and distance) of the components for a given specification and its number and position, but can even produce an estimate of the weight and cost of the system. It is clear from the foregoing that an objective is more than the sum of its parts; it is self-evident, therefore, that with an objective in which the front-lens is missing a totally distorted - if any - image will be formed. Fig. 3.1 shows a rather simple type of objective for medium

Fig. 3.1. Technical drawing of an achromatic objective, containing five lenses. On the mount are mentioned: magnification factor, numerical aperture and thickness of the cover glass, for which the objective has been calculated.

power; the composition of objectives with different degrees of correction is shown schematically in fig. 3.8.

40 OBJECTIVES

A simple or compound lens is characterized not only by its focal length, but also by the cone of light it can accept for refraction. As the image distance is fixed by the position of the intermediary image (chapter 1), this cone is described completely by the size of its top angle (fig. 3.2). This socalled angular aperture of an objective is a very important parameter for describing an objective, and as will be shown later in many respects an even more essential characteristic than the focal length. The lens aperture will indicate with any well-designed objective: 1) How far the given focal length can be used for resolving details in the object and 2) The amount of light which can pass through the objective when the aperture is filled with light (light gathering power).

In microscopy the numerical aperture (abbreviation N.A.), as introduced by Abbe, is used generally as a measure for the opening of an objective. The N.A. is the product of the refractive index (n) of the medium between the specimen and objective lens times the sin of the half-angle (u) of the cone of light entering the objective: N.A. = n sin u (fig. 3.2).

The optical object distance (the height of the cone between specimen and front lens of the objective) is not of very great importance in microscopy. The distance between the under-border of the objective mounting and the specimen, the so-called/ree working distance (PQ in fig. 3.2) is, however, in

A __ ---1.--.:..~_ B

Fig. 3.2. Schematic view of the aperture cone of an objective, showing the angle u (half the top angle); AB object plane, PQ free working distance.

practice a very important qualification of an objective. As this distance cannot be made much smaller than about one-tenth of a millimeter, it is a serious limiting factor in the design of objectives for high numerical apertures. It should be noted that the free working distance is also diminished

NUMERICAL APERTURE 41

by the cover glass and the mounting medium lying over the specimen in preparations for transparant light microscopy.

Generally it can be stated that in the case of standard objectives the N.A. increases (but not linearly) with decreasing focal length. The maximal N.A. admissible with an objective of given focal length depends not only on the functional design of the objective, but also on its degree of correction. Numerical aperture and magnification should be considered as independent quantities within certain limits. A 40x objective can exist, therefore, with a N.A. of e.g. 0.65, 0.80 or 0.95. As the numerical aperture is a measure for the diameter of the light bundle which can pass through the objective, the lighting intensity of the image will change, in the series of objectives just mentioned with the same focal length, with the square of the N.A. In contrast to the situation with a photographic objective in which the aperture is adjusted to the circumstances (e.g. the brightness of the object), the N.A. of a microscopic objective is fixed as a rule at the highest admissible value, as the resolving power of an objective lens depends on the N.A. which can be used effectively (chapter 5). Some microscopic objectives exist which have a built-in diaphragm enabling a certain variation of the N.A. It should be emphasized that the practical value of such a device (e.g. in dark-field microscopy, see chapter 8) differs totally from that in a photographic objective; it cannot be used, in the former case, for changing the light-gathering power without interfering rather drastically with a.o. the resolution in the image. In this connection, it should be remembered that in a photographic camera the objective always forms an image which is reduced in size compared with the object; moreover, a large image distance exists as a rule in relation to the focal length. In microscopy this situation is reversed; the image is always magnified, although, in the last few years 1 x objectives have been made.

As explained in chapter 1, spherical aberration, curvature of field and chromatic aberrations will increase with the aperture of a lens, as will other lens errors. It is self-evident that the largest problems will exist therefore in the correction of objectives with higher apertures. As will be explained in detail in chapter 5, the resolving power of an objective depends on the effectively used N.A. Although, due to optimal computerized calculation of lens combinations, it is now possible to construct objectives with comparatively higher admissible apertures than were known in the past, the upper limit of the N.A. effectively to be used has virtually not changed much from the value of 1.25-1.30 attained by Abbe in 1886. It has appeared that the numerical aperture which can be used effectively with light, is subject to an absolute maximum, determined by an optically admissible (cor-

42 OBJECTIVES

rection!) size of the lenses and a minimum practicable free working distance. This will be explained in some more detail in the next section.

IMMERSION -OBJECTIVES

When air fills the object space between specimen and front lens of an objective, the most oblique rays from the specimen to the objective which can leave the surface of the cover glass are those which touch upon the glass-air interface at the critical angle of refraction: 41.5° when the refractive index of the glass is about 1.5. This corresponds with rays entering the air-filled part of the object space at an angle of 90°, i.e. brushing the surface of the cover glass. It is clear that the free working distance cannot be made so small, or the front lens so large (the practical limit with high-power objectives is 1 mm) that this purely theoretical value, which would correspond with a N.A. of 1.0 could be reached. The upper limit of the N.A. in such a situation is therefore 0.95, corresponding with a value of the angle u (fig. 3.2) of about 72°, when n = 1. This limit is an absolute one; only when an immersion fluid with a refractive index greater than I fills the object space the use of an aperture of 1.00 or greater is possible.! This is not a question of a real enlargement of the angle u as is sometimes thought, but depends on the increase ofn in the formula N.A. = n sin u. This can be iIIustrated by the following facts. With the immersion fluid most commonly used having a refractive index near that of glass, the theoretically maximal value of N.A. of an objective is something near 1.40; this would correspond with a value for the angle u, (when n is 1.515 for average immersion oil) of 67°. This means that in the case of the maximal N.A. with an immersion objective, the value of u would be lower than in the case of the highest N.A. with a 'dry' objective! The significance of u, of course, alters with various immersion media and is the real origin of the gain in light gathering power where immersion media with a refractive index greater than 1.00 are applied (fig. 3.3). With the use of other immersion media, such as water (maximal N.A. 1.15-1.20) or glycerin (N.A. up to 1.25) the value for u remains well below 70°.

Even if it is not the determining factor in the increase of numerical apert-

1. The elaboration of this fundamental principle - already tried empirically by Hooke with a primitive water-immersion - is mainly due to Amici, who performed a series of experiments around 1850 with water-, glycerin- and oil-immersion. In 1878 the first cedar oilimmersion objectives, as calculated by Abbe were put on the market by Carl Zeiss. In the U.S.A. the principle had been developed and brought to practical realization as early as 1874 byR. B. Tolles.

IMMERSION -OBJECTIVES 43

ure, the use of an immersion medium makes more oblique entry of light rays possible. In the case of immersion with an oil having a refractive index near that of the material of the front lens, the rays can enter with virtually no refraction at all (fig. 3.3). This has certain technical advantages both with

c

I. II.

Fig. 3.3. The effect of oil immersion. In the situation I without immersion, of three rays with an ever increasing angle towards the optical axis only ray A reaches the objective in such a way that it can be presumed to take part in image formation, whereas ray C does not even reach the upper part of the object space, being totally reflected at the surface of the cover glass. In situation II the same three rays reach the object space (virtually without being refracted) and both rays A' and B' probably can take part in image formation C' reaching at least the upper part of the object space. It should be noted that the angle of ray A and B with the optical axis is greater than of their counterparts A' and B'.

regard to the correction of the objective and in the fact that reflection phenomena, leading to stray-light in the object space, are reduced. The useful effect of the immersion is not limited, therefore, to the increase in admissible aperture; a further advantage is the virtual disappearance of the effect of the cover glass on the course of the light rays (see page 45).

With oil-immersion no layer may occur in the object space with a refractive index lower than that of glass or immersion oil, say around 1.5 (hence the term homogeneous immersion). If such a lowering occurs, the advantage of the immersion system with regard to the gain in aperture may be lost, as now rays of larger angle to the optical axis are refracted or reflected so that they can no longer take part in image formation (fig. 3.4). Consequently, with a specimen in air under a cover glass, filling of an ob-

44 OBJECTIVES

Fig. 3.4. In using oil immersion, a mounting medium (MM) with a lower refractive index than glass or oil will cause a loss in aperture (from u, to u l ); CG cover glass.

jective aperture of 1.00 or higher is not possible; with an object in water some loss in aperture will occur also in using e.g. an 1.30 N.A. objective. As will be shown in chapter 5, an image can be formed quite well here but with a loss in resolving power. For correct adaptation of the light cone entering the objective the conditions under the object slide are of importance too, as will be explained in chapter 6.

The situation with the less common objectives for immersion with glycerin (n = 1.47) or water (n = 1.33) which are used for special purposes, is of course quite analogous to that with the widely used oil immersion. Some refraction will occur in the object space of such objectives at the interfaces between immersion fluid and objective and cover glass; it is not a homogeneous immersion as oil immersion is.

Theoretically one might argue that, in using an immersion fluid with a refractive index of over 1.5, the maximal value of the N.A. of an objective could be further increased. Experiments have indeed been made in this direction; the effect of such an immersion would be nullified, however, if a lower refractive index existed somewhere in the object space (compare with the situation of fig. 3.4). Objectives for use with monobromo-naphthalene (n = 1.66) as immersion fluid, with a N.A. of even 1.6 have been constructed for use with incident illumination without a cover glass, e.g. in metallurgy.

It is clear from what has been explained so far, that an immersion objective can only be designed for use with a single immersion fluid; it can even be said that the immersion fluid forms a part of the objective. It is very obvious, therefore, that an immersion objective cannot be used without immersion, or a 'dry' objective with immersion; similarly, a water-immer-

IMMERSION -OBJECTIVES 45

sion objective cannot be used with oil immersion and vice versa. When an unsuitable immersion medium is used some image is formed in most cases, but it is distorted and often cannot be brought into focus. It is of primary importance, therefore, to know at once, especially with an unfamiliar microscope, which objectives are to be used with immersion and with which fluid. With all modern makes this is marked on the mount with the indication 'OIL' or 'OEL'; with older German objectives with 'H.I.' (Homogene Immersion) and in the case of water-immersion 'W.I.' or 'WAS', or waterimmersion in full. Finally, it should be pointed out in this connection that apart from the refractive index of immersion fluid, also the dispersion characteristics of the immersion medium should be taken into account for high performance work. For optical results, it is to be recommended, therefore, to use the immersion fluid provided by the manufacturer; this is only of some importance, however, when using high-power objectives of the highest degree of correction. In the German optical industry, a standardization of the optical properties of immersion oil has been agreed upon (DIN-norm).

Apart from a contingent immersion medium, the other indications engraved on the mount are mostly limited to the values for N.A. and magnification; in many older types of objective the focal length is given in mm or inches from which the magnification can be calculated with the formula on page 16. The standardized tube length (160 or 170 mm) is seldom mentioned on modern objectives. Recently objective series for infinite tube length have been developed, indicated with the sign ~ on the mount. These lenses do not produce a real intermediary image, the intermediary image being formed only in combination with a built-in lens system in the tube. These objectives (generally of the highest degree of correction) can be used only with special stands. The magnification of these objectives (which have no I:!../f) is calculated with the formula for the magnification of the loupe (page 7).

Finally, the thickness of the cover glass for which the objective has been calculated is sometimes engraved on the mount under the value for the N.A. (fig. 3.0. This refers to an important problem which has not been dealt with so far and will be treated in the following section.

THE COVER GLASS EFFECT

Microscopic specimens which are studied with transmitted illumination are mostly affixed to a slide, stained and mounted with some kind of resin

46 OBJECTIVES

under a cover glass (chapter 7). This cover glass exerts a function that is primarily a protective one, but influences as well the course of the light rays coming from the specimen. When a dry objective is used, it is obvious that light rays coming from the object will be refracted at the glass-air interface, so that they seem to come from another point than is in fact the case. The greater the angle between such rays and the optical axis, the more pronounced this effect will be (fig. 3.5); this phenomenon thus causes an effect which has much in common with the spherical aberration of a lens (strictly, it is a negative spherical aberration). Apart from the cover glass, the resin beneath the cover glass (with generally a refractive index near to that of glass, see chapter 7) also plays a role. Generally, however, this layer is so thin that the cover glass is by far the more important in bringing about this optical phenomenon.

From an inspection of fig. 3.5 it is obvious that the thickness of the cover glass will considerably influence the effect. It is clear, therefore, that correction of an objective is possible only for a given thickness of cover glass.

CG

MM

Fig. 3.5. Influence of the cover glass on the rays from the specimen; A, Band Creal course of rays, A', B' and C' apparent course; OS object slide, MM mounting medium, CG cover glass.

The thickness of the cover glass has been standardized internationally at 0.17 mml; all modern objectives have been calculated for this thickness, even if the number 0.17 is not given on the mount. When used without a cover glass, objectives with higher N.A. will give an image which is hazy and unsharp, due to overcorrection of the spherical aberration. Special objectives designed for use without a cover glass are marked as such, e.g. with the mark 0 on the mount. As a matter of fact, the real thickness of

1. Apart from the thickness, the refractive index of the glass from which cover glasses are made has also been normalized; fluctuations of this are of minor importance. It appears that the results of all these normalizations are rather disappointing (Norris, 1961). Cover glasses made from quartz, as used in ultraviolet microscopy, have quite other specifications, and generally have a thickness of 0.35 mm.

THE COVER GLASS EFFECT 47

cover glasses varies considerably around the normalized value; with a random sample from 10 different boxes of cover glasses from a good brand the real thickness, as measured by the author, appeared to vary between 0.145 and 0.185 mm (fig. 3.6).

30

20

10

I h 150 160 170 180

Fig. 3.6. Frequency-distribution in [Lm of the thickness of 108 cover glasses as measured with a technical precision-micrometer.

These variations are not, in practice, important with objectives with a N.A. up to about 0.5 or 0.6; as explained earlier, even the cover glass effect itself becomes unimportant with apertures below 0.4. With higher apertures (0.75-0.95) of dry objectives, however, the variations shown in fig. 3.6 around the normalized thickness, begin to playa role; moreover, the layer of resin can no longer be neglected. With a N.A. of 0.90, deviations in thickness in the refractive layer over the object of the order of 0.01-0.02 mm are clearly expressed in the image; on the other hand, with a N.A. of 0.50, even differences of 0.05 mm are hardly noticeable. High-quality dry objectives of higher aperture, therefore, often are provided with a special device to compensate for variations in cover glass thickness, the correction collar. The rotation of the knurled collar on the mount of the objective is translated in a change in position between the rear components of the objective along the optical axis, so that cover glass thickness variations in the range 0.15-0.25 can be compensated for (fig. 3.7). The collar is graduated and may be pre-set for an exactly known cover glass thickness; even in this case the correction may not be optimal due to the layer of mounting medium beneath the cover glass. In many instances it is best to set the graduated collar at the mark of a cover glass thickness of about 0.17, select a dark speck or opaque portion of the object and focus up and down with the fine adjustment. With a

48 OBJECTIVES

I [~ II

Fig. 3.7. Effect of change of position of certain lens components, as applied in an objective with a correction collar; I situation with a thin cover glass, II situation with a thick cover glass.

correct setting, the change in unsharpness and expansion of the dark outline of the spot in the object will change in both directions in about the same way. When the unsharpness is accompanied with a considerable loss of contrast in focussing down, the collar should be set at a higher value and vice versa. If no correction device is provided in the objective, an extensible tube (fig. 2.5) can be applied for cover glass correction for a certain degree: the draw-tube should be shortened for too thick covers, and extended somewhat for too thin covers.

All these considerations are of importance only with high-power dry objectives; in using oil immersion objectives, the cover glass effect plays virtually no role in image formation, as between specimen and objective the object space has practically everywhere the same refractive index. Theoretically, when an oil immersion objective is used without a cover glass, a special oil with a higher refractive index should be used to compensate for the missing optical effect of the cover glass; in practice, however, this is seldom applied. Quite another consequence of the homogeneity (or quasi-homogeneity) of refractive index in the object space with oil immersion is a considerable diminishing of stray-light in the object space, as compared with a high-power dry objective. This is due to a reduction of reflection phenomena, not only in the space between the front lens of the objective and the cover glass, but also in the specimen itself, as a consequence of virtual absence of total reflection at the upper surface of the cover glass. This gives a considerable gain in image contrast (quite apart from the cover glass effect) for an oil immersion objective over a high-power dry objective of similar quality, when used with a condensor of high aperture. A tendency exists, therefore, in the last few years to construct oil immersion objectives in the

THE COVER GLASS EFFECT 49

magnification range of high-power dry objectives (40-70 x) with apertures in the range of 0.8-1.2.

TYPES OF OBJECTIVE

With virtually all modern microscopes, the sum of the free working distance and the length of the objective is standardized at a fixed value. This length adjustment of objectives is made somewhat easier by the fact that stronger objectives have many lenses, making them longer, and have a short object distance. With a set of objectives adjusted in this way, so-called para/ocal objectives, change of objective with the revolving nosepiece necessitates only some focussing with the fine adjustment to obtain a sharp image. It should be noted, however, that the value of the length of the objective + working distance, or adjustment length is not standardized in any way (such as tube length or cover glass thickness). Parafocal adjustment of objectives, therefore, only holds true for a given manufacture, and often only for a specific series of objectives. Because of the large number of lenses required for flattening the object field, series of plan-objectives often are standardized for a greater adjustment length.

Apart from qualifications as dry, immersion, magnification and N.A., objectives are classified in a number of types on the basis of their correction grade. As this division in achromatic, fluorite, apochromatic, plan-achromatic and plan-apochromatic objectives is not subject to a strict international standardization, nominally similar objectives from different manufacturers may have a somewhat different degree of correction.

1. Achromatic objectives: These objectives are corrected for chromatic aberration in such a way that the image points for two colours (mostly at 486 nm in the blue and at 656 nm in the red) coincide; they contain the classical achromatic doublets (fig. 3.1 and 3.8). The correction for spherical aberration is made for one colour only, usually in the yellow-green part of the spectrum. When mixed light of all wavelengths is used, colour fringes may appear along the outer borders of parts of the object; this phenomenon forms part of the so-called residual aberration (secondary spectrum), which is due to the fact that the variation of focal length with wavelength has the shape of a curve. When using light from the green or yellow-green range, the residual chromatic aberration is much less obvious to the eye; moreover, spherical aberration is also minimal in this wavelength region. At any

50 OBJECTIVES

§

8 0 c:J g ~ a ~ ~ c::=:::::.

CJ a b c

Fig. 3.B. Schematic view of the optical components of high-power dry objectives of about equal N.A.; a achromatic, b plan-achromatic and c plan-apochromatic. It should be noted that both objective band c have a concave front lens. Components made from fluorspar or special types of glass (not shown separately) are particularly numerous at c.

wavelength a considerable degree of curvature of field exists with these objectives (fig. 3.9 I); up to objective magnifications of about 30-40 X and with normal (i.e. not wide-field) eyepieces this is not necessarily very disturbing in the observation.

Achromats are the most common type of objective used for routine observation; objectives without other imprint on the mount than magnification and N.A. generally belong to the achromatic category.

It should be noted that photographic emulsions generally have a sensitivity for the different colours which differs from the human eye. Consequently microphotographs (both black and white and coloured) made with achromatic objectives often do not match the expectations of the observer due to the manifestation of colour dispersion phenomena not apparent during observation.

2. Fluorite or semi-apochromatic objectives (mostly indicated with FL or Fluorite on the mount). Fluorite or fluorspar (CaF2) is a mineral with optical characteristics which differ considerably from that of glass (see table I on page 11); its low degree of colour dispersion can be used in lens combinations to reduce chromatic aberrations more effectively than is

Fig. 3.9. Photomicrograph of a haematoxylin-phloxin stained section of the renal papilla of a dog at a final magnification of 120 x, as photographed with 10 x objectives with identical focal length (16 mm) and comparable N.A. (0.24-0.32) but different correction grade (I achromatic, II plan-achromatic, III plan-apochromatic). -+

52 OBJECTIVES

possible with combinations of glass lenses only. Fluorite objectives are especially well corrected for chromatic aberrations and their correction for spherical aberration is generaily also better than with achromats. Fluorie systems are often used for photomicrography and for high-power observation. They still show, however, a certain curvature of field, although this can be compensated for to some extent with the eyepiece (see next chapter). There is no sharp limitation between objectives of this frequently used modem type and true apochromats, which must meet rather severe demands.

3. Apochromatic objectives (APO on the mount): This type of objective, already manufactured near the end of the nineteenth century, is corrected for chromatic aberrations in the three primary spectral colours of red, green and blue; triplets appear here instead of the doublets of achromatic objectives (fig. 3.8). It is corrected for spherical aberrations for two colours (blue and green); in the apochromat of the classical type a considerable amount of curvature of field is, however, present. It is a system which is optimally corrected for chromatic aberrations, the secondary spectrum being virtually absent; the apochromat is particularly suited for colour photomicrography and optimal resolution of fine details. Due to high cost of material and manufacture, apochromatic lenses are very expensive as compared with achromats, fluorite lenses being intermediate in pricel • It should be noted that also apochromatic objectives often contain fluorite; the designation only refers to the specification of its optical properties (apo = away from, chromatic = showing colour dispersion). Both fluorite and apochromatic lenses should be combined with so-called compensating eyepieces to achieve their best performance (see next chapter). The high degree of correction of both these types of objective enables them to be designed for a higher N.A. than with an achromatic objective of the same focal length. This means that both a broader band of light can take part in image formation - entailing a greater lighting intensity of the image - and a greater resolving power can be attained.

4. Plan-achromatic objectives (PL or PLAN on the mount): These objectives are corrected primarily for curvature of field, in all other respects they correspond to the qualifications of good achromats. The differences with ordinary achromats, both in construction and performance may be striking (fig. 3.8 a and b; 3.9 I and II). For many situations in which correction for

1. Fully apochromatic objectives without plan-correction are virtually no longer made; when present (e.g. of older make) their field of application is about the same as that for fluorite objectives.

TYPES OF OBJECTIVE 53

colour dispersion is not of primary importance, this is a convenient modern type of objective. It can be used to advantage with wide-field eyepieces in combination with which most of the objectives of the preceding types suffer badly from curvature offield towards the periphery.

5. Plan-apochromatic objectives (PLAN APO or PL APO on the mount) have, as the name suggests, a combination of correction for curvature of field with a correction for chromatic and spherical aberration on the apochromate level. These objectives, in which the degree of correction has been brought to the limit of which is technically possible, are commercially available only since 1955. It is self-evident that these objectives are highly complex and costly; in the high-power versions they contain 8-15 individual lenses of different materials, in which the typical triplets of the apochromats still can be recognized (fig. 3.8c).

Although the image quality of these lenses can be superior to any other type of objective, their use for observation, black and white photography etc. seldom justifies the high price in comparison e.g. with plan-achromatic objectives. The differences are often very slight indeed with e.g. planachromatic objectives (fig. 3.9) and become totally nullified e.g. with an illumination which has not been correctly adjusted.

With no single objective, including the plan-apochromats, a complete correction of image errors can be reached; some residual errors will always remain. As will be explained in the next chapter, these can be compensated for, at least to some extent, with the eyepiece. On the other hand, it should be kept in mind that e.g. a certain degree of curvature of field is not necessarily very disturbing in the microscopic observation; one only relatively seldom needs to scan the entire field of view sharply with one glance. In many cases a well-considered choice of eyepieces and the use of adequate filters (see chapter 7) can make the application of objectives of a more complex type superfluous. Moreover, as pointed out before, a badly adjusted illumination can nullify entirely the effect of a costly objective.

The following general remarks can be made in conclusion with regard to the application of objectives of the described different correction grades. With differences which are as small as illustrated in fig. 3.9 and a rough relation in price of something of the order 1 :2:4 for achromats, plan-achromats and plan-apochromats respectively, there should be some reason for a wellconsidered choice.

Achromatic objectives are suited for routine observation work in the

54 OBJECTIVES

middle of the spectrum, without high demands for resolution. Plan-achromatic objectives: black and white photomicrography and observation especially at lower magnifications, using a yellow-green filter. Fluorite objectives: colour photomicrography, in particular when demands of contrast and resolution are stringent in the centre of the field, but less important at the periphery. Plan-apochromatic objectives: photomicrographic (especially colour) and observation work with high apertures or at the extremities of the spectrum; all cases in which sharpness, contrast and resolution of the image over the entire field must be on the highest level that can be obtained.

QUALIFICATIONS AND PERFORMANCE OF AN OBJECTIVE

The testing of the performance of a microscopic objective is a rather complicated matter, all details of which cannot be treated here. It should be pointed out first and foremost that with a brief observation of a routine object, such as a stained section, rather serious defects are not necessarily revealed. In order to say more about a given objective, special test-objects and measuring equipment are necessary.

The classical Abbe test plate consists of a layer of silver deposited on an object glass, in which parallel lines have been drawn; it is mounted with resin under a cover glass. The objective to be tested is focussed on the border of such a line; the sharpness of the image and the eventual occurrence of coloured fringes around the borders of the silver layer enable certain conclusions to be drawn about the presence of spherical or chromatic aberration in comparison with other objectives. As the coverslip is wedge-shaped in transverse section (with a thickness generally from 0.08-0.23 mm, with graded divisions which can be read under the microscope), the thickness of the cover glass at which the objective gives its best performance can be determined. A more simple test object for testing the degree of spherical aberration which can also be obtained commercially but may - unlike the Abbe plate - easily be made, is an artificial object providing minute points of light. To this end, a thin layer of silver or a film of a dark stain like nigrosin, indian ink or the like, is prepared on a slide and mounted with a cover glass. As these dark layers will have local defects of all sizes and kinds, it will be relatively easy to focus a minute light spot as a source of light practically without size; it will be observed as a bright point surrounded by rings of light decreasing in brilliance towards the periphery. With the so-called startest the objective is focussed up and down through such a bright point and the differences are noted. With e.g. an excess of spherical aberration (i.e. a

QUALIFICATIONS AND PERFORMANCE OF AN OBJECTIVE 55

badly corrected objective or a correction-mount set for too Iowa value for cover-glass thickness) the bright point will change into a ring when focussing up and into a dark spot with lighter centre when focussing down. With optimal correction, virtually the same changes take place uniformly in both directions in focussing through. All this resolves to the method explained for the correct setting of a correction collar of a high-power dry objective and amounts to finding the position with minimal spherical aberration (page 47). It is sometimes stated that the optimal setting for such a collar should be tested with a star-test. In a given situation, with an object with unknown thickness of cover glass plus mounting material, such a star-like point in the object will seldom occur, however, and collateral adjusting with a standard test preparation will be of limited value. The setting of a correction collar (page 48) therefore has been explained with a contrast-rich small detail in the object, which can always be found; the changes in focussing up and down are, however, not so clear as with a luminous point.

Other data which are relevant for the qualification of an objective are curvature of field and numerical aperture. The curvature of field can be tested most easily with a regular and flat object, e.g. an object micrometer or an object finder with its regular subdivision of the field of view in squares (see page 131), or just a stained thin blood smear. The differences in depth of focus at the border and in the centre can be measured with the calibration of the fine focussing control and so compared with other objectives. For the precise measuring of the numerical aperture a so-called apertometer is necessary. It is possible, however, to make a rough estimate of the light cone accepted by an objective by illuminating an objective in 'reverse' direction with a parallel band of light and comparing this cone with that of another objective.

Finally, the resolving power as a result of the effective N.A. of an objective is an important measure for its practical performance; this can be tested directly using special test preparations in which linear systems with decreasing distance can be focussed. A frequently used rather simple type of commercially available test preparation is that in which a series of diatoms are mounted under a cover glass. These organisms show highly regular structures in their shells, the dimensions of which are exactly known, so that it can be predicted that they should be resolved by a given objective, when this meets certain specifications (more details about this will be given in chapter 5).

An informative review of average values for common objectives of the different types described is presented in table II. Objectives of different makers can vary in the details of their design; it is obvious, therefore, that

56 OBJECTIVES

for a certain objective the precise data from the manufacturer should be consulted. As a general principle, improvement of the correction in an objective with given focdllength, clearly gives a considerable increase in the N.A. wich can be attained; this is often accompanied on the other hand by a reduction in free working distance (PQ in fig. 3.2).

TABLE II. SOME AVERAGE DATA OF COMMON OBJECTIVES WITH DIFFERENT DEGREES OF CORRECTION.

magnifi- N.A. focal free cation length working factor (mm) distance

(mm)

achromatic 4x 0.08 33.0 20.0 plan-achromatic 4x 0.10 36.0 20.0 plan-apochromatic 4x 0.16 35.0 2.5

achromatic lOx 0.22 16.0 6.0 plan-achromatic lOx 0.25 16.0 4.8 fluorite lOx 0.30 16.0 4.0 plan-apochromatic lOx 0.32 15.0 0.40

achromatic 40x 0.65 4.0 0.40 plan-achromatic 40x 0.65 4.0 0.23 fluorite 40x 0.80 4.1 0.33 plan-apochromatic* 40x 1.00 4.0 0.22

achromatic* 100x 1.25 1.9 0.09 plan-achromatic* 100x 1.25 1.8 0.10 fluorite* 100x 1.30 1.9 0.12 plan-apochromatic* 100 x 1.32 1.7 0.10

* = oil immersion

SPECIAL OBJECTIVES

After the objectives discussed so far which belong to standard series, some special types of objectives will be reviewed briefly. Objectives for use with particular optical techniques (e.g. phase contrast, strain-free objectives) will be dealt with in chapter 8 and 9 together with the observation techniques in question. The spring-mount, a telescoping system for objectives with a short working distance to avoid damage of the objective in focussmg, will be dealt with for its practical aspects in chapter 7.

SPECIAL OBJECTIVES 57

Mirror- or reflecting objectives So far, all objectives discussed are designed on the principle of optical refraction; in some objectives, use is made of the phenomenon of reflection in image formation. Optical systems which consist only of lens combinations, as is mostly the case with microscopic objectives and oculars, are called dioptric (the greek dia means through). Catoptric systems (kata = onto) are mirror combinations, catadioptric systems are lens-mirror combinations.

The construction of mirror objectives in any form is based on the principle, long recognized, that the use of a hollow spherical mirror for image formation produces no chromatic aberration as no refraction (entailing dispersion) but reflection occurs in changing the course of the rays.1 Errors such as spherical aberration, coma and curvature of field are present, however, and must be taken into account. It is possible to produce low-power objectives that are entirely catoptric; fig. 3.10 gives an example of such an

Fig. 3.10. Course of the rays in a mirror objective of the Schwarzschild-type.

objective of the so-called Schwarzschild type. In the mirror objective according to the Newton principle the primary reflecting surface is in the optical axis. The great advantage of the absence of chromatic aberration is counterbalanced by the fact that (even with catadioptric systems) it is very difficult to reach numerical apertures higher than 0.5-0.6. Moreover, the

1. On the basis of rather limited experiments, the famous English physicIst Isaac Newton (1643-1727) had wrongly concluded that refractive index and dispersion would always change in a fixed relationship, which would mean that correction of chromatic aberrations in a lens would be impossible. As a consequence of this incorrect opinion (which made, however, considerable impression due to his great authority) he took up the study of the formation of images by curved mirrors and soon described their fundamental principles.

58 OBJECTIVES

maximal size of the object field remains rather small. The application field of mirror objectives in microscopy is limited therefore to the medium and lowpower range.

The relative long free-working distance with most catoptric systems, in comparison with dioptric objectives, can sometimes be used to advantage; some objectives constructed especially for long working distance, even are designed on a catadioptric principle primarily for this reason. With such objectives, e.g. that of the Dyson-type, a 'primary' image is formed near the focal plane of an objective of more conventional design. Catoptric and catadioptric systems are frequently applied in astronomical apparatus (e.g. the Kellner-Schmidt reflector) in which some of the disadvantages of these systems are not so important as in microscopy, where their application field has remained rather discrete.

Objectives for use with invisible light It is obvious from what has been previously explained that with so many difficulties to overcome in the formation of an image, e.g. the difference in refraction of red and green light, a series of problems will arise in the construction of an objective corrected for light down to the ultraviolet region. Moreover, virtually all types of glass are totally opaque for light of a wavelength below 340 nm; consequently use has to be made of quartz and other substances transparent for light at these wavelengths.

It is only a few years since the construction of objectives having coinciding image points in a certain region of the ultraviolet and in the visible region has been succesful. With differences in wavelength of a factor of two or more, considerable differences in image distance cannot be avoided; for the far ultraviolet and infrared catoptric objectives are used, where such a difference in focus does not occur and focussing of the image can be made in the visible region. As for high-power ultraviolet objectives, if they are designed for use with an immersion fluid, this is mostly glycerin; both immersion oil and water become totally opaque in the near ultraviolet.

In using infrared light, as this is applied in absorption photometry of organic material, up to a wavelength of 1-2 !Lm dioptric objectives can be used. With longer wavelengths (2-20 !Lm) catoptric systems are used. In the ultraviolet, dioptric systems can be applied down to a wavelength of about 250 nm. Further details of this aspect will be given in chapter 12.

Photomicrographic objectives As a rule photomicrography employs the same objectives as are used for observation. Special objectives for photomicrographic work are in use,

SPECIAL OBJECTIVES 59

however, where the image is formed through a single lens complex. These lenses, designed as 'macro-lenses', 'luminars' or with other names have much in common with an ordinary photographic objective, but they are designed exclusively for microscopy; they have a high degree of correction for curvature of field. They are applied without a projective or eyepiece in specially designed stands for exposures in which a very large object field (several centimeters in diameter) is to be photographed with low magnification. In quite a few photomicrographic stands they cannot be used where the photographic equipment is attached to the eyepiece. The newly designed flat-field 1 x objectives, in which the intermediary image is of the same size as the object, opens up the possibility of making review photomicrographs of areas up to about ± 1 cm diameter with a compound microscope.


E. Abbe: The relation of aperture and power in the microscope. J. Roy. Micr. Soc. 2 (1882),300-309 and 460-473.

S. Bradbury: The development of the reflecting microscope. J. Queckett Micr. Club 31 (1968),1-19.

D. P. Feder: Automatic optical design. Applied Optics 2 (1963), 1209-1226. K. P. Norris: Some observations on microscope cover glasses. J. Roy. Micr. Soc. 79 (1961),

287-298. H. Osterberg and L. W. Smith: Effects of numerical aperture on contrast in ordinary

microscopy. J. Opt. Soc. Amer. 51 (1961),709-714. M. Uhlig: Priifung der einzelnen Abbildungsfehler von Mikroobjectiven an verschiedenen

Testplatten. Mikroskopie 17 (1962), 273-284.

CHAPTER 4

EYEPIECES OR OCULARS

MAIN TYPES OF EYEPIECE

The function of the eyepiece can be defined as the magnification of the visual angle at which the intermediary image is observed. As explained in chapter 1, this is attained by focussing the object in such a way that the intermediary image is formed just within the focal plane of the eyepiece-lens, so that a final virtual image is formed at some distance from the eye (fig. 1.8). When the intermediary image is formed extactly in the focal plane, the final image is theoretically at infinity. In practice, however, this is not comfortable for observation (if indeed it were possible), as the less strained position for the eye is that of slight accommodation (cf. Baker, 1966).

Accordingly, in focussing one brings the intermediary image slightly within the focal plane of the eyepiece. If this situation is to be maintained with alternating use of objectives and oculars of different focal lengths, the only solution is to fix the intermediary image plane at a given place in the tube, while the image distance and object distance of objective and ocular respectively are adjusted accordingly.

The distance of the magnifying lens of an eyepiece to the intermediary image is not identical, as a rule, with the focal length of the eyepiece minus the small factor just discussed. This is due to the fact that apart from the magnifying lens a second lens is placed in the light path between objective and eyepiece, a so-called field lens. This lens has only a slight effect on magnification, but its position (generally not far from or in the intermediary image plane) leads to a larger field of view than with the proper eyepiece lens alone. The (generally weakly positive) field lens ensures that oblique light rays which would not otherwise reach the eye, can take part in image formation (fig. 4.1).

The position and size of the intermediary image is influenced by the field lens; as stated before, the latter plays no important role in the magnification of the eyepiece, actually even reducing the intermediary image somewhat in size, as can be seen from fig. 4.1. From the same figure it is clear that in designing eyepieces, the field lens can be used to bring the intermediary

MAIN TYPES OF EYEPIECE 61

Fig. 4.1. Schematic view of the effect of the field lens in an eyepiece.

image plane in any desired position with regard to the eyepiece lens proper. Eyepieces built according to the principle that the intermediary image is

formed behind the field lens, are called negative eyepieces. The most important representative of this type of eyepiece is that designed by Christiaan Huygens as early as 1690 and which is still often used. The less common Dollond-eyepieces also belong to the same category. Fig. 4.2 A and B show a schematic transverse section of a low-power and a high-power Huygens ocular. At the plane of the intermediary image a circular stop is located which determines th~ size of the ultimate field of view. With positive eyepieces the intermediary image plane is in front of the field lens; the eyepiece designed by the English instrumentmaker Ramsden in 1783, shown in fig. 4.2 C is a representative of this type. The front lens of the Ramsden eyepiece is generally at a certain distance from the end of the tube, so that shis ocular protudes somewhat from the border of the tube. The Kellner ocular is practically the same as that of Ramsden, only it has a compound front lens. In these positive oculars, the intermediary image plane is about 1 cm from the free end of the eyepiece tube; as in the case of the Huygens eyepiece, a diaphragm limits the field of view (fig. 4.2 C). The field lens has also an entirely analogous function, apart from the fact that the intermediary image is left in its original position and size. This latter circumstance is sometimes mentioned as an advantage of the Ramsden and Kellner eye-

62 EYEPIECES OR OCULARS

pieces, particularly in performing measurements. This is open to criticism as far as measurement of the total magnification is concerned, as with good calibration with an object-micrometer (see chapter 11) the actual size of an intermediary image is unimportant. Moreover, the position of the stop in e.g. a Ramsden eyepiece is not very convenient for bringing in a measuring scale.

A disadvantage of the Huygens eyepiece is its rather limited field of view and - especially with magnifications of over 8 x - a rather small eye clearance. For higher magnifications the Ramsden or the better corrected (especially for chromatic aberration) Kellner eyepiece is used; alternatively the more complex, likewise positive, orthoscopic eyepieces (fig. 4.2 D) can be applied.

A 8

-

!lJ D

c

I- -

Fig. 4.2. Longitudinal sections of some characteristic types of eyepiece; A low-power Huygens eyepiece, B high-power Huygens eyepiece, C Ramsden eyepiece, D wide-angle eyepiece.

The fact that the eyepiece consists, even in its most simple form, of at least two lenses, opens the possibility for a certain degree of correction for image errors. With a given relation of the focal lengths and mutual distance

MAIN TYPES OF EYEPIECE 63

of the two lenses such a simple system can be compensated reasonably well for chromatic aberration. For low-power routine work and with objectives which do not suffer from a high degree of curvature of field, eyepieces of the Huygens or Ramsden type can produce quite satisfactory results, while for higher magnifications the Kellner type is to be preferred. All these eyepieces will keep the longitudinal chromatic aberration in the intermediary image introduced by the objective so that coloured fringes can be seen at the periphery of the image, when looking with a Huygens eyepiece in combination with e.g. a high power achromatic objective.

With apochromatic objectives the longitudinal chromatic aberration is corrected in a very high degree; they retain, however, some chromatic difference of magnification (see chapter I), leading to an impairment of the image quality. With so-called compensating eyepieces a negative transverse chromatic aberration is introduced which antagonizes these chromatic differences of magnification. These eyepieces should be used in combination with apochromatic and fluorite objectives. The aberration introduced could - in contrast with an eyepiece of the more simple type - even deteriorate the image of e.g. a low-power achromatic objective, which does not produce the type of chromatic difference of magnification for which the compensating ocular has been designed; for high-power achromates, however, it is often advisable to use compensating eyepieces, but this does not hold true for all makes. Compensating eyepieces, therefore, are not necessarily always 'better' than other eyepieces, as is sometimes thought. Compensating eyepieces can be recognized by the presence of a yellow fringe at the edge of the image of the field stop when held up to a bright surface. They are mostly marked on the mount with the letter C or 'Comp.'. Some manufacturers use their own designation of what is essentially a compensation eyepiece. Theoretically, a curvature of field in the secondary image could also be adjusted to a certain extent by an opposite effect in the eyepiece. Although some efforts have been made in this direction, they have been only partly successful, however, due to the difficulty in bringing such a correction for different objectives in a single eyepiece. Moreover, the need for this compensation is less felt in more recent years with the newer types of plan-objectives with their flat field.

With non-compensating eyepieces the only designation on the mount is

the angular magnification/actor 250 . An eyepiece of an older type often f

bears a number (without x) only, the so-called eyepiece number, which originally represented the tube length divided by the focal length; some


eyepieces of older manufacture are designed entirely arbitrarily with I, II, III, IV; A, B, C, D or I, 2, 3,4 etc. It should be kept in mind only that whatever these numbers may mean, they do not designate the magnification. As a rule the earlier the letter or the lower the number, the lower is the magnification. This holds true, of course, also for the more exactly defined eyepiece number, as used by the older continental microscope makers.

It is clear from the foregoing that (even apart from the question of tube length) correction- and compensation-effects of an eyepiece should be attuned to the correction pattern of the matching objective series. Although standardization has been reached up to a certain degree, it is nevertheless advisable to combine only objectives and eyepieces of the same manufacture; in the case of e.g. low power achromates and Huygens eyepieces this does not hold so strictly, however. Compensation eyepieces should be used only when prescribed: as stated before, generally with high-power achromatic objectives and with more highly corrected objectives even of lower magnifications. In this, a general rule for all makes cannot be given. On the other hand - as long as the correct tube length is respected - there is some room for experimentation; if one makes no high demands, eyepieces of virtually all modern microscopes can be exchanged.

Apart from a few exceptions all eyepieces are standardized to an external diameter of 23.2 mm. Stereo microscopes and certain microscopes constructed for a larger field of view (fig. 2.7) are often provided with a tube with larger diameter. This enables the use of field lenses with a large surface, resulting in an extended field of view.

EXIT PUPIL AND THE EYE

The band of light leaving the eyepiece of a microscope which is correctly illuminated and focussed, has the shape of a diabolo, which can be visualized with smoke or a fluorescent transparant material (fig. 4.4). The point above the eyepiece where the emerging rays cross, is called the exit pupil or Ramsden circle, sometimes also called the eyepoint. In cross-section this area is disc-shaped and it has usually a diameter of I-It mm; it is optically the image formed by the microscope of the objective aperture, strictly the entrance pupil of the microscope (cf. chapter 1). The diameter of the exit pupil is proportional to the (effectively used) N.A. of the objective and inversely proportional to the total magnification of the microscope. It is, therefore, a quantity which is determined by the combination of objective and eyepiece (this will be commented upon further in chapter 5). The

EXIT PUPIL AND THE EYE 65

height of the eyepoint, that is its distance to the front lens of the eyepiece, is dependent only on the design of the eyepiece. As the exit pupil is the place where the pupil of the eye is brought in order to receive all rays of the

----.::::::-~-.-------- ~ -- -.... ....---~~--- ------ ------

Fig. 4.3. Position of the exit pupil with a feeble and a strong eyepiece-lens (the effect has been somewhat exaggerated).

bundle of light emerging from the eyepiece the height of the exit pupil is of great practical importance. The position of the exit pupil is related to the inclination of the rays at the periphery of the emerging cone of light and will be dependent, among other factors, on the magnification of the eyepiece. The higher the magnification, the lower the height (fig. 4.3); this holds only true for oculars of a given type, as the design of the eyepiece as a whole is also of importance.

With a classical eyepiece of the Huygens type of 6 x, the height of the eyepoint (sometimes called the eye clearance) is 11.6 mm and with a 10 x ocular this is only 7.6 mm. This value is at the limit of the practical eye clearance, in view of the length of the eyelashes and small movements of the eye made unconsciously when looking down a microscope. With eyepieces of the Ramsden type there is a somewhat more favourable clearance, with an eyepoint height of e.g. 10-11 mm for a 10 X ocular.

In some circumstances, e.g. when spectacles are worn even an eye clearance of 10 mm can be insufficient. Special oculars have been designed (sometimes with a &:-" on the mount) with an extra high exit pupil. In fig. 4.4A the light band leaving a 10 X Huygens eyepiece with an eye clearance of about 7 mm is shown. In fig. 4.4B, where all other factors for the microscope are similar, the eye point height is more than twice as great (16 mm) with an eyepiece of even higher magnification for spectacle wearers. As a rule, these eyepieces are of more complicated construction and thus more expensive; they are often of the orthoscopic type (fig. 4.20). Some further remarks on the practical aspects of microscopy for spectacle wearers will be given in chapter 7.

With microprojection or photomicrography, the eyepoint height is immaterial; for these purposes, therefore, special eyepieces (or better: pro-


B

Fig. 4.4. Exit pupil of a Huygens eyepiece 10 x (A) and a 12.5 x wide-angle eyepiece with high eyepoint (B), made visible with a block of fluorescent glass.

jectives) have been designed with a very low eyepoint height, or even a negative one (i.e. an exit pupil within the projective). These special lenses cannot be used, therefore, for direct observation.

EYEPIECE AND FIELD OF VIEW

When one eyepiece is exchanged for another, while the objective remains the same, not only the total magnification will undergo modification. In the first place, the size of the retinal image, the field of view will change. In the second place, the part of the microscopical object which can be overseen, the object field (sometimes, somewhat confusingly, also called field of view of the microscope) will change. The maximal size of the object field is of course determined by the objective, but the part of this field which actually can be overseen, depends on the eyepiece used. The so-called field number of an eyepiece gives a measure of the field of view of that particular eyepiece. It expresses the diameter of the intermediary image in the eyepiece field stop in mm. As the maximal size of this intermediary image must fall within the inner diameter of the eyepiece (generally, slightly over 20 mm), the field number cannot exceed 18 or 19 mm, even with a maximal diameter of the field stop. With a given focal length, the field number may vary considerably with different types of eyepiece; with eyepieces of a given type, the field number decreases with increasing focal length (table III). When the field number is divided by the magnification factor ofthe objective,

EYEPIECE AND FIELD OF VIEW

6x

12.Sx

12.5x Wide-angie

8x

67

Fig. 4.5. Photomicrographs from the same area of a stained section of cat kidney, made with objective 10 x and eyepieces 6 x, 8 x , 12.5 x and 12.5 x wide-angle. Note the changes in field of view and object field.


the diameter of the object field can be calculated, with sometimes (e.g. a binocular tube) an extra tube-factor to be taken into account. As a consequence of this, the diameter of the object field is reduced (as is the field number) with increasing magnification of the eyepiece. As can be seen from 4.5, this is accompanied with an increase in the diameter of the field of view. The latter factor is a function of the angle of vision cr under which the borders of the image are seen. This angle is related to the field number Z and

focal length of the eyepiece/, according to the formulacr = ~. f

With conventional eyepieces, the angle of vision seldom exceeds 36°. Wide-jield eyepieces are characterized by a cr of up to 50°, with a corresponding high value for the field number (table III). The difference between a conventional eyepiece and a wide-field eyepiece is impressive (fig. 4.5). These wide-angle eyepieces - often with a high eyepoint and, therefore, also suitable for use with spectacles - form a comparatively new development in eyepiece construction. They can be of the orthoscopic type (fig. 4.2D) or be constructed according to more classical principles. In the latter case the inner diameter of the eyepiece limits the diameter of the field lens and field stop. This can be solved by using a tube of greater diameter (e.g. 30 mm 0) with a corresponding increase of the inner diameter of the eyepiece. As it is not necessary to enlarge the front lens of the eyepiece to observe the larger intermediary image in full, the mounts of those eyepieces are often tapered with the part protruding from the eyepiece tube (fig. 2.7, page 29); needless to say these eyepieces do not fit a conventional tube.

The greatest angle of vision with most high power wide-field eyepieces is about 50°, near the maximum that can be overseen without strain on the eye. Although in many instances the use of this type of eyepiece can be of advantage, a very large object field is not always necessary or even desirable, e.g. in the systematic search of certain small details in a specimen. In the second place it can be stated that such an addition to the object field is only useful if it is of any real value for observation. For with an average achromatic objective, in combination with an eyepiece of even medium magnification, the image at the border of the field of view is already so distorted by spherical aberration and curvature of field (fig. 3.9 I) that it makes no sense to enlarge it further on the periphery. One would always have to focus separately on the border region with the fine adjustment to make this area visible at all, with simultaneous loss of focus for the centre. Only with a well-corrected objective, in particular for curvature of field,

EYEPIECE AND FIELD OF VIEW 69

could the enlarged field of view be overseen; the illustrations of fig. 4.5 were made with a plan-apochromatic objective.

TABLE III. SOME TYPICAL VALUES FOR COMMONLY USED EYEPIECES.

magnification factor f (in mm) field number angle of vision

6 x * 40 20 24° 8 x* 32 18 30°

10 x 25 16 34° 12t x * 20 12.5 36° 12t x * wide-angle 20 18 50° 20 x 12.5 8 36°

THE MEANING OF EYEPIECE MAGNIFICATION

Table III lists a few average values for commonly used eyepieces. Those marked with * have been used for the series of photomicrographs of fig. 4.5. The type (Huygens, Kellner, orthoscopic etc.) has not been specified, as this is of less importance for the field number and angle of view with a given focal length than for the eye clearance or degree of correction.

Apart from the special situation with the wide-angle eyepieces, it can be stated in general that the field number - and consequently the diameter of the object-field which can be covered with a given objective - decreases with increasing magnification of the eyepiece. Consequently, as also can be seen from fig. 4.5, there is always some gain in object field diameter with decreasing eyepiece magnification; this is lost for a considerable part, however, with the diminishing field of view in that same series.

In the past, until about 1950, only few objectives with a magnification of under 10 X were manufactured. The great difficulty with these low-power objectives was to reach an efficient correction of the high degree of curvature of field and spherical aberration occuring in lenses with a comparatively great diameter. With unsharpness and distortion of the image at the periphery, the gain in the object field was of limited value only. Moreover, most low-power objectives had a very long working distance, so that the tube had to be racked up with the coarse adjustment. In order to obtain as large an object field as possible for searching a specimen with a 10 x objective, it had to be combined with a low-power eyepiece. Now that with all modem microscope makes parafocally adjusted flat-field objectives with magnifications between 2-!- and 4 x are made, a much larger gain in object field (with


unchanged field of view) is possible with one movement for low-power searching, making the use of weak eyepieces for this purpose very unpractical. As will be explained in chapter 5, this is one of the reasons why a single eyepiece will do as a rule for most routine work in the present situation. In spite of this, microscopes with a 4 x objective are often still delivered with the two or three eyepieces dictated by tradition.

Many circumstances exist, however, in which a change of eyepiece really does make sense, such as in certain kinds of observational work and in photomicrography. This always requires the pulling out of one eyepiece and the insertion of another; in the past microscope stands have been constructed with so-called revolving eyepiece-changers. This rather complicated construction at the top of the tube made the stand very top-heavy and clumsy, so that these devices have fallen into disuse, in contrast to the revolving nose-pieces of objectives. In recent times, the somewhat cumbersome exchange of eyepieces has been simplified in some larger microscope stands with the development of the so-called magnification changer. This consists of a revolving disc with different lenses between objective and eyepiece, enabling stepwise changes in magnification and field number of the final image. This system, which brings about the effect of an eyepiece change (although it is strictly a modification of the objective image) greatly facilitates a change of magnification during observation.

A further development is the continuous magnification changer, based on a zoom-system which permits a variation in focal length without defocussing the image. Fig. 4.6 shows an example of the effect of a three-lens

-b-=U==========-Fig. 4.6. Change in focal length with a three-lens zoom-system.

zoom-system consisting of a pair of positive lenses coupled together to move in tandem, with a stationary negative lens between them. The effect of such zoom systems in an image changer is spectacular, but does not always

THE MEANING OF EYEPIECE MAGNIFICATION 71

counterbalance its rather high cost. Moreover, there is always some deterioration of image quality due to the extra refractive surfaces in the light path, although this has become very slight with newer technical developments. Generally, these zoom-devices have been applied so far rather unfrequently to regular microscopes. With stereoscopic microscopes they are more often seen; an eventual slight loss in image quality can be considered not so important, in view of the lower magnifications generally used with these microscopes. As objective changes are less easily performed with most stereoscopic microscopes, some kind of magnification changer, stepwise or zoom, is found with virtually any of the modern larger stands.

SPECIAL TYPES OF EYEPIECE

In practical microscopy, it often occurs that a certain image has to be observed by two or three persons, where the reproduction of the image with television microscopy or projection microscopy (cf. chapter 10) would go too far. Different possibilities exist in this respect which do not regard the eyepiece only, but will be dealt with in this section for practical reasons.

The most simple solution is obviously to use alternatively the same microscope and eyepiece. It saves much time to have a pointer eyepiece at hand for these occasions. These can be bought as such, mostly consisting of a 8 x or 10 x Huygens eyepiece, with a movable pointer at the plane of the intermediary image. A simple form of pointer eyepiece can be made easily by glueing a small needle, an eyelash or eyebrow hair (more pointed at the free end naturally than a hair from elsewhere) at the upper side of the field stop (fig. 4.2) of any Huygens eyepiece after having screwed off the front lens; care has to be taken that the point of the needle or hair comes exactly in the plane of the stop.

As it is often embarrassing looking one after another and not together (the resulting head-banging has been a recognized hazard for two centuries), several devices have been developed to enable simultaneous use of a microscope by two observers. An ordinary binocular tube is useless in this respect, as the two eyepiece tubes are too close to one another. The same type of image splitting and bending prisms can be used for bringing the two eyepieces much further (40-50 cm) apart, so that two observers can each look down a monocular (or binocular) eyepiece at the same intermediary image; if a pointer can be moved in the common intermediary image, a very confortable way of microscopy with two observers is possible. Another possibility is to divide the light beam in the tube so that two observers can look


Fig. 4.7. Discussion head with a double binocular tube, provided with an illuminated movable pointer projected into the image (extension of the tube, upper left).

at the same intermediary image opposite one another; in many instances this is both optically and mechanically a very good solution with a high-quality image for both observers (fig. 4.7). This discussion head with its image splitting device becomes rather heavy when fitted with two binocular heads and calls for a very stable stand and a high lighting intensity of the intermediary image, since its light is divided over not less than four eyepieces. Problems of leftjrightjupperjlower in the image (which is reversed for both observers) can be effectively solved with a pointer. In the situation of fig. 4.7 this is the image of an illuminated arrow, projected into the plane of the intermediary image by an accessory optical system. With all these new developments the older tubes with an oblique side-arm in which at least one of the two observers has to make his observation in a rather awkward position and often with an unsatisfactory image have fallen out of favour.

SPECIAL TYPES OF EYEPIECE 73

In the case of 3-5 observers, the use of a projection head with ground glass can be considered; generally this consists of a screen of about 15 cm diameter on which the image is projected with a projective and a mirror. In fig. 11.11 on page 264 such a screen is shown, and an unusually large one in fig. 2.8 on page. 30. Generally, a satisfactory image can thus be obtained using ordinary low-voltage illumination with objectives up to 20 to 25 x in a non-darkened room. Loss in image detail due to the grain of the screen is unavoidable with all these devices. For the demonstration of microscopic images to large audiences, televized images may be the answer to the problems rising with

Fig. 4.8. Binocular comparison tube mounted on two identical microscopes and provided with a common mechanical stage.


microprojection; this will be discussed at some length in chapter 10. An instrument with an effect exactly opposite to that of the discussion heads

just described, is the comparison-eyepiece or tube (fig. 4.8). This instrument permits the observation of the images from each of two microscopes, placed side by side, seen in one field of view. Generally, these two images form two halves of a circular field of view through half stops, but this may be often varied; sometimes the two images can be made to coincide. As in the case of the discussion devices, extensions of the tube length require compensation. The comparison tube, which of course calls for two identical microscopes, is applied in the systematical comparison of e.g. microscopic slides of the same material treated with different staining techniques. It has been provided in the situation of fig. 4.8 with a 'home-made' common mechanical stage which makes possible a simultaneous and identical movement of both specimens at the same time (van Eek, 1975). Such a device is not necessary in all circumstances, although it is extremely cumbersome to move two slides in conjunction with two separate mechanical stages, especially at higher magnifications.

Other special types of eyepiece will be dealt with in the different sections on UV-microscopy, photomicrography, measuring techniques etc. in the chapters 9-12.


J. R. Baker: Experiments on the function of the eye in light microscopy. J. Roy. Micr. Soc. 85 (1966) 231-254.

J. R. Benford and H. E. Rosenberger: Zoom systems in microscopy, in: Advances in optical and electron microscopy, Vol. 3, ed. R. Barer and V. E. Cosslett. Academic Press, New York- London 1969.

W. H. van Eek: A mechanical stage for the combined use of two microscopes with a comparison eyepiece. Microsc. Acta 77 (1975) 157-161.

E. Lau: Das Iichtoptische Doppelmikroskop. Naturwiss. Rundschau 14 (1961) 156-158. c. Metz: Neue Okulare zur Ebnung der Gesichtsfelder der Apochromate. Z. wiss. Mikr.

37 (1920) 49-52.

CHAPTER 5

RELATION OF OBJECTIVE AND EYEPIECE

RESOLVING POWER

The details of the fine structure of the object resolved by the objective are present in the intermediary image and have to be made discernible to the eye by the eyepiece. With regard to this cooperation of both lens systems which will be treated in more detail in this chapter, it is evident from the outset that the resolving power (see chapter 1) of a given objective - eyepiece combination will be determined by the objective, as details not present in the intermediary image cannot be brought out by the eyepiece.

As has been discussed in the first chapter, the wave character of light comes out more obviously when phenomena near the dimensions of its wavelength are studied. The smallest objects which can be observed with a light microscope are indeed of the order of the wavelength of visible light, i.e. around 0.5 [Lm. Before dealing in somewhat more detail with the fundamental problems of resolving power in microscopy, some basic facts of wave optics have first to be considered. They are treated as far as microscopy is concerned in considerable detail in the reviews by Fran~on (1961), Michel (1964) Martin (1966), and Beyer (1973) and will be summarized only very schematically here.

In general, it can be stated that the wave theory shows that a point emitting light forms the centre of spherical waves, and the classical comparison is that with a stick which is moved up and down in a pond, emitting concentric circular waves travelling on the water surface. It should be noted in passing that all parts of such a circular wave are in the same phase of movement. Light rays starting from the light emitting source just mentioned are fictive lines drawn in the direction of the propagation of the light; they are just lines, they cannot be isolated experimentally. When a wavefront is allowed to pass through increasingly smaller apertures, it appears that at a given. moment, when the aperture is already very small and is made yet smaller, the pencil of light emanating from the aperture begins to widen again. This is due to diffraction, a typical wave-optical phenomenon. Another fundamental principle of wave optics is interference, the phenomenon that waves,depending on how they overlap,intensify or weaken each other.

76 RELATION OF OBJECTIVE AND EYEPIECE

Coming back to the problem of resolving power, it can be stated that an essential fact is that objects are observed via the wave fronts they emit. Two light sources separated by a certain distance A will be observed as two separate points, when their wave fronts (after modification by the lenses used) can be untangled as separate entities. This can be shown clearly if again a comparison is made now with two sticks, being moved up and down in a pond; they will both generate a wave front and an observer who does not see the sticks but can observe the circular waves could tell, even if they are fairly close together, that two sticks are being moved up and down. When the sticks are brought more closely to one another, they will finally give rise to a wave-front pattern which no longer enables the observer to tell that two sticks are being moved (fig. 5.1). On the basis of this very

B

Fig. 5.1. Two sticks moved up and down at distance A from each other in a water surface will generate two wave-front patterns; when the sticks are brought together at distance B, they will generate a single wavefront only.

simple experiment it can be understood that the wave character of a radiation will place a limit on the size of the details which can be observed with it; this does hold true for all kinds of electron magnetic radiation and wave movements behaving as such which are used in microscopy (cf. chapter 12).

When an image is formed of a luminous point of very small size, the image will not be congruent with the object: due to the wave character of the light, the image will be that of a bright disc-like maximum (principal or zeroth order maximum), surrounded by a number of fainter rings with decreasing brightness (first, second order etc. maxima separated by minima, which soon become invisible). The entire configuration is called the diffraction disc or Airy disc. A diagram of the light distribution in a transverse section of such a disc is given in fig. 5.2. This phenomenon is brought about by diffraction and interference, leading to mutual enhancement and destruc-

RESOL VING POWER 77

tion of light waves; it could be called a defect of the lens preventing the formation of a perfect image. The theoretical background of all this is rather complicated (cf. Martin, 1966; Beyer, 1973); from a practical point of view, however, it can be stated that a lens cannot form a perfect image, because only a (small) part of the wave front emitted by the object can pass through the lens. It is not difficult to see, therefore, that the larger the angular

III II II III

Fig. 5.2. Distribution of light intensity in a transverse section through an Airy disc; o zeroth order or principal maximum; I, II and III first-, second- and thIrd order maxima, forming luminous rings of decreasing intensity around the central maximum.

aperture of the lens (the objective), the larger the part of the wave front passing through the lens, the better will be the reproduction of the object. In fact it can be shown that, all other circumstances remaining equal, the diameter of the central maximum of the Airy disc (which acts as the imaged point) in the intermediary image varies in inverse proportion with the numerical aperture of an objective, although this does not hold exactly true for higher apertures. The minimal size of the Airy disc is thus limited by the numerical aperture of the objective. We touch here on the very fundamental question of resolving power, as the ability to distinguish two separate points as entities will depend on the relation of both corresponding Airy discs. If we consider two identical minute light-emitting points on a black ground, they will appear discretely only (independent of the enlargement by the eyepiece) if their corresponding diffraction discs are not too close to each other. It is obvious that a separation will be reached when both first-order minima coincide (fig. 5.3A); this is not the limit by far, however. Resolution of two discs is usually considered to be reached when the principal maximum in one of the diffraction patterns corresponds to the first order minimum of the other and vice versa. This situation is called Raleigh's criterion (fig. 5.3B), the light intensity half-way between the two


principal maxima being 74% of that at these maxima. It can be shown mathematically that such a situation corresponds with a minimum resolvable distance of the object plane a which equals:

a = 0.61 A N.A.

III which A is the wavelength of the light used and N.A. the numerical aperture of the objective. The greater the N.A., the finer the details that will thus be made visible with the eyepiece.

A B

Fig. 5.3. A Diffraction patterns in a transverse section of two luminous points at such a distance from each other that the first order minima of both Airy discs have come to coincide; B Raleigh's criterion: the two luminous points have been approached so that a zeroth order maximum of one Airy disc coincides with the first minimum (dark ring around the zeroth order maximum) ofthe other.

The circumstances in practical microscopy are, however, quite different from those of the self-luminous points of the theoretical reasoning and this makes the situation much more complicated. The light from the object does not come from the object itself - except in very special circumstances - but is derived from a light source, an image of which is formed in the object (see chapter 6) and this entails a very fundamental difference in the image formation. The point sources in a self-luminating object will behave independently of each other, i.e. there is no relationship between the phase of the waves; they are said to be incoherent. In an object which emits light from an external source, different parts of the object may receive light from the same point on the light source; a fixed relation will exist then between the phase of their vibrations. They will behave as so-called coherent light sources. Two pin-point luminous sources on black ground will not be as discrete under these circumstances as with incoherent illumination, because coherent light sources are capable of interference, resulting in mutual enhancing or destroying of light waves. As a consequence of this, the minimal resolvable distance under coherent conditions will be considerably greater, to be exact

RESOL VING POWER 79

by a factor of about 1.63. It has been shown that this factor may theoretically be considerably reduced by focussing the light source onto the specimen with a condenser which brings an aperture angle into the illuminating beam. Generally speaking, it can be stated that the smaller the aperture of the illuminating cone the more coherence will exist in the object plane, while the larger the aperture, the less coherent the light in the specimen will be. With an aperture of the light beam of about 1.7 x that of the objective, the situation theoretically closely approaches that with incoherent illumination. As such a high aperture of the illumination cone would give rise to a substantial amount of stray light (see next chapter), entailing decreased image contrast, this is not practicable; generally therefore the condenser aperture is held at the same - or a slightly lower - level than that of the objective. The minimum resolvable distance then will be about 1.22 greater than that with self-luminous, i.e. incoherent, points (cf. Franr;on, 1961). When the condenser is diaphragmed, the criteria of coherent illumination will gradually apply and the resolving power will finally be reduced so that:

8 = 1.63 x 0.61 A =_A_

N.A. N.A.

which is quite sufficient to render some details in the object no longer discernible (fig. 5.4A and B); this will be treated in more detail in chapter 6.

Fig. 5.4. The fine ridges in the shell of the diatom Grammatophora marina can be resolved at N.A. 1.00 and a magnification of 400 x (A). When the numerical aperture of the objective is stopped down to 0.7, the details in the surface scale are lost (B). Oblique illumination at the same aperture (C) brings back the ridges, but the image as a whole shows a certain distorsion.


The situation can be summarized by stating that the minimal resolvable

d · '10 '11 . . b 0.74 A d 1.00 A d lstance 0 WI vary III practIce etween an , correspon -N.A. N.A.

ing with condenser apertures between 0 and the same value as the objective aperture, respectively. When an oblique illumination is used with an incident angle of the order of the aperture of the objective, the effect is that of increasing the aperture asymmetrically. The resolving power is increased, but only in one direction in the specimen, leading to acertain distorsionin the image (fig. 5.4C). This is a system of illumination which nowadays is seldom used (cf. chapters 6 and 8).

TABLE IV. MINIMUM RESOLVABLE DISTANCE 1) WITH DIFFERENT OBJECTIVE APERTURES.

N.A. magnification 1) for self-luminous range of 1) in fl-m to bc range of the points in fl-m for reached with absorbing

objective 'A = 550 nm objects under optimal

(~) conditions for 'A = 550 nm

N.A. (0.74'A _ 1.00 'A) N.A. N.A.

0.10 3-5 x 2.75 4.10-5.50 0.20 8-12 x 1.38 2.00-2.75 0.30 10-16 x 0.92 1.35-1.83 0.60 25--40 x 0.46 0.67-0.92 0.80 40-60 x 0.35 0.51-0.69 1.25 60-100 x 0.22 0.33-0.44 1.32 ±100x 0.21 0.31-0.42

As explained a little earlier, the theoretical resolving power using an illumination with a condenser will, depending on its aperture, vary between

the two limits 0.74 A and 1.00 A . In table IV these values have been N.A. N.A.

calculated for a number of commonly encountered objective apertures, on the base of A = 550 nm (green light). It should be noted, however, that these values are primarily of theoretical value; in a thick section with low contrasts even the highest number will be far from attainable. A clear distinction

RESOLVING POWER 81

should be made between resolving power and actual resolution, in the sense that a fast car in rush-hour traffic does not necessarily progress more quickly than its slower brother. Roughly, it can be stated that optimal conditions exist only when the object is not thicker than about ten times the minimum resolvable distance and when a sufficient degree of contrast with the background exists. It is totally useless to make a rapid calculation with this or any other magic formula to calculate 'the' resolving power in a given case. Even theoretically, Raleigh's criterion has been criticized and called overgenerous. On the other hand, the paramount influence of a change in the objective N.A. (all other circumstances remaining the same) on the resolution in the image from a routine specimen is clear from a single glance (fig. 5.5).

With regard to resolving power and the eye, a clear distinction has been made in chapter I between resolving power and minimum resolvable distance;

Fig. 5.5. Haematoxylin-phloxin stained section of human skin; objective 40 x, eyepiece 8 x, final magnification 400 x. The lower image has been photographed at the full objective aperture of 1.00; the fine intercellular connections (desmosomes) between the epithelial cells are clearly shown. The upper image shows the same area photographed with the same objective stopped down with a built-in diaphragm to a N.A. of about 0.6: the desmosomes are no longer resolved as a part of a general deterioration of the image.

one factor is inversely proportional to the other, a high resolving power means a small minimum resolvable distance. In practical microscopy, however, this difference is often forgotten and so one can read (e.g. about an

82 RELA TION OF OBJECTIVE AND EYEPIECE

electron microscope) of a resolving power of so and so much nm or Angstroms, whereas the minimum resolvable distance actually is meant; this is quite confusing.

When the minimum resolvable distance amounts to a certain value, as listed in table IV, its meaning is often misunderstood. An object does not need to have the outer dimensions of the resolving power in order to be discernible under the microscope; as will be shown in chapter 8, it is indeed very well possible to observe e.g. fine particles with dark field light microscopy down to a size of 5 nm (0.005 [Lm), provided there is a sufficient degree of contrast between such a particle and its background. The same situation - although to a much lesser degree - exists with dark particles against a light background. One is unable to say, however, anything about the shape of the particle; the object has been made visible to the eye, but is not resolved. A comparable situation exists when a ship is observed at a very great distance, so that is can just be discerned as a minute spot and one is yet unable to recognize its oblong shape. In microscopy, the image of objects which approach the minimum resolvable distance, is not reliable with regard to the geometric impression they make. The contrast is of paramount importance with objects near to, or under, the resolving power; objects which are easily resolved cannot be visualized, of course, without a certain amount of contrast, which is due principally to light absorption, or phenomena giving this impression. Ultimately, the image formation of resolved details in the object is the result of a complicated pattern of diffraction- and interference -phenomena in the back focal plane of the objective and intermediary image. The theory of this was developed in 1873 by Ernst Abbe, a German physicist and partner of Carl Zeiss Optical Works at Jena (cf. Michel, 1964; Martin, 1966; Beyer, 1973). Forreasons unclear to the author, his name is often misspelled Abbe in the anglo-american literature.

OBJECTIVE, EYEPIECE AND THE EYE

As the eyepiece has no other function than to make details resolved by the objective discernible to the eye, the relation intermediary image/eyepiece should be considered primarily from this point of view. It is possible to demonstrate that a total magnification (eyepiece x objective) of about 250 x the N.A. of the objective is necessary in order to observe the details resolved by the objective under an angle of I' (cf. chapter 1). To facilitate the observation of the smallest details it is advisable to set the limit of this necessary magnifying power at about 500 x the N.A. to hold a larger margin of

OBJECTIVE, EYEPIECE AND THE EYE 83

safety. It should be noted that in this final magnification objective and eyepiece magnification can compensate for each other up to a certain degree; again, the N.A. is the determining factor and not the objective magnification.

On the other hand, it is quite clear that increasing the eyepiece magnification can be compared to a certain extent with the making of ever increasing enlargements of the same photographic negative: from a given moment on no new details are added to the image. When two resolved points are observed under an angle of 4', the optimum has been reached amply; a further increase of the magnification beyond this limit does not provide any new detail and even tends to make those already present less distinct, the image becoming more and more hazy (fig. 5.6). This is called empty magnification, and it is reached at a total magnification of about 1000 x the (effectively used) N.A. of the objective!.

An optimal cooperation between objective and eyepiece is reached therefore in the region where the product of the magnification of objective and eyepiece falls in the range from 500 to 1000 x the N.A. of the objective. This is called the region of useful magnification; in many cases, however, it is wise to remain under the upper limit.

Other reasons, apart from those already explained, exist to avoid extremely high or low eyepiece magnifications. The diameter (in mm) of the exit pupil or Ramsden circle (chapter 4) is determined by the formula:

500 x N.A.

V

in which N.A. is again the (effectively used) numerical aperture of the objective and V the total magnificatiQP. As the diameter of the pupil of the human eye with a sufficiently illuminated retinal image is about 3 mm, it can easily be calculated that with a magnification of 170 times the N.A. of the objective, the diameter of the exit pupil will equal that of the diameter of the eye pupil. This means that the field of view can be limited by a keyhole-effect if the eye pupil is somewhat narrow and that such an effect will also occur with small eye movements. With a magnification of 500 times the N.A. of the objective, the exit pupil is kept at the practical value of around 1 mm. For a number of technical-optical reasons, a diameter of the

1. Empty magnification does not mean meaningless magnification in all cases. In different measuring conditions (microphotometry, microspectrophotometry, see chapter 11), magnifications in the range of empty magnification even are applied systematically. It should be emphasized that the range of 'useful magnification' only applies for visual observation (directly or via photomicrography) and that in a measuring procedure or automatic image analysis with an electronic sensor the circumstances can be totally different.


exit pupil of t mm or less is undesirable. More or less by coincidence, the region of useful magnification therefore falls in with that which causes a practicable diameter of the exit pupil.

In recording a microscopic image with photography, the diameter of the exit pupil - as the height - is immaterial, as the image is projected onto the photographic plate (chapter 10). The same arguments for holding the total magnification between 500 and 1000 X the N.A. of the objective likewise hold true, be it for non-identical reasons. For the lower limit, the situation is different in so far, that, unlike that with direct observation, by using a fine grain film, 'hidden' details in the negative can be brought out by an adequately enlarged print. For the upper limit, it should be noted that this is in fact ultimately based on the wave character of the light and not on any property of the human eye. Therefore empty magnification occurs likewise when a print is studied at a viewing distance of 25 cm, when the final magnification in a print comes to exceed 1000 x N.A. (fig. 5.6C). Because

Fig. 5.6. Lymphocyte in a blood smear, stained according to May-Griinwald-Giemsa; photomicrographs made with a objective 100x, N.A. 1.32; A final magnification 650x (= 490 x N.A.); B 1150 x (870x N.A.); C2250x (1700x N.A.). Two closely associated azurophilic granules (arrow in image B) cannot be seen as separated entities in fig. A, whereas fig. C, revealing no more detail than fig. B, has become unclear because of empty magnification.

of the grain of the film, it is sometimes practical to put the upper limit somewhat higher than with direct observation; this does not mean, of course, that the resolving power would be any greater.

OBJECTIVE, EYEPIECE AND THE EYE 85

In table V the final magnifications for a series of common objectives and eyepieces have been calculated in relation to the objective-N.A. It is manifestly apparent that in order to remain within the limits of useful magnification, the eyepiece magnifications must be held much higher with lowpower objectives, whereas with high-power objectives increasingly longer focal length eyepieces are necessary to remain outside the bounds of empty magnification.

TABLE V. REVIEW INDICATING WITH x THE COMBINATIONS BETWEEN MORE COMMONLY USED OBJECTIVES AND EYEPIECES FALLING

WITHIN THE RANGE OF USEFUL MAGNIFICATION (500-I000x N.A. OF OBJECTIVE).

objectives eyepieces

6x 8x lOx 12 x 15 x 20x 25 x

2! x, N.A. 0.08 x x

4 x, N.A. 0.12 x x x

10 x,N.A.0.22 x x x

20 x, N.A. 0.45 x x x

40 x, N.A.0.75 x x x

40 x, N.A. 1.00* x x x

60 x, N.A.0.85 x x x

63 x, N.A. 1.30* x x x

100 x, N.A. 1.25* x x x x

* oil immersion

This 'shift to the left' of eyepiece magnification with increasing objective N.A. enables a compromise to be reached, in which the 12 x eyepiece in table V never brings the magnification into the empty region. While even a 20 x objective can be exploited to the full, this is no longer the case with the 4 x and 2-t x objectives. That some resolving power would be lost in using these low-power objectives which are mainly applied for screening purposes, is of little importance, however. It can thus be stated that with an eyepiece in the 12 x range, virtually all standard objectives lead to final magnifications in the useful region. Only the classical 100 x oil-immersion lens with an aperture of 1.25-1.30 approaches the region of empty magnification with a 12 x eyepiece. Newer developments have led to the manufacture of an oil-immersion objective of about 60 x with an aperture around 1.25, which


has a much more favourable position in table V than the 100 x objective. Even an oil immersion of 40 x with a N .A. of 1.00 does not yield much less information in the image than a 100 x N.A. 1.25 objective (the theoretical difference in resolving power is often illusory) and has a much greater depth of field, as will be explained in the next section. For fluorescence microscopy, special low-power oil immersion objectives with high apertures have been manufactured, which will be dealt with in chapter 8; they are of no special advantage for use in conventional microscopy.

In conclusion, it may be stated that it seems oflittle sense to use more than a single eyepiece in the 12 x range for all routine work. Leaving one and the same eyepiece in the tube all the time has the additional advantage that there is less chance that dirt and dust, which always accumulate at the bottom, i.e. at the back lens of the objective, become trapped in the tube when changing eyepieces.

Under different circumstances, such as in photomicrography, with certain types of quantitative work and also in special cases of observation, it is sensible to use different eyepieces, or different settings of a magnification changer (cf. chapter 4). As explained in the previous chapter, change in eyepiece magnification (or -type) entails a change both in field of view and object field. It is possible, e.g. in photomicrography, to make a curvature of field less obvious, or to take just a given detail in the frame at an optimal magnification without objective change etc. The choice of a certain eyepiece-objective combination for a given magnification can also be determined by consideration of the depth of field. This important problem will be dealt with in the next section.

DEPTH OF FIELD

If the projected image of a microscope is received on a screen placed some distance away from the actual image plane, the image is said to be out of focus and will be of poor quality. The maximum movement away from the ideal image plane which can be made without serious deterioration in the image is called the depth of focus. The permissible movement of the object plane with a stationary receiving screen is called the depth of field. Depth of field is sometimes loosely called depth of focus. This is confusing as these two values should be considered as separate, though conjugate, distances in object and image plane, respectively. In microscopy, the depth of field is important, because it determines the thickness of the layer yielding a reasonably sharp image. This factor is a summation of three factors, wave-

DEPTH OF FIELD 87

optical and geometric-optical and finally the quality of the human eye. These three factors are:

1. axial resolving power

2. geometrical depth offield

3. accommodation range of the eye.

re 1. The axial resolving power. The resolving power of points beneath each other on the special axis is subject to rules other than that for points in a plane perpendicular to the optical axis, the so-called lateral resolving power, as dealt with ill the beginning of this chapter. The axial minimum resolvable distance 3a is inversely proportional to the second power of the N.A., according to the formula

n ), 3a =-----

2 (N.A.)2

in which n is the refraction index of the medium in which the points are beneath each other in the object, A the wavelength of the light used and N.A. the effectively applied N.A. of the objective (i.e. with adequate illumination).

re 2. Apart from the axial resolving power, which is determined by waveoptical factors, the geometrical depth of field T is determined by rules of linear optics. It can be calculated with the formula

0.34n T=-----

V X N.A.

in which V = total magnification and nand N.A. as with 1. re 3. As discussed in chapter I, the human eye can, by changing the

power of its lens, accommodate for observation of object details at finite distances. The maximum change in refractive power possible, corresponding with an adaptation from the infinite to 250 mm, would amount to about 4 diopters (it may be much more when the near point is closer, as in children). This is of importance in microscopy as the image is generally viewed in slight accommodation, i.e. at a not too near, finite distance. Theoretically, the maximal change in depth which can be reached, the depth of accommodation A, would amount to:

in which n and V have the same significance as in the two previous formulae. It appears that this third factor is the least predictable, as the degree of


accommodation is dependent on the individual observer and the way he uses his eye.

In table VI some values are given for the depths of accommodation, as calculated with this formula for different commonly used values of V for n = 1.50. These numbers hold for a classical microscopical specimen for transmitted illumination, e.g. a section in a mounting medium under a cover slip. For an object in air, observed with incident illumination, these values have to be reduced by a factor of about t.

TABLE VI. DEPTHS OF ACCOMMODATION CALCULATED FOR n 1.50.

total magnification of the microscope

depth of accommodation in [Lm

50 100 250

150.00 37.50 6.00

500 1000 2000

1.50 0.37 0.09

As follows from these data, the depth of accommodation (which is a maximum value) can be of importance with lower magnifications; with higher magnifications it rapidly becomes negligible in comparison with the depth of field from physical-optical conditions, as will be shown in the following.

The purely 'instrumental' depth of field al, which is of importance e.g. in making a photomicrograph can be calculated by adding the values for axial resolving power and geometrical depth of field, therefore:

a} = n [ A + 0.34 ] 2 (N.A.)2 V (N.A.)

When A is expressed in !Lm, the value for a1 which ensues, also comes out in !Lm. In table VII this factor a1 has been calculated for some common N.A. values and magnifications. It has been assumed that the refractive index in the object is 1.50, as is often the case. It should be emphasized that in studying surface structures of objects in air (e.g. in preparation microscopy) the depth offield will be only t of the values in table VII.

As different theoretical calculations in the optical literature lead to results which show considerable differences (e.g. in the formula for the geometrical-optical depth of field), the values for a1 have been calculated to a single decimal only. The numbers in table VII are meant principally to

DEPTH OF FIELD 89

give a general impression for practical microscopy and to show the differences in influence of aperture and magnification.

The instrumental depth of field is very small with higher apertures and magnifications; Abbe has introduced in this respect the notion of an 'optical section' from a specimen. With ordinary sections for the study with transmitted light (e.g. 4-7 [km thick tissue sections), this optical section will be of the order of the thickness of the specimen when studied with an effectively used N.A. of 0.3-0.4. With a photomicrograph made with such an aperture with a final magnification of e.g. between 100 and 200 x, the entire section thickness can be sharp when focussed in the middle (fig. 3.9). Any eventual loss of definition at the border is due to curvature of field, which will be more pronounced, of course, when the depth of field approaches or becomes less than the section thickness. With the highest magnifications, such a 4-6 [km thick section can be focussed in several layers.

TABLE VII. TOTAL INSTRUMENTAL DEPTH OF FIELD IN [.Lffi FOR SOME COMMON VALUES FOR N.A. AND MAGNIFICATION

(CALCULATED FOR n = 1.50).

N.A.ofthe final magnification

objective lOx 50x 100x 250x 500 x 1000 x 2500 x

0.05 1186.6 370.5 0.10 143.6 92.6 0.20 61.4 35.9 20.6 0.30 21.6 11.4 8.0 0.40 15.3 7.7 5.2 0.65 4.1 2.6 1.8 0.85 3.0 1.8 1.2 1.00 2.5 1.4 0.9 1.30 1.0 0.6 0.4

From the formula for 01 on page 88 and the numbers in table VII it is obvious that the influence of the aperture of the objective on the value of Ol is much greater than the magnification, although it is manifestly not correct to take into account the objective only for an estimation of the depth of field, even apart from the accommodation factor.

In practice, it is important to bear in mind that with a given magnification (which fixes the maximal accommodation depth), the depth of field is greater with a combination of a low-power objective and a high-power eyepiece than in the case of an objective with higher magnification in combination with a correspondingly longer focal-length eyepiece. This is evidently


accompanied by differences in minimum resolvable distance; it is a general rule that depth of field can only be increased at the expense of resolving power. In most circumstances, however, the full resolving power cannot be used (e.g. because of the thickness of the specimen), so that this loss is purely theoretical. This is of importance not only for photomicrography, but also for visual work. As a consequence, in some instances a high aperture can be more of an inconvenience than an advantage, when a very small and often unusable extra lateral resolving power has to be paid for with a sensible loss in depth offield, which varies with the square of the objective N.A.

The users (and consequently the manufacturers) of microscope objectives seem in the past often to have concentrated on a maximal exploitation of theoretical lateral resolving power; moreover, some fear seems to have existed in using the oil immersion principle unless strictly necessary. This has resulted in the time-honoured situation that 'the' oil immersion objective (90-100X, N.A. 1.25-1.35) usually was followed by a dry system 40 x, with a N.A. of 0.65-0.75, depending on the correction grade with a more rarely used dry system of about 60 x (N.A. 0.8-0.9) in between. In the more recent period the newer oil immersion objectives already mentioned in the 40-60 x range with a N.A. between 1.00 and 1.40 have quickly won favour on the basis of their position with regard to empty magnification (table V) and depth of field. Consequently, they gradually come to replace high-power dry objectives (such as those in the 60 x, N.A. 0.85 range) which suffer from much stray-light in the object space and call for correction of the variations in the cover-glass thickness. Apart from very thin specimens, such as smears of blood cells or bacteria or 'semi-thin' sections of plastic-embedded material, these oil immersion objectives in the medium magnification range compare favourably with the standard 100x oil-immersion objective.

Some medium-power oil-immersion objectives in the 40-60 x range are provided with an iris diaphragm enabling stopping down of the aperture to a certain value (e.g. 40 x, N.A. range 1.0-0.6). Such a system, which can also be made from an ordinary objective by screwing on a so-called Davies shutter, is designed mainly for use with dark-field microscopy (cf. chapter 8). It is also possible to apply this in photomicrography to reach a compromise between depth of field and lateral resolving power. As has been discussed before, however, the margin is much smaller in using the microscope than with an ordinary camera with usually much greater dimensions of the object in comparison with the image. Consequently, in stopping down the objective N.A., the loss in resolving power often overshadows the increase in depth of field (fig. 5.6) although the latter is theoretically always present.

DEPTH OF FIELD 91


E. Abbe: Ober die Grenzen der geometrischen Optik. Sitzber. Jen. Ges. Med. Naturwiss. (1880) 71-109.

J. E. Barnard: Resolution and visibility in medical microscopy. J. Roy. Micr. Soc. 50 (1930) 1-40.

H. Beyer: Handbuch der Mikroskopie VEB-Verlag Technik, Berlin 1973. H. Boegehold: Das optische System des Mikroskops. VEB-Verlag Technik, Bedin 1958. R. Bouyer: Microscopie optique a resolution maximale. J. Microscopie 4 (1965) I-S. M. Fran<;on: Progress in microscopy. Pergamon Press, Oxford-London-New York 1961. A. Grabner: Objektiv, Okular und Auge; eine Bilanz ihres Zusammenspieles beim Mikro

skopisieren. Mikroskopie 10 (1955) 83-119. L. C. Martin: Theory o/the microscope. Blackie, Glasgow and London 1966. J. R. Meyer-Arendt: Introduction to classical and modern optics. Prentice-Hall, Englewood

Cliffs 1972. K. Michel: Die Grundlagen der Theorie des Mikroskops. Wiss. Verlagsgesellschaft, Stutt

gart 1964. H. Osterberg and L. W. Smith: Effects of numerical aperture on contrast in ordinary

microscopy. J. Opt. Soc. Am. 51 (1961) 709-714. M. Uhlig: Einflusz der Deckglasdicke und des nD Wertes der DeckgUiser auf die sphiirische

Liingsabstimmung bei Mikroobjectiven. Mikroskopie 19 (1964) 161-164.

CHAPTER 6

CONDENSER AND ILLUMINATION

THE FUNCTION OF THE CONDENSER

As discussed in the previous chapter, the object in microscopy is not, except in a few special cases, self-luminous and has to receive light it emits from an outside source. In fact it may be even stated that the microscopic image is formed by light from the light source, as modulated by the specimen. It has also been explained that the best approach to a self-luminous object (which would be ideal from an optical point of view) is reached by forming the image of the light source in the object. As will be shown this is reached most effectively with a light source of finite dimensions, although diffuse daylight can be used in some cases.

In illuminating an object with transmitted light the most simple solution is the substage mirror, used as early as the eighteenth century (fig. 2.3). As virtually no angular aperture can thus be brought in the light bundle passing through the object, the rules for parallel illumination hold true (chapter 5). In using a hollow mirror for forming an image of the light source, the situation is somewhat more favourable, but obviously this is insufficient except for the lowest magnifications. The use of a hollow mirror for illumination goes back to the Dutch scientist Christiaan Huygens (the inventor of the eyepiece named after him), who proposed in 1678 the use of a positive lens to concentrate the light of a candle onto the object to be investigated with a simple microscope. This has led ultimately to the development of the illuminating apparatus. In the pioneer period of the compound microscope illumination with incident light was often used with a glass sphere to 'condense' the light onto the object (fig. 2.1). In later periods, the hollow LieberkUhn mirrors with a central hole for the objective lens became widely used for incident illumination; the stand of Cuff (fig. 2.3) was provided with such mirrors (not shown in the picture) which could be attached to the objective mount.

All modern microscopes are provided with a lens-system which can be moved vertically along the optical axis. It is called the condenser, after the

THE FUNCTION OF THE CONDENSER 93

old idea that its main function would be the concentration of light onto the object but, as has been briefly discussed in the previous chapter, the situation is much more complicated. Under the condenser a mirror fork is mounted with a mirror having a flat and a hollow side which can be rotated around an axis (fig. 2.5). In the case of built-in illumination a (flat) mirror is mounted in the interior of the foot (fig. 2.6). The main function of the condenser is to form an image of the light source in the object, so that the aperture in the illuminating cone reduces the coherence of the illumination, bringing the situation with regard to the resolving power more closely to that of a selfluminous object (cf. chapter 5). The systematic use of a condenser goes back only to the last period of the nineteenth century. It is interesting to note that the improvement in image quality and resolving power by the mere use of the condenser has made significant progress possible, e.g. the discovery of the tubercle bacillus by Robert Koch in 1882, in which he used a condenser as designed by Abbe and brought out by Zeiss in the beginning of that same year.

All condensers are designed for receiving almost parallel light rays, so that the flat mirror should always be used. With the condenser in place the hollow mirror does not fulfill any function at all, except for a particular use with low power, as will be explained later in this chapter. With most modern microscope stands, the condenser cannot be swung out, leaving the mirror as the sole illuminating aid, as was the case with the original illumination apparatus of Abbe. The hollow mirror can be considered as a remnant of this system; misunderstanding of this situation is frequent. Perhaps misled by its designation (Abbe tried in vain to replace the name condenser with 'illuminator'), it is a common mistake of inexperienced observers to use the hollow mirror together with the condenser, thinking that the effect of the condenser is enhanced in this way. In fact, the hollow mirror only enlarges the image of the light source formed by the condenser with a totally uncorrected system, so that the field of view may become irregularly illuminated.

Illumination using the condensor and the flat mirror is illustrated in fig. 6.1. Starting from this scheme, the basic features of illumination will now receive comment and explanation.

The condensor should be capable of being moved along the optical axis to focus the image of the light source exactly in the object plane with different thicknesses of object slide. In most cases, the cond~nser will then be at or near its highest position in the rack, with its front lens in the round opening in the stage. The free working distance determines in those cases

94 CONDENSER AND ILLUMINATION

where this is critical (i.e. with a short-focus condenser) the maximum tolerable thickness of the object slide. In most cases this working distance is 1-1.5 mm which is much larger than with objectives of a comparable aperture and length of focus.

The substage iris diaphragm D under the condenser, which is housed in the same mount as the condenser, is mostly located just outside of the focal plane of this lens system, forming the entrance pupil of it. When this diaphragm is gradually closed, the diameter of the illuminating bundle will become smaller, entailing a reduction of the aperture of the light cone coming from the object. In view of this function, this condenser iris diaphragm is also frequently called aperture diaphragm. In so far border rays are shielded which would not have reached the objective anyhow (S' and S" in fig. 6.1), this can reduce stray-light, resulting in better contrast in the image.

Fig. 6.1. Schematic view of the illumination apparatus in a standard microscope; closing of the diaphragm until the position shown with the dotted line screens the rays emerging as S' and S" from the specimen, reducing the aperture of the illumination cone. To keep the drawing as simple as possible, the image formation of the light source has been shown for one point of the lamp surface only.

As can be seen clearly in fig. 6.4, the emanating light cone as a whole also shows a sharper outline when the N.A. of the condenser is reduced with the aperture diaphragm. As this is closed, the aperture of the light cone entering the objective is, however, reduced accordingly, so that from a given point on the aperture of the objective is no longer filled with light (fig. 6.3). In this case, the stray-light has been controlled very effectively, with as a consequence high image contrast ,but the effect of the condenser is reduced. With a stained section as used in routine light microscopy, the condenser aperture


Fig. 6.2. Polytene chromosomes in a cell nucleus of a salivary gland of the insect Chironom us sp.; magnification 400 x (objective 40 x, N.A. 0.65). A fully opened condenser diaphragm: hazy image due to presence of much stray-light in the specimen. B closing of the diaphragm so that about 75% of the objective aperture is filled with light: well-balanced image, in which the banding patterns of the polytene chromosomes are sharply outlined. C fully closed diaphragm: loss of resolving power, distorsion of the image by diffraction and interference phenomena due to the illumination being virtually coherent.

The situation in A, Band C corresponds roughly with the three drawings of fig. 6.3.


OP OP

AD AD

Fig. 6.3. Effect of different openings of the condenser aperture diaphragm on the light cone reaching the objective, the aperture of the cone being too large, about equal to that of the objective and too small, respectively. The optical effect would be something like the three images of fig. 6.2. OP object plane, AD aperture diaphragm.

is usually stopped down to a slightly lower value than that of the objective; practical rules for this will be given in the next chapter. In this situation, a good compromise is usually reached between a hazy image due to glare and an image deteriorated by too Iowan aperture of the condenser; the latter not only leads to a loss in resolving power, but the image also tends to be distorded by interference effects (see fig. 6.2) which arise from the decrease in illumination cone aperture due to the more coherent nature of the illumination (cf. chapter 5).

In general it may be stated that the condenser diaphragm has its main function in the adapting of the N.A. of the condenser to that of the different objectives with the changing optical circumstances in various objects.

All that has been said in chapter 3 concerning the maximal N.A. with dry objectives, will also hold true for condensers. This means that, also with a condenser with a N.A. of 1.20 engraved on the mount, all rays with an inclination greater than that which corresponds with a N.A. 0.95 cannot reach the object on the other side of the slide. An illumination cone with a N.A. of 1.00 or more is only possible with so-called condenser immersion, in which a drop of a fluid (as a rule immersion oil) is brought between the front lens of the condenser and the underside of the object slide. As the demands for image quality of the light source are not very high, virtually all condensers with higher apertures can be used with or without immersion; this is in sharp contrast to the use of objectives. It is self-evident that the


application of immersion does not make sense with a condenser of e.g. 0.9 N.A., but even the immersion of a condenser with N.A. 1.20, to fill the aperture of an objective with a N.A. of e.g. 1.30 is not always without problems. It can be shown that the light cone rising from the condenser front lens is enlarged (fig. 6.4C) and also has much less sharp outlines than without immersion, which could generate a good deal of stray-light in the object.

A B c Fig. 6.4. The light cone emanating from an aplanatic condenser (N.A. 1.25) illuminated with a parallel beam of light as shown with blocks of fluorescent plastic under different conditions of illumination. A aperture diaphragm of condenser opened; B aperture stopped down to about 0.7; C diaphragm opened again, but now with immersion oil between condenser frontlens and the block of fluorescent plastic.

This can be controlled, of course, by stopping down the condenser somewhat, but this would led to virtually the same situation as before immersion. This unsharpness of the light cone is dependent on the degree of correction of the condenser. If this is not particularly good, it makes virtually no sense to use immersion with a condenser (if designed for higher aperture) in order to bring the N.A. of the illumination to higher values than 1.0 despite perfectly sound theoretical reasons for so doing.

The image of the light source as formed by the condenser with a diameter of roughly a few mm will be as a rule much larger than the object field of the objective. This no longer holds true, however, with objectives of a magnifi-


cation of 3-4 x and below, where as a consequence only the central portion of the object field will be illuminated. The size of this disc is determined by the focal length of the condenser (not its N.A., of course) and the distance and size of the light source. As it is obviously very impractical to replace the condenser by another with longer focal length when changing from one objective to another, other solutions have been soughtl. One of the most commonly employed is that of a condenser in which the front lens can be screwed off or swung out around an axis. This enables an instantaneous change in refractive power of the condenser and an enlargement of the image of the light source (fig. 6.SB); this is accompanied by a desirable

A B c

Fig. 6.5. Scheme of the illumination beam with a condenser with swing-out front lens. A frontlens in position; B frontlens swung out; C enlargement of the illuminated objectfield by lowering the condenser; in contrast to the situation at B, the image of the light source is no longer in the object plane OP.

lowering of the aperture of the condenser. A disadvantage of this widely used system is that the correction of image errors in the condenser is often less good, especially in the case of a swing-out front lens where some distance must remain between the front lens and the rest of the condenser.

With some types of microscope, the illuminated area in the object can be

1. In the early thirties Richter has developed the principle of the pancratic condenser (Greek: pan = all and kratos = power), in which the size of the illuminated spot in the specimen could be varied continuously in accordance with the aperture of the illuminating cone by a system in which the mutual distances of optical components could be varied mechanically In spite of the theoretical advantages, this rather large and costly system has not found general acceptance. In the last few years, newer versions of the Richter condenser have been applied in some large research stands.


enlarged by the other factor which determines its size, i.e. the light source itself. An auxilary lens beneath the condenser in some larger types of microscope with built-in illumination permits enlargement of the image of the light source without interference with the aperture of the illuminating cone. Its effect which is often misunderstood (it serves only a useful purpose with lower magnifications) is comparable to that of a hollow mirror, used in combination with a condenser.

With very low objective magnifications (1-2~ x) it is often not possible to achieve the conditions of a homogeneous illuminated object field, even with a condenser with a swing-out front or auxiliary lens. For observational work it is then advisable to take out the condensor entirely and work with the hollow mirror only, or an auxilary lens in the case of built-in illumination. For photomicrography which requires a perfectly even illumination of the object field, this is often not satisfactory. Specially designed longfocus condensers ('spectacle-glass condensers'), which fit in the slide or sleeve in which the ordinary condenser is fixed, have then to be used.

CRITICAL ILLUMINATION AND KOHLER ILLUMINATION

In most cases with routine microscopy, a diffuse light source is used, such as an ordinary high voltage opal bulb or a ground glass illuminated from behind by some kind of bulb. By the method which has been called critical illumination, the luminous surface of the opal bulb or the ground-glass screen is imaged directly into the object. The aperture cone can be controlled by the aperture diaphragm in the entrance pupil of the condenser (fig. 6.6, upper image), the size of the illuminated area eventually with a field diaphragm. This widely used system for illumination is, notwithstanding its name, not particularly critical and nothing will change in the performance of the microscope when the condenser is put into a slightly higher or lower position. This is very convenient, as the surface of the opal lamp or the grain of ground glass may disturb the image of the specimen; this can be corrected, therefore, by moving slightly the focussing control of the condenser without impairing the quality of the illumination. Two main disadvantages of this system of illumination remain, however: the unevenness of the light intensity in the object field and the fact that the light source must be rather large to produce a sufficiently extended image when lower magnifications are used.

One would like to have at one's disposal a light source of sufficient homogeneity and extension to ensure an even lighting intensity throughout the


FD AD CD OP

FD AD CD OP

Fig. 6.6. Schematic view of the principal differences between critical illumination (upper drawing) and Kohler illumination (lower drawing). CL collector lens, FD field diaphragm, AD aperture diaphragm, CD condenser, OP object plane.

entire object field with all magnifications. Such a light source cannot be manufactured, but it can be 'made' with optical means. When a weak positive lens!, a collector or auxiliary condenser, is placed in front of the light source, so that an image of the light source is focussed in the back focal plane of the condenser (i.e. in the plane of its aperture diaphragm), an image of the surface of the auxiliary condenser is focussed in the plane of the specimen (fig. 6.6, lower image). It appears that the surface of the collector and the region immediately in front of it - which function as a secondary light source - have a homogeneous light distribution, even if the light source is a lamp with a dense coil in non-opalescent glass. This principle, which also has some other advantages, is called the Kohler-illumination, after its inventor A. Kohler, who published the theoretical basis for this illuminating system in 1893. The principal difference between this system and critical illumination is that not the lamp itself, but an optically made homogeneous field is used as the light source By closing the field diaphragm (FD in

1. It should be mentioned in passing that the collector is usually corrected to a certain degree at least for spherical aberration so that a complex of lenses is involved which can be separate or cemented together.

CRITICAL ILLUMINATION AND KOHLER ILLUMINATION 101

fig. 6.6), its border can be imaged in the object; subsequently, the diaphragm can be opened in such a way that only the actual object field is illuminated. In limiting the illumination of regions of the specimen which take no place in image formation, glare in the preparation is reduced, resulting in an image with higher contrast. Closing of this field diaphragm (which can exert a similar function in critical illumination, but can not so easily be controlled) does not influence the aperture of the illuminating cone. The aperture diaphragm (AD in fig. 6.6) does this in the same fashion with both types of illumination.

Although the microscopic image with a Kohler-illumination can impress as a consequence of the homogeneity of the illumination and the low level of stray-light, the resolving power is theoretically not higher than with critical illumination (Hopkins and Barham, 1950). The image may be qualitatively better, however, by the sharper contrasts thus enabling the perception of fine details. Another advantage of the Kohler-illumination is that it allows the use of small low-voltage bulbs with a dense coil and a high lighting intensity. This is especially useful for built-in iIlummation; the larger stands of research- and photomicroscopes are provided as a rule with the devices necessary for mounting a Kohler-illumination, such as a collector in front of the light source and a field diaphragm (4 in fig. 2.6, page 28) facing the aperture diaphragm (6 in fig. 2.6) under the condenser, which is provided with a centering adjustment. It is not necessary at all, by the way, that these two variable diaphragms are united in a single stand; it is very well possible to realize a Kohler-illumination over a mirror with a detached light source provided with a collector and a field diaphragm. It is with such an equipment that Kohler has demonstrated his new illumination system, and for many years it has been used as the preferential illumination for e.g. photomicrography. With this set-up, it is important to fix the microscope lamp and the microscope with respect to each other, as the slightest movement of the lamp or the microscope would necessitate readjustment.

For both illumination systems, some kind of centering device of the condenser towards the optical axis is necessary. This is mostly arranged with two screws and is included in virtually any modern microscope with built-in Kohler illumination. With critical illumination this is not so important, as long as the light source is sufficiently great for the object field to be illuminated. In most routine microscopes a centering device is missing, therefore, although something can be done about a badly centered condenser by moving it somewhat in the sleeve of the holder. The practical side of setting up the illumination will be dealt with further in the next chapter. Some

102 CONDENSER AND ILLUMINA nON

special types of image formation are possible only with a Kohler-type illumination; this will be dealt with in chapter 9.

TYPES OF CONDENSER

With most routine microscopes manufactured in continental Europe, a condenser according to Abbe is mounted. This is a simple type of condenser consisting of two lenses which transmits much light and gives good results with lower magnifications. With higher apertures than about 0.6, this simple complex shows quite a deal of chromatic and spherical aberration. It is an old dispute in how far such aberrations in the illuminating system are of importance; traditionally, microscopes manufactured in England and the U.S.A. have a condenser with a smaller diameter, but somewhat better correction than the classical Abbe condenser, as is usually provided with routine microscopes on the European continent and those of Japanese manufacture. Such a smaller condenser has a less extended area which it can illuminate; as long as critical illumination is applied with a sufficiently large light source the difference will not be large. Anyhow, these simple condensers have rather poor optical qualities in comparison with a simple achromatic objective. In using a condenser of the Abbe type with Kohlerillumination, it appears that the borders of the field diaphragm can hardly be brought into sharp focus (fig. 6.7).

According to the most recent views, the optical properties of the system that forms the image of the light source are not of primary importance for the resolving power; condensers of somewhat better correction grade give rise to a lower degree of glare in the specimens, however. It is for this reason that in the larger stands of research microscopes and photomicroscopes condensers with a better correction grade are mounted; the best achromaticaplanatic condensers (aplanatic means: corrected for spherical aberration and coma) give a sharper image of the border of the field diaphragm (fig. 6.7); they have the correction grade of no more than an average achromatic objective, however. Image contrasts are - especially when Kohler illumination is used - often considerably better with these more highly corrected condensers and an even illumination can be attained more easily.

A still higher degree of correction of the condenser makes no sense; the apochromatic condensers which have been constructed in the past have become museum pieces. On the other hand, there is not the slightest objection against the use of objectives as a condenser. For observational purposes, however, the fixed N.A. of most objectives is a draw-back when they

TYPES OF CONDENSER 103

Fig. 6.7. Transverse section of a condenser of the Abbe-type (left) and an achromaticaplanatic condenser (right), with corresponding images of the border of a field diaphragm, as observed with a 10 x objective at a 100 x final magnification of the same area from a section of human liver, stained with haematoxylin-phloxin. Note the differences in sharpness of the image of the diaphragm border, as weIl as in the contrasts in the specimen.

are used as a condenser; moreover, the free-working distance of all objectives with a magnification of over about 20 x falls below the thickness of an object slide (0.9-1.2 mm) so that the specimens should be mounted between two covers lips in order to use a high power objective as condenser. As no real advantages stand against all these practical problems (notably not in resolving power) objectives are seldom used for this purpose, except in certain measuring microscopes (e.g. microspectrophotometers, see chapter II) where objectives are used as condensers with a view to the prevention of stray-light. As in these situations low apertures of the illumination are generally used (also in view of the generation of stray-light), the free-working distance does not form a problem of primary importance.

SPECIAL TYPES OF ILLUMINATION; INCIDENT ILLUMINATION

Particular problems occur when e.g. living cells on the bottom of a culturing flask, a petri dish etc. have to be studied under a layer of fluid covering the


object proper. Except in the rare cases where the objective can be dipped into the fluid over the cells (water-immersion, see chapter 3), it is often more convenient to study the cells from beneath through the bottom of the vessel with an illumination coming from above. The only alternative - use of special objectives with long-working distance, the illumination coming through the bottom of the vessel - is always less favourable in these oircumstances, as aperture loss due to the thick layer of fluid is much less serious for the illumination than for the objective.

A stand in which such an exchanged position of microscope and illumination system has been realized is called an inverted microscope or plankton

Fig. 6.8. Inverted binocular microscope stand with as object a specimen (here a piece of white paper) at the bottom of a petri dish. Note the practical solution for bringing the (co-axial) controls for the high positioned mechanical stage more closely to the coarse and fine adjustment.

SPECIAL TYPES OF ILLUMINATION; INCIDENT ILLUMINATION 105

microscope (fig. 6.8). It is often used in tissue and cell culture for control of the cell growth and also in hydrobiology for the study of water samples (hence its second name) up to objective apertures of 0.4-0.5; with higher apertures, the thickness of the bottom of the vessel and its optical properties come to playa role. Apart from the stand, such a disposition does not differ essentially from a conventional microscope for transmitted illumination. The optical axis has to be deviated with a prism over 135 0 to enable the study of the image with a tube which has the usual 45° forward inclination. The condenser, which should be movable along the optical axis, generally is of the long focus type for reasons just explained.

Apart from the rather atypical conditions of the inverted microscope, circumstances arise with the upright microscope in which the illumination bundle must traverse a thicker layer than the 0.9-1.2 mm of the standard object slide in an otherwise conventional situation (e.g. special observation chambers for single cells). Special condensers with long working distance exist for these purposes, which can cope with a distance between condenser front lens and object of 5 mm and over, while enabling a condenser aperture of up to about 0.5. These condensers cannot compensate, however, for the irregularities in thickness which often occur in thicker glass bottom or plastic culture chamber bases, so that some loss in resolving power at higher magnifications is often unavoidable.

Totally different to the situation of the inverted microscope is that in which both objective and light source are at the same side of the object, which is called incident illumination. Although in metallurgy and mineralogy the principle of incident illumination is sometimes applied with a microscope of the inverted type, because of bulky specimens, the term microscopy with incident illumination is generally linked with the conditions in which the object is approached from above by both the objective and the illumination. This type of illumination is used for the study of surfaces; for materials intransparent for light or specimens which cannot be cut into thin slices (metals, rocks) it is the most common technique for microscopic observation. It used to be applied on a rather small scale only for biological materials, but the situation has changed somewhat with a.o. new developments in fluorescence microscopy (chapter 8).

The incident illumination with low-power objectives (e.g. in preparative microscopy with stereomicroscopes) does not give rise to great difficulties, as the long free working distance usually enables the focussing of a spot light onto the object to be studied. With rising N.A. of the objective, however, the free working distance is rapidly diminished to a few mm or less, so that


the objective itself becomes an impediment for the light bundle reaching the object. At the same time, the illumination has to meet higher demands. The best method to illuminate the object would of course be to image the light source on to the object by the lenses of the objective itself. This can practically be realized, but it is then necessary to separate between the light 'on the way' to the object and the light reflected by the object. Essentially, two different systems exist to achieve this, known as vertical illumination or normal incident illumination; they are illustrated in fig. 6.9.

-IP -11'1

l C

B

Fig. 6.9. Scheme of two systems for vertical incident illumination. A with totally reflecting prism, B with semi-reflecting plate, L light source, C condenser, P prism, SP semi-reflecting plate, 0 objective, IP intermediary image plane.

With the system illustrated in fig. 6.9A, the light source is imaged in the object plane over a reflecting prism or a mirror via one half of the objective. The other half of the objective is used to converge the image forming a light band towards the intermediary image plane. As only half of the N.A. of the objective is used in image formation, a loss of 50% in resolving power occurs with this system, in comparison with transmitted light. When the bundle from the light source is led through the objective via a semi-transparent plate instead of a totally reflecting surface, the reflected light can fill the entire aperture of the objective (fig. 6.9B). The image forming light bundle has, however, undergone a considerable loss of energy. Only a

SPECIAL TYPES OF ILLUMINATION; INCIDENT ILLUMINATION 107

certain percentage of the light 'on the way' to the object, is reflected by the semi-transparent plate, the rest is transmitted and is lost for image formation. Vice versa, only a portion (in the ideal situation just 50%) of the light on the 'way back' is transmitted; here the reflected light is lost for image formation. This means that even under the most favourable circumstances (which can never absolutely be attained) only 1 x 1 x 100% = 25% of the available light can be used for image formation. In practice, even 20~~ is rather high, as some light is also lost by reflection at other surfaces, such as that of the objective. Special low-reflection objectives are manufactured for use with incident light and higher powers, in order to minimize these losses (see chapter I). Apart from the light loss, the system with the semi-transparent plate enables the setting up of an illumination system totally comparable with that in transmitted light. With a field diaphragm in front of the collectorlens, it is also possible to realize an illumination of the Kohler type. Different special techniques usually applied with transmitted light, such as phase contrast, polarization and fluorescence microscopy (see chapter 8 and 9), can be applied also to microscopy with incident illumination via this system.

For realizing a more oblique illumination, such as with dark field microscopy (see chapter 9), this vertical illumination system cannot be used. The hollow cone of light for dark field can be obtained by a condenser with a parabolic mirror surface in a concentric position around the objective (fig. 8.6 on page 152). Although it is usually applied for these special techniques the principle of the circular mirror condenser can be considered as a third system (after those in fig. 6.9 A and B) of incident illumination.

A very special type of illumination, which has a place of its own is the quartz-rod illuminator. This is a more or less bent rod of fused quartz in which light can be retained because the refractive index of quartz is such that all light is reflected back into the rod. Hence light can be delivered deep into accessible organs of living animals and a microscope can be focussed on free borders of such an illuminated organ without further preparative measures. This method for diffuse illumination had already been tried out in the nineteenth century, but was worked out in detail by Knisely between 1936 and 1954. More recently, it has been used by Bensley (1960) in the microscopic investigation of the iris in the eye. Apart from the fact that this illumination is a diffuse one, so that the aperture of a highpower objective cannot be filled, this technique is again subject to the cardinal problem of studying three-dimensional structures with higher magnifications: the small depth of field, which limits the part of the object to be overseen to a thin curved disc.


THE LIGHT SOURCE

The very old controversy between artificial illumination and daylight - both of which were already in use in the seventeenth century - is now of historical interest, as daylight is only used exceptionally nowadays. Ordinary daylight of course is readily available, but it is a very diffuse form of iIlumination; the light source cannot be imaged in the object; this entails quite a deal of glare in the object, lowering the contrasts in the image. With low-power observation this can be limited to some extent with the aperture diaphragm; in many cases a satisfactory illumination can be reached on a clear day using the flat mirror near a large window without frames. In the eighteenth century when only feeble artificial light sources existed, the sun has been used very often as a powerful source (e.g. for microprojection, very popular in that period with groups of amateur microscopists); mirrors were placed outside the house to bring the sunlight via a hole in the wall to the projection microscope in a dark room. In the pioneer period of photomicrography the extremely low speed of the materials also demanded a more intense light source than the industry could provide then; one of the first succesful permanent photomicrographs was made by W. H. Fox Talbot (1839) by means of a solar microscope. An artificial light source, however, has obvious advantages as all circumstances can be controlled more efficiently.

The primary requirements for an artificial light source are a. a sufficient and sufficiently homogeneous luminance and b. a sufficiently large surface. The unity for luminance is, as explained in chapter 1, the stUb (abbreviation: Sb). A light source with a surface of 1 cm2 which gives a luminous intensity of 1 candela (the modern version of the formerly used 'international candle'), has a luminance of 1 stilb. Luminance and surface can virtually not be too great: the former in view of the higher magnifications, the latter for lowpower observation. With a variable resistance and/or a neutral density filter (which absorbs light of all wavelengths to the same extent), a surplus of brightness can be compensated for; the effective size of a light source can always be regulated with a field diaphragm. Irregularities in the luminance of the light source can be smoothed away with a Kohler-type of illumination and/or a ground glass filter in front ofthe light source.

In practice, a compromise has to be made between surface and luminance of the light source, which factors are not independent from one another. The most commonly used light source in routine microscopy is the incandescent 40-60 Watt high voltage tungsten filament lamp with opal bulb of the domestic type. It has a relatively large surface with a luminance of a few hundred Sb; they are best suited for use with a more simple type of

THE LIGHT SOURCE 109

critical illumination. A variable resistancel will seldom be of any use, as the brightness of the image will not often be too great (it would, moreover, change its spectral properties in an unfavourable direction). In contrast to what one might think in first instance, it hardly makes sense with these high voltage lamps to use e.g. a 100 Watt lamp instead of a 40 Watt lamp in case of insufficient brightness of the image with high-power observation. The light-emitting surface will increase correspondingly resulting in a virtually equal luminance. The only - rather paradoxa! - advantage of such a 'stronger' light source would then be its larger surface, which could be useful with lower magnification. Moreover, these large high-voltage lamps give off a considerable amount of heat.

The low-voltage lamps, as used with the larger research-types of microscope (nowadays virtually always with built-in illumination) are mostly 12 V (more rarely 6 V) lamps, made in a wide selection of wattages, generally 15-60 W, sometimes higher; they have a great luminance (up to 2000-3000 Sb) over the region of their tightly coiled tungsten filament, as projected on the bulb wall. In spite of their smaller dimensions, they thus have a greater total effective luminous flux than the larger high-voltage lamps just mentioned. The proper light-emanating surface with this high luminance is only a few mm2 and therefore too small for use with critical iIIumination; with Kohler-illumination this can be compensated with the collector. The surface of the light source should not become too small, however; in using the Kohler-principle the aperture of the collector has to increase with a smaller light source in order to image the filament in the aperture diaphragm of the condenser, so that its opening is filled by this image. Although the collectors in modern microscopes with built-in ilIumination are compound lenses and usually have a fair degree of correction (at least for spherical aberration) their aperture cannot be made too large. Moreover, there is little sense in using extremely small light sources; in contrast to what is often thought, a point-shaped light source is far from ideal in image formation as has been explained in chapter 5.

The low-voltage tungsten lamp with a variable resistance, easy to handle and not very expensive, gives satisfaction with regard to light yield for most kinds of conventional microscopy and photomicrography. The tungsten incandescent lamp in general has a few typical disadvantages, however, which emerge so strongly under certain circumstances that one is forced

1. In the most recent period, built-in thyristor resistances have been succesfully applied for this purpose with some types of student-microscopes having a rather bright highvoltage light source.


to look for other light sources, even apart from situations (e.g. ultraviolet microscopy) where it is clear from the beginning that an incandescent lamp will not be the appropriate source. The luminous energy given off by any incandescent lamp has a spectral distribution which is very unfavourable for microscopy, causing a very inefficient yield. The largest part of the luminous energy given off by the glowing filament is in the region of the infra-red or invisible heat radiation; also the light given off in the visible region under 750 nm is mainly of longer wavelengths (fig. 6.10). In charging a lamp at overtension the yield in the visible range can be made somewhat greater; this reduces the life of the lamp considerably and the difference in light yield is not substantial (See also page 231-232).

300 400 500 600

_0 --.--700 nm

III

II

Fig. 6.10. Spectral energy distribution with I high pressure mercury arc, II low voltage incandescent lamp, III xenon-burner.

Another problem involved with tungsten lamps is that the glass envelope gradually becomes blackened with age, due to the fact that evaporated tungsten from the heated filament is deposited at the inner side of the glass. This effect causes a gradual reduction in light yield and a change in the spectral distribution of the light energy given off. This problem has been effectively met with in the so-called tungsten-halogen lamp, a recent development, to be considered more as an improvement of an existing system than as a really new type of lamp. With these halogen lamps a tungsten filament is still the light source proper, but the glass envelope is filled with halogen


gas (e.g. iodine) which combines temporarily with the tungsten, as given off in gas form by the heated filament. Subsequently, the bound tungsten is redeposited on the filament, the halogen gas is released and the cycle can begin again. Tungsten-halogen lamps have become quite commonly used in recent microscopes, especially for photomicrography. They are manufactured for a tension of 12 Volt and a power up to 100 Watt; at the moment these are light sources with the highest light yield of all incandescent lamps used in microscopy with a bulb life of up to several hundred hours. They have a small and dense filament, are very convenient to use, but due to a high temperature of the filament they develop quite a deal of heat so that the housing needs some kind of cooling by air slits and a heat absorption filter. The temperature of the glowing filament cannot exceed 3000-3100°C; higher temperatures than this can never be reached with heated bodies, the melting temperature of tungsten being about 3300°C. More elevated temperatures are necessary, however, for still higher levels of light yield, especially in the lower regions of the visible spectrum and ultraviolet radiation, which is not given off by any incandescent lamp. Only light sources based on the principle of glowing gasses as source of radiant energy can yield substantially more in this direction.

The oldest representative of this type of discharge burner is the carbon are, in which an arc of light is generated by an electrical discharge between two pointed carbon rods. Using continuous current, a temperature of about 400QOC is developed in the 'crater' of such an arc and in this region a luminance can be reached of more than four times that of a low-voltage incandescent lamp. This light source has now practically disappeared, not only because it is difficult to keep the arc constant between the two tips of the carbons which gradually melt away, but also because of its unfavourable spectral properties, with an isolated peak at 400 nm, but for the rest most of the energy output in the red and infrared region.

High pressure mercury-vapour lamps have an energy emiSSIOn which is more dispersed over the visual spectrum; in contrast to the continuous spectrum of e.g. an incandescent lamp, this arc between two electrodes in a discharge tube made of quartz has a so-called band-spectrum, a rather low basis continuum, superimposed by narrow and high emission bands at definite wavelengths (fig. 6.10). When (e.g. for fluorescence microscopy) special peaks, such as that at 546,436 or 365 nm, are selected with appropriate filters it can be applied as a very powerful light source. As a lamp for general use it is of limited value as a consequence of its band-spectrum, which does not enable good contrasts with stains to be obtained; notwithstanding this, it is


a powerful light source with a considerable deal of the energy output in a desirable part of the spectrum.

A more - though not entirely - continuous spectrum is generated by what is considered as the most powerful universal light source of the present day, the xenon high pre.ssure burner. This rather new type of gas-discharge light source has many advantages when an extreme brightness is required; it has a virtually continuous spectrum in the visible range and proximal ultraviolet (fig. 6.10); only in the infrared has the spectrum more ofa bandcharacter. It yields the full output shortly after having been started with an adequate discharge apparatus and gives a rather stable crater of very high luminance.

In table VIII a few average data are given about the main types of light source described in this section. Facts and problems concerning colour temperature and light filters will be dealt with in chapter 10.

TABLE VIII. REVIEW OF SOME AVERAGE VALUES FOR DIFFERENT LIGHT SOURCES USED IN MICROSCOPY.

nominal power average area voltage used lighting radiating (Volts) (Watts) intensity surface

(Stilb) (mm)

high-voltage bulb 220 50 200 14 x2

low-voltage lamp 6 15 900 3 x 1.2

low-voltage lamp 12 60 1600 4.5 x2

low-voltage halogen lamp 12 100 3400 3.5 x2

carbon arc 60 360 16000 40

high-pressure mercury lamp 60 200 25000 2.5 x 1.3

xenon burner 14 75 40000 0.5 xO.25

xenon burner 20 460 35000 2.4 x 1.2

High pressure gas-discharge burners as mentioned in table VIII with their housings, heat filters, transformers and power units are complicated and costly equipment and in quite another category to incandescent lamps of any form, which will remain the main sources for routine microscopy. The ad-


vantages of a low-voltage illumination over the time-honoured 220 V-lamp (be it in a separate stand or as an attached device below the condenser) are so evident, however, that even in routine conditions it should be considered to leave the high voltage bulb altogether.


E. Abbe: Uber einen neuen Beleuchtungsapparat am Mikroskop. Schulzes Arch. 8 (1873) 469-480.

S. H. Bensley: Microscopic studies of the living iris. Anat. Rec. 138 (I960) 39-48. F. Habermalz: Die Verteilungstemperatur der Niedervoltquellen fUr die Mikroskopie. Z.

wiss. Mikr. 68 (1967) 73-83. H. H. Hopkins and P. M. Barham: The influence of the condenser on microscopic resolu

tion. Proc. Phys. Soc. 63 (1950) 737-744. M. H. Knisely: The fused quartz rod technique for transilluminating living internal organs

in situ for microscopic study. Anat. Rec. 120 (1954) 265-275. M. H. Knisely: Fused quartz rod livmg tissue illuminators, in: In vivo techniques in histology,

ed. G. H. Bourne. The Williams & Wilkins, Baltimore 1967. A. Kohler: Ein neues Beleuchtungsverfahren flir mikrophotographische Zwecke. Z. wiss.

Mikr. 10 (1893) 433-440. K. Larche: Moderne Gliih- und Entladungslampen. Naturwissellscha!ten 56 (1969) 429-

434. J. Rienitz: Uber Koharenzverhaltnisse bei der Abbildung im Mikroskop, Microsc. Acta 73

(1973) 217-233. A. Westphal: Eill!iihrullg in die Re/fexmikroskopie und die physikalischen Grundlagen mi

kroskopischer Bildelltstehung. Thieme, Stuttgart 1963.

CHAPTER 7

SPECIMEN, MICROSCOPE AND OBSERVER; MICROSCOPY IN PRACTICE

THE MICROSCOPIC OBJECT AS AN OPTICAL MODEL

As has been stated in the previous chapter, it may be said that the image is nothing but the radiation from the light source, as modulated by the object. It is not surprising, therefore, that the type of illumination is of the greatest importance for the way in which the image is formed; in relation with this, the optical demands for the specimen will differ greatly in the case of incident and of transmitted illumination. With incident illumination, image formation occurs by the light reflected from the specimen. On the other hand, with transmitted illumination, the reflected light is of no importance for the image and even forms a source of glare reducing the clarity of the image.

In transmitted illumination, contrasts are usually brought about by differences in absorption of light and, apart from this, by refraction. Contrasts in the latter case come about by diffraction and reflection phenomena at refracting boundaries. A transparent object which shows virtually no absorption causes quite a deal of changes in the passing light, which cannot be observed directly, but can be made visible by special techniques (see chapter 9). The contrasts observed with conventional microscopy in such an object, on the basis of secondary changes in amplitude are generally very weak; it is clear that a high degree of coherence in the illumination (as a consequence of a more closed position of the condenser diaphragm, cf. chapter 6) will raise the image contrasts, which will remain anyway rather low and somewhat distorted by diffraction phenomena. The specimens which have to be studied with conventional light microscopy with transmitted illumination have to be prepared in such a way that the contrasts due to differences in absorption are enhanced artificially.

The preparation of an object for study with transmitted illumination is consequently more complicated than in the situation of incident illumination, where (e.g. in metallurgy) preparation of the specimen is often limited to rather simple techniques like cleaning or polishing. This does not hold true for some newer developments, e.g. incident fluorescence microscopy,

THE MICROSCOPIC OBJECT AS AN OPTICAL MODEL 115

but generally it may be stated that the complex of techniques for the preparation of microscopic specimens, or microtechnique is mainly concerned with objects for study by transmitted illumination. This technique, developed since the second half of the nineteenth century, has become an applied science in itself. Only some aspects which are of importance for the optical image formation will be dealt with here; in the references at the end of this chapter some general books on microtechnique will be listed.

From an optical point of view, the following demands should be made upon a microscopic specimen to be studied with transmitted illumination. 1. A thickness, such that as much as possible of the resolving power in the

optical system used can be brought into effect and which corresponds moreover with the depth of the field of this system;

2. A sufficient degree of transparency; 3. A sufficient degree of contrast formation, preferably on the basis of local

differences in absorption.

It is possible with virtually all biological materials, by cutting thin slices, smearing, flattening or dipping to obtain a specimen which meets the neccessary demands for thickness and transparency, whereas the contrasts may be brought in by staining procedures. Particular situations, such as cells which are completely opaque (e.g. by the accumulation of pigment) or tissues which have an unusual hard consistency (bones, teeth) call for special measures.

The tissue sections, as commonly used in biology and medicine, are made virtually without exception after the material in question has been embedded in a supporting material (paraffin, plastic). Their thickness in conventional light microscopy mostly varies between 3 and 7 fLm; when table VII on page 89 is consulted, it appears that the depth of field corresponds in the medium power range with such an object thickness. With an oil-immersion 100 x objective, the depth of field is only a fraction of the thick)1ess of such a section (fig. 7.1, right). Only by a continuous movement with the fine adjustment can an impression of the section in its totality be gained. With a 10 x objective such movements are superfluous once the section has been brought into focus, as the depth of field is sufficient to oversee the whole section thickness (fig. 7.1, left).

It is not difficult to see that parts of the object which are out of focus could nevertheless exert some influence on the formation of the image as a whole. It appears that a sensible loss in resolution occurs by this effect when the section thickness comes to exceed something like ten times the minimum resolvable distance (see chapter 5). The time-honoured 5-7 fLm

116 MICROSCOPY IN PRACTICE

10 x N A 025

100 x N A.125

Fig. 7.1. Depth of field (grey) with a low-power and a high-power non-flat field objective; section thickness 6 [Lm; diameter of field, free working distance and size of objectives not drawn to scale.

thick routine section can therefore be considered as a compromise between the ideal situation for lower magnifications and that for higher magnifications, both with regard to depth of field and resolving power. For several reasons, this compromise meets many needs of every day practice; in the first place, the finest details of an object are not always of primary importance, whereas with very thin (1-2 [Lm thick) sections the spatial relationships in a biological object often become disturbed. On the other hand, sections of a thickness of 8-10 [Lm and over, as they are used sometimes for reconstructions, suffer from the fact that different layers of the object overshadow each other, apart from the loss in resolution already mentioned.

After the different procedures of fixation, sectioning, smearing etc. most animal or botanical specimens are sufficiently thin and transparent, the contrasts are very slight and are determined mainly by refraction and diffraction. These objects, still far from ideal with regard to contrast formation, are converted into true 'absorption-objects' by reducing the refraction effects and enhancing the occurrence of contrasts by absorption. Refraction in and around parts of the specimen can be reduced by mounting the object in a medium like Canada balsam and similar natural resins or one of the modern synthetic medial, The refractive index of all these media, after

1. Clarite, Caedax, Eukitt etc. These synthetic resins are not used primarily for their optical properties, in which mainly they have no definite advantages over natural resins, apart from the less important fact that they do not have the latter's tendency for the slow development of a yellow hue over the years. The main reason for the use of these synthetic media is the fact that some delicate stains keep better in them. For particular purposes or objects a choice can be made from the list of mounting media in appendix I. In using a fluid non-hardening mounting medium, the cover glass has to be sealed off with a lacquer which is not miscible with the mounting medium.

THE MICROSCOPIC OBJECT AS AN OPTICAL MODEL 117

hardening by evaporation or polymerization, comes in the range of the refractive index of the main components of fixed and dehydrated tissues; in most animal tissues this amounts to 1.51-1.54 (Goldstein, 1965). Moreover, reflections at the underside of the coverslip as an important source of glare can be virtually cancelled out in this way, as the refractive index of the medium also is not much different from that of glass. Under certain circumstances (e.g. in microphotometry, see chapter 11) it may be necessary to have a still closer match between the refractive index of the medium and certain parts of the object than can be reached with the standard mounting media which form again a compromise.

The method of choice which has been used for over a century to bring contrasts into a microscopic object, is the application of dyes. The contrasts in the image of an average unstained biological specimen (especially when cleared and mounted in a medium with matching refractive index) are generally so low that, strictly speaking, there is not so much question about enhancing the contrasts: the image is formed in fact on the basis of selective distribution of bound dye (fig. 7.2).

Fig. 7.2. Section through the epiphyseal disc of the tibia of a rat, 160 x. A Unstained section, mounted in Canada-balsam, photomicrograph made with small opening of condenser diaphragm; B same area, after staining with haematoxylin-phloxin, mounted likewise in Canada-balsam.

The specimen is usually mounted on a glass slide, which generally has standardized dimensions of about 26 x 76 mm with a thickness varying


between 1.1 and 1.3 mm. As long as both sides of the slide are perfectly plan-parallel, the thickness is optically not critical; in some instances, however, problems may arise with thick slides exceeding the free working distance of some of the more highly corrected condensers so that it becomes impossible to focus the light source or the field diaphragm into the object. Object slides with known thickness (e.g. 0.9-1.0 mm) are commercially available for these occasions. Problems around cover glasses and their standardization have been dealt with in chapter 3.

THE TECHNIQUE OF MICROSCOPIC OBSERVA nON

Now that the most important points regarding the construction and function of the microscope and its object have been treated, a few general rules will be given for the practice of microscopic observation. Only the use of the conventional microscope for observation will be dealt with; techniques for the recording and reproducing of images and the application of special illumination- and observation-methods will be treated in the chapters 8-12.

Position of the observer and the placing of the microscope The sitting position for observing with a microscope (the standing position with a high bench used in former years has disappeared now) can be considered to be an interplay between length of the upper part of the body, the forward inclination of the tube with the eyepiece, the height of the microscope and the height of the bench and the seat. As with virtually all modern microscopes the forward inclination of the tube is fixed at 45° and the standard microscopes do not vary much in height, variations in the length of the upper part of the body should be compensated for in the difference between the height of the seat and that of the working table. In most cases, use is made of a standard height of 82-83 cm of the table surface and a stool with a seat height of about 50 cm from the floor, which can be raised some 20 cm. These measures have long been standardized in the furniture industry; with young men and women becoming increasingly tall nowadays, these domestic standard measures are no more adequate, however. A young man with e.g. a total body length of 1.86 m cannot look down a microscope tube of 45° inclination with a difference of maximally 32 cm between height of the seat and working table without curving his back (fig. 7.3A). A lower stool is no way-out of the difficulty, as even if such a seat can be obtained, leg-room becomes the problem. With the classical hinged stand which enables the variation of tube inclination, better results

THE TECHNIQUE OF MICROSCOPIC OBSERVATION 119

can be obtained with a more upright position of the tube (fig. 7.3B), although a higher working table appears to be the real solution (fig. 7.3C). A similar situation can be reached by placing the microscope on one or two books; this will not be beneficial to the stability of the position of the microscope, however!. All these problems are met with to a lesser degree with larger types of stand, as the distance table-top/eyepiece is generally greater than with the smaller routine stands.

Fig. 7.3. A Young person with a length of 1.86 m on a standard seat with standard table height has a most unhappy position when looking down the eyepiece in a tube with 45° inclination; B under identical conditions, a more upright position of the tube can give rise to a better working position; C a satisfactory position with the same stand as in the first photograph can be reached by raising the table top 8 cm.

General rules for setting up a microscope A description of the correct way for setting up a microscope with its illumination takes more time than the actual procedure. It is, moreover, not always necessary to start each time anew, especially not when the microscope has a

1. This is the remedy, applied by anyone who has come into the situation of fig. 7.3.A. It is sad to note that this happens over and over again, due to the fact that in building new laboratories a table-top height of 82-83 cm is generally applied for microscopy rooms, not taking into account that a) most, if not all new microscopes have a fixed tube angle, b) a considerable proportion of young people now reach a height of 1.80 m and over.


permanent position. The setting up will be described separately for a simple critical illumination (A) and for an equipment with a built-in Kohlerillumination (B).

A 1. Adjust seat height (and inclination of the tube, when possible) so that the eye can be brought easily before the eyepiece; the draw-tube, if fitted, should be adjusted to the correct tube-length.

A 2. Turn on a low-power objective, e.g. lOx, with the revolving nosepiece; bring the condenser in its highest position.

A 3. Switch on the lamp and manipulate the (flat!) mirror until the front lens of the condenser lights up.

A 4. Move a stained object in the light beam from the condenser, using the mechanical stage (object lights up).

A 5. Look in eyepiece and bring the object into focus with the coarse adjustment so that an image appears.

A 6. Remove the eyepiece from the tube and manipulate the mirror and the height adjustment of the condenser so that a disc with even illumination is seen when looking down the tube. Close the substage diaphragm so that the outer border (about t-t of the diameter) of the disc, actually the entrance pupil of the microscope, is screened off. This is no more than a rule-of-thumb to obtain a tentative adaptation of the numerical aperture of the condenser to that of the objective, so that with an average stained specimen not too much glare is generated and no appreciable loss in aperture occurs.

A 7. Re-insert the eyepiece and adjust the condenser so that maximal intensity of illumination is obtained. When the image of the surface of the opal bulb is found disturbing, the condenser may be moved slightly up or down.

In setting up a microscope with a built-in Kohler-illumination, the steps 1-5 are the same; the subsequent steps are:

B 6. Close the field diaphragm and focus the border of the illuminated disc sharply in the specimen (see fig. 6.7, page 103).

B 7. Bring the illuminated disc to the center of the field using the centering screws of the condenser, which should be present with any microscope with Kohler-illumination.

B 8. Open the field diaphragm until its image fills the field of view, so that the border is no longer visible.

B 9. Adjust the aperture of the illumination cone with the substage diaphragm, as described in A 6. As the Kohler-illumination in itself


already reduces glare, an excessive aperture of the illumination becomes less easily manifest than with a critical illumination; this adaptation is required nevertheless in order to obtain an optimal result.

B 10. In changing an objective, both field diaphragm and aperture diaphragm should be adjusted, while the condenser has not unfrequently to be re-aligned with the objective.

Use o/immersion objectives Oil-immersion objectives are by far the most commonly used immersion objectives; consequently the term immersion objective virtually has come to mean oil-immersion objective, while objectives designed for immersion with water, glycerin and methylene-iodide are designated as such. The theoretical aspects of immersion have been treated in chapter 3 and 6; some specific practical problems will be dealt with here.

With regard to the setting up of a microscope with an oil-immersion objective, the main practical point to consider is not so much the introduction of an oil film between the cover glass and the front lens, but the combination of a very short working distance with a very thin depth of field. Missing the image plane in focussing is a far from imaginary event; in the assumption that the image still has to appear, the objective can be forced without noticeable resistance against the object with the advancement of the fine adjustment. This can have serious consequences, as the front lens of the objective might get damaged or become loose in its mounting because of the pressure (which often leads to a crushing of the cover-glass too): due to the high costs of individual manufacture the reparation of a damaged front lens is often more expensive than a complete new objective of the same type.

A few technical devices exist for this very old problem. The most universally applied guarding device is the so-called spring-mount; this consists of a telescoping system in which the objective proper can be pushed against the pressure of a spring in an outer tube-mount. This type of mount is not only applied with oil immersion-objectives - for which it has become more or less the standard practice - but also for some high-power dry objectives l .

When the correct image plane has been missed, the telescoping of the springmount, when the objective is forced down onto the slide with the fine ad-

1. An extra advantage of a spring-mount is that it is often possible to fix the lens in a shortened form by rotating it over a quarter of a turn in the telescoped position. The objective thus shortened cannot be used for observation, but it is easy to keep it in the revolving nosepiece without the risk of touching the specimen with this objective when the nosepiece is rotated.


justment, becomes evident only when the lens is inspected from the side, for there is no image! When the spring-mount has reached the end of its movement, it has no more effect and thus the system is far from fool-proof. The same is the case with other guarding devices, such as a slipping of the fine adjustment when a certain pressure is exceeded, or an adjustable mechanical stop to the fine adjustment. These latter devices can even give a false feeling of security; with any appreciable variation of the thickness of slide + mounting medium + coverslip they fail completely.

The following general rules can be recommended for maximal safety, although caution should always be exercised.

I. First select the area to be studied with a low-power dry objective. II. Apply one, and not more than one, medium drop of oil on the cover

glass exactly where the specimen is illuminated by the condenser, e.g. with a plastic bottle with a long spout. It is important to let any air escape first from the spout, as air bubbles in oil on the cover glass are very difficult to remove.

III. In the case of parafocally adjusted objectives (see chapter 3) the immersion objective can be rotated with the nosepiece into position in the oil without changing the height adjustment of coarse or fine adjustment.

IV. When the immersion objective has snapped into position, further adjustment should be made with the micrometer until the image comes into focus. When the same low-power objective is always used first, the necessary correction will be identical each time (e.g. half a turn forward with the fine adjustment).

With objectives which are not parafocally adjusted such as may be the case with an older microscope type, or a set of objectives of various makes), the focussing of an immersion objective cannot be performed in the way just described. When a suitable area has been selected with a low-power lens, the oil immersion objective is rotated into position and racked up 8-10 mm above the object. When the oil has been applied as described previously, the eye level should be placed near the top of the object slide, so that the objective can be inspected from the side. The oil-immersion objective should now be lowered carefully with the coarse adjustment until it touches the oil. At the moment of contact, the oil spreads with a clearly visible jerking movement in the slit-like space between the lower surface of the objective mount and the coverslip. The objective is now lowered towards the object with the fine adjustment, while looking in the eyepiece until the image appears. This is a very important point, which may also apply when an oil-


immersion lens can be set up in the easier way discussed at the beginning of this section, with a para focally adjusted low-power objective. In selecting an area to be focussed with the oil-immersion objective, one has to make sure that under any circumstances an image can be expected to appear. Often the rather small object field of a high-power objective is overestimated, so that one may try to focus on an empty field (fig. 7.4). Moreover, the centering of

Fig. 7.4. Object fields (with a 10 x eyepiece) of a few standard objectives, as projected over a section of human lung.

the objectives is seldom perfect, so that the prospective field of the highpower objective cannot be estimated as precisely as would seem from fig. 7.4. It is important, therefore, to select a homogeneous area with contrast-rich details for setting up the high-power objective, if necessary even outside of the area to be studied. When the correct level of focus has been found, it is relatively easy to pass a few 'empty' areas in the specimen with the mechanical stage. Uncertainty whether one is under or over the level of focus is a situation which should be avoided; when this state of affairs has been reached (and movements with the mechanical stage do not yield anything like an image either) it is best to start anew with a low-power objective. In some situations, e.g. a smear of scattered cells of low contrast, it can be difficult to find the correct level of focus with the oil immersion lens. In some circumstances it is even recommended to mount a very small piece of cigarette paper or thin metal foil which is visible to the naked eye with the specimen under the coverslip, so that an approximate level of focus can be found at once at the border of this object in the specimen.

With parafocally adjusted objectives, it is easy to turn back eventually to


a low-power objective without removing the oil. In contrast to what is often thought, the film of oil does not interfere very much with the formation of an image at low power, as long as the front lens does not touch the oil (which will not easily occur with e.g. a 10 x objective with its long free working distance). The oil film on the cover glass will cause, of course, an exaggeration of the cover-glass effect (cf. chapter 3).

With regard to the different immersion fluids, such as water (n = 1.333) used e.g. especially for wet specimens without a cover glass, glycerol (n = 1.473) as used with ultraviolet objectives, methylene-iodide (n = 1.740) and oil (n = 1.515), only some remarks about the latter, because it is not a sharply defined substance.

When, just over a century ago, Abbe developed his immersion objectives for use with cedar oil with a refractive index from 1.512 to 1.518, the chemical industry simply could not supply anything better to meet Abbe's demands for a transparent fluid matching more or less the refractive index and dispersion characteristics of glass. Cedar wood oil, which can easily be recognized by its typical resinous smell, has the property of polymerizing slowly when exposed to the air during which process the viscosity increases; when left for long periods on an oil immersion objective, it forms yellow crusts which are difficult to remove. Virtually all microscope manufacturers now provide a non-resinifying oil with a refractive index which varies from 1.515-1.518 and suitable dispersion characteristics. It makes no sense to-day - even with older objectives - to apply natural cedar wood oil as an immersion medium. At the other hand, as stated in chapter 3, there is not yet a generally accepted standard (both for refractive and dispersive properties) of immersion oil, so that it is advisable in general to apply the oil provided by the manufacturer of the lens for high-quality work. Small deviations in optical dispersion and/or refractive index in the immersion medium (which should formally be considered as a component of the objective) can influence the image, e.g. in colour photomicrography. The fluid anisol, a volatile benzol derivative with a refractive index of 1.517 which has been recommended as an immersion fluid some time ago, unfortunately has a dispersion index which differs greatly from the usual immersion oils. Apart from this, indications have been obtained that frequent use of aniso1 might weaken the lens cement in some objectives. After a brief popularity this fluid has fallen now into oblivion as immersion fluid; in itself, a quickly evaporating immersion fluid could have been very useful.

Even in using a non-resinifying oil both the oil immersion objective and


the slide should always be cleaned carefully after use!. When a condenser immersion has been applied, the condenser should be cleaned, moreover, and the slide on both sides. As the use of a condenser immersion is seldom justified, even from a theoretical point of view (see chapter 6), the rather tedious immersion of the condenser and the cleaning it entails (often also of the stage) is kept for a few selected situations with a highly corrected condenser and a contrasty object, so that the full condenser aperture can effectively be used.

Light and illumination in practice In describing the standard-procedure for setting up a microscope, a position of the condenser diaphragm has been indicated which, in the case of a stained specimen with average contrast, leads to a good adaptation of the N.A. of objective and condenser. In many cases the illumination cannot be considered as optimal under these conditions, however: the aperture of the condenser may need further adjusting, or the intensity of illumination may be too high or too low. These two factors have to be dealt with independently; the use of the condenser diaphragm for tempering the brightness of the image is one of the most common mistakes of beginners in microscopy2. It is true that in closing the substage aperture diaphragm the amount of light passed is reduced, but this is accompanied by a change in the aperture cone of the illumination, which quickly brings about a loss in resolving power and an increase in diffraction phenomena in the specimen as discussed in chapter 6. The best way to change the brightness of the image is a variable resistance on the lamp tension, as can be easily installed with a low voltage lamp. When such a control is not feasable (e.g. with a simple high voltage illumination) or the lamp current should be kept constant for certain

1. In one respect, a warning should be given against these new immersion fluids, which almost invariably contain polychlorinated biphenols (PCB's) as a major constituent. This class of compounds also used as lubricants and plasticizers for paints, has recently been curtailed by the consumer-product regulations of some countries due to certain effects on liver metabolism. It is not generally known (and microscope manufacturers seldom seem to be aware of it) that these new immersion oils contain a component which might possibly have harmful effects even in small doses (cf. A. Kappas and A. P. Alvares, Scientific American 232, 1975; 22-31). It is in any case recommended to avoid skin exposure as much as possible in using these oils and to clean the skin carefully even after trivial contamination. 2. In the view of the author this common misconception is partiy due to the generally known use of the diaphragm in a photographic camera, placed behind the objective. It is customary in photography to reduce the diaphragm-opening with a brightly lit subject; loss in resolving power by loss of aperture of the image-forming objective is generally unimportant, the object usually being reduced in the formation of the image; the concomitant increase in depth of focus is often of great value, on the other hand.


purposes (e.g. in photometry or photomicrography), an eventual change in image brightness should be brought about with filters. In view of the low luminance of the high voltage bulb poverty of image brightness at high power observation will be a more probable event than a too intense illumination, although this may occur with low-power work. With high-power observation (e.g. using a 100 x, N .A. 1.25-1.35 objective) the high-voltage bulb will certainly fall short when a binocular tube is used.

The uninitiated observer generally has a tendency to use a too low level of image brightness with a stained specimen, underestimating the consequences of this. A dimly illuminated image reduces the actual resolution

(not the resolving power) considerably, however, certain details can be made clear merely by increasing the image brightness. Local differences in light absorption which determine the contrasts in the image of a stained specimen, come out only with a certain degree of lighting intensity. With monocular observation, a too low brightness of the image will increase moreover the habit of tightly closing the eye which is not in use. The tense attitude which this brings about will promote fatigue and headache, which phenomena are often ascribed too easily to monocular observation. A somewhat more dim lighting of the image, on the other hand, is sometimes preferable with unstained objects which are poor in absorbing details and in which the contrasts are determined mainly by diffraction at boundaries. The condenser diaphragm should be in a much more closed position than with a stained specimen, in view of the fact that a higher degree of coherence

Fig. 7.5. Interface water-air (air in the centre) A with an opened, B with a more closed position of the condenser diaphragm.


(see chapter 6) in the illumination beam, in combination with a minimum of glare in the specimen is desirable in these situations. In the case of fig. 7.5, the contrast of the boundary line is visible only by the occurrence of reflection and diffraction at the air-water interface. The image A has been photographed with the condenser diaphragm in the standard position so that the outer third of the entrance pupil has been stopped; in fig. 7.5B a much sharper outline has been obtained by further reducing the aperture of the illumination cone, partly by better control of glare, but certainly also by a higher degree of coherence of the illumination as obtained "hen the condenser diaphragm is closed (cf. chapter 5). The loss in resolving power which the situation B theoretically entails, is not evident at all in this image which does not reveal any fine detail; with most objects of low contrast in which more details are present a compromise must be sought. If such an object is of a more complicated nature, and/or certain fine details are important, it is often preferable to leave conventional microscopy altogether and use e.g. phase contrast, interference contrast or dark field microscopy (chapters 8 and 9) when staining is not possible.

As discussed in chapter 6, the light emitted by an incandescent lamp has for a large part too great a wavelength to be perceptible to the eye and is consequently of no use for image formation. The radiation given off in the visible spectrum is also relatively poor in light of shorter wavelength (uninterrupted line in fig. 7.6). It is possible to compensate for this by using a filter with a transmission curve with an opposite slope to that of the lamp, e.g. curve I drawn in fig. 7.6. It is clear that such a filter, which is represented

II

400 500

\. \

\ \

600

\. '-.

700

Fig. 7.6. Schematic (and somewhat simplified) view of the spectral energy distribution of an incandescent lamp (continuous line), with I spectral transmission curve of a blue filter and II of a green filter with a broad transmission in the central region of the spectrum. Vertical axis: light intensity, horizontal axis: wavelength in nm.


more or less by the well-known blue glass supplied with most microscopes, sometimes called 'daylight filter' will cause a flattening of the lamp curve, but at the expense of a huge loss of light. Under most routine conditions the illumination cannot afford this loss with higher magnifications, so that the filter is taken out and 'ordinary' (i.e. red) light is used, which falls outside of the range of optimum sensitivity of the eye and yields a rather low contrast with most dyes used. Moreover, even with a sufficiently powerful light source, one may wonder whether the mark is not overshot with a filter which has an already considerably decreased transmission in the middle range of the spectrum, where the eye has its greatest sensitivity. A filter, showing maximal transmission in the yellow-green range around 550 nm (curve II in fig. 7.6) and consequently having a better correspondence with the sensitivity curve of the human eye, will have a much more favourable effect, keeping the light loss within an acceptable limit so that it can also be used with a simpler type of illumination. With most stains the contrasts will show to more advantage than with a blue filter or no filter at all. There is another reason why a filter with a transmission curve as in fig. 7.6 (curve II) often is useful. As explained in chapter 3, achromatic objectives have been calculated in such a way that they show the best correction for spherical aberration in the middle of the spectrum. Moreover, the blue borders around highly refractile object details seen when unfiltered incandescent lamp light is used as remnants of chromatic aberration, are no longer perceptible with such a yellow-green filter.

On the way through the object The study of a microscopic specimen starts - after an inspection with the unaided eye - by focussing a low-power objective on it as described on page 120. With some experience this can be performed with a few quick actions. When the microscope has been set up and the object focussed with e.g. a lOx objective, the observation proper can start. This will differ so much from case to case, that it is virtually impossible to make any sensible general remark in this respect. Problems which will often recur, however, will be those of a systematical search for certain details and marking and re-Iocating of a particular field, once such a detail has been found.

In searching a microscopic specimen, a comparatively arbitrary way can be followed when a structure to be studied with high-power can be found easily. The situation is quite different, however, when a specimen has to be examined systematically, e.g. in making a differential count of various components or in finding (or excluding the occurrence of) a certain rarely occurring detail. It is often the best in these situations to search the specimen


meander-like, in which use is made of the fact that it is possible to pursue straight tracks through the specimen with the mechanical stage. The track is followed in a given direction up to the border of the preparation, after which the position of the object is shifted with the other control until a detail which could just be observed at one side still remains visible at the other side. When the track is followed back after this shift, the strips of object-field just overlap, so that a complete inventory of the specimen is possible if the same shift (of slightly less than the diameter of the object field) is always made when the border of the preparation is reached.

In surveying quickly large series of preparations in the search for certain details, it is sometimes easier to move the specimen by hand (depending on the magnification, entirely free on the stage or with one side under a spring clip) than with a mechanical stage. In many cases the mechanical stage is detachable; a built-in mechanical stage can be moved often to an extreme position so that the object can be put on the stage outside of its object holder. When quick screening of larger specimens is often required, the mounting of a gliding stage can be considered.

When a certain area in the object has been found which has to be marked, e.g. with a view to making a photomicrograph with another microscope, different methods exist. In the first place the readings on the scales of the horizontal and vertical movement from the mechanical stages can be noted. With the nonius generally an accuracy of 0.1 mm can be reached in both coordinates, which is generally sufficient to re-locate a characteristic detail in the specimen, when the specimen is put back in the same position in the specimen holder. This method of marking an area in a microscopic specimen has a very serious drawback, however; the divisions of the two coordinates are completely arbitrary and are of value only for the same microscope. Even with two microscopes of the same make and type it is not possible to bring over a not too large area from one microscope to another on the basis of these coordinates without a preceeding gauging. In order to get free from the divisions of the mechanical stage, a coarse marking of a certain field is often made with spots of drawing ink, feltpen ink or special glass ink on the cover glass (ordinary writing ink will not hold on glass). This is best made with a low-power objective (in view of the long free working distance) in such a way that e.g. three spots are laid on the coverslip around the area to be marked under microscopic control. It is not possible, however, to mark and re-locate a small detail amidst a large number of similar configurations; moreover, ink spots on the coverslip can be troublesome when the marked detail has to be studied with oil immersion.


The placing of ink spots at the underside of the object slide can avoid the latter difficulty. It can be rather troublesome (because of the illumination apparatus) to reach the underside of the slide, whereas the ink spots become located in a plane about 1 mm under the object proper, so that they cannot be focussed together with the specimen area concerned. This does not provide more than an approximate indication of the area to be re-Iocated.

A so-called object marker or field marker, which can be screwed in the revolving nosepiece in the place of an objective, enables the marking of a small circle in the upper side of the cover glass. This is made by rotating with a collar a diamond tip with an excentric position (fig. 7.7) so that a

Fig. 7.7. Specimen marker (= slide ringer) with the diamond tip lowered onto the cover glass of a mounted specimen. By rotating the under part of the device, a small circle is cut in the cover-glass surface by the excentric position of the diamond tip which can be regulated by rotating the lower knurled disc.

variable circle can be scored on the cover glass. Damage to the cover glass can be avoided by lowering the marker slowly onto the object so that it comes to rest on the cover glass with only the weight of the moving part of the device. The marks made on the cover glass can be re-Iocated most easily with a low-power objective and a closed aperture diaphragm; when oil has been used which has filled the circular score, it can be rather difficult to find the marking.


Another technique for marking areas in a specimen makes use of a socalled object-.finder. This is a pattern of squares etched microphotographically onto the entire surface of an object slide, mounted with a cover glass. The different squares are numbered consecutively; they are so small that they can be read under the microscope. With their subdivisions into smaller squares which can be numbered A, B, C etc., the centre of the object field of a low-power objective or the entire field of a high-power objective can be indicated with some precision (fig. 7.8). When an area in the specimen has

718 719

748 749

778 779

Fig. 7.B. Two types of object-finder; photomicrographs at a magnification of about 20 x.

to be marked with this device, the slide should be firmly clamped into the mechanical stage, making sure that the slide is in contact with its base stops and that the label is in a standard position (e.g. left). The slide should be replaced now by the object-finder, without changing the position of the mechanical stage (if possible, the stage should be blocked); the label of the finder should be again in standard position (e.g. again left). Focus the pattern of the finder and note letter and number in the center of the field of view. This would be something like 748/F or V 21/4 for a high-power field from the two examples offig. 7.8.

To re-Iocate the selected field, place the object-finder (the same one) on


the stage with the label in standard position and clamp firmly. Find the square with the letter and number noted with a similar, not necessarily identical, objective as used for the marking and then replace carefully the object-finder by the original preparation with the label again in standard position. If everything has been carried out correctly, the selected area should appear more or less in the center of the field; some inaccuracy will occur due to variation in the centering of the objectives. If the area cannot be found back at all (or the area appears to be outside the section etc.) some mistake has been made with the left-right position of the slide or the finder on the stage. It should be noted that a mechanical stage with good fitting clamps is essential for the use of an object-finder.

Microscopy for observers wearing spectacles An old question in microscopy is whether those wearing glasses should retain them while making microscopic observations, or whether they should be discarded. In general, the following can be said about this problem which is of some practical importance. As the pupil of the eye is mostly 12-14 mm behind the spectacle glass, the exit pupil of the microscope should have at least this height, plus the thickness of the glass, to enable the eye to oversee the full field of view. The optical effect of the spectacle glass is such that negative glasses (with myopy) enlarge the height of the exit pupil of the microscope; on the other hand, positive glasses will reduce this height. The eye clearance of most ordinary eyepieces generally is no more than 6-12 mm, depending on the magnification and type of the eyepiece (see chapter 4). Consequently, the field of view is reduced considerably, as the border of the pupil of the eye comes to limit the diameter of the light cone emanating from the eyepiece. Moreover, the chance exists that scratches will be formed on the spectacle glasses and/or the eyepiece front lens when they are touching each other, as one is inclined to approach the eyepiece as closely as possible.

As with a correctly focussed microscope the image is in such a position that there is only very slight accommodation of the eye, reading glasses (which only serve to compensate for a failing accommodation) need not be worn. When the study of a microscopic image has to be alternated with reading a text or the taking of notes for which the glasses are necessary, it is, however, extremely cumbersome to look into the microscope without glasses unless they are provided with a special construction enabling the folding upwards of the lenses. With higher degrees ofmyopy or hypermetropy with observers who permanently wear glasses, it is possible to compensate the refraction anomaly by adjusting the position of the intermediary image


in such a way that a sharp image can be obtained without spectacles. With persons wearing positive glasses, the tube has then to be moved up from the object, whereas in the case of negative glasses, the objective should be moved slightly towards the object with the fine adjustment. With stronger degrees of aberrations of ocular refraction, this compensation with the microscope becomes insufficient. Special eyepieces with adjustable front lenses as used formerly for this purpose have been superseded by the development of special high-eyepoint spectacle eyepieces, which enable a comfortable observation through spectacle glasses (chapter 4). These eyepieces generally have a concave upper lens (fig. 4.2D), whereas the upper border of the mount is often protected by a rubber ring, so that the spectacle glass and front lens of the eyepiece cannot scratch or even touch each other. The pupil height of 16-20 mm of these high eyepoint oculars can appear insufficient with very strong positive eyeglasses, so that some degree of compensation by changing the position of the intermediary image may still be necessary.

With astigmatism, a frequently occurring aberration of the eye, the refraction power in different sectors of the eye is different; this can be compensated by so-called cylindric eyeglasses. These can be recognized easily by the fact that when such a lens is turned before a page of a book, height and width of the page and the letters change. With astigmatism of any degree, spectacles should always be worn in performing microscopic observation, preferentially with spectacle eyepieces, as not the slightest compensation is possible here with the focussing of the microscope.

The use of high-eyepoint eyepieces by observers not wearing spectacles is of course very well possible; it is often felt as comfortable and is preferred by many microscopists nowadays. With persons with very long eyelashes (either natural or artificial), spectacle eyepieces may be the only solution when using stronger eyepieces. Wide-angle and spectacle eyepieces are frequently united in a single design, often of orthoscopic type.

MAINTENANCE AND SMALL TECHNICAL DIFFICULTIES

Care of the stand and the cleaning of optical components A microscope stand of good make hardly needs any maintenance for long periods; the moving parts, such as the rack-work, only need cleaning and new grease at very long intervals. Care should be taken never to use thin oil on rack-work, as this may cause the spontaneous sinking of a tube or condenser.


A microscope should be kept as much as possible free from dust in a case or under a plastic cover; dust particles do not only adhere to the stand and its moving parts, but accumulate especially on lenses and other glass surfaces (cover plates of built-in illumination, mirrors). The stage should be cleaned regularly; if anything is spilled on the stage it should be wiped off. Especially mounting material (Canada balsam and the like) should be removed at once (when necessary with some xylol) as it can form hard cakes and be difficult LO remove afterwards.

The following general rules can be given for the cleaning of optical surfaces, they hold true especially for objectives. It should be kept in mind that external surfaces of optical parts, treated with anti-reflection coating are relatively hard, but that those coverings are very thin. Some of the internal coatings are soft and easily damaged. I. Non-adhering dust can be removed best with a dry soft brush (if shortly

warmed against the surface of a bulb it picks up dust particles more easily) or lens paper. Dust should never be removed with the fingers from optical surfaces; the skin which is moist and greasy will usually take up the dust, but leaves a greasy substance instead. Fingermarks or other adhering grease or dirt should be removed with a soft cloth or lens paper barely moistened with xylol, or petrol if available. Never soak or immerse a lens in xylol; avoid under any circumstances the use of methylated spirits or other alcohols, ether or acetone in view of the cementing material. When water is used, aqua de~t. should always be taken instead of tap-water, which leaves a deposit of salts on evaporation.

2. Always take lens paper, linen etc. double over the finger to prevent infiltration by moisture or grease from the skin. When the border of the mount is protruding over the lens and/or the front lens is concave (as is often the case with plan-objectives) it can be difficult to reach the entire lens surface with lens paper or a cloth. The cleaning should be performed then with a piece of cotton wool impregnated with some xylene around a match stick. Pabst (1973) recommends the use of polystyrol-foam, as is often used as packing material, for the cleaning of objective lenses from small remnants of dirt, and especially grease. When a freshly broken surface is pressed against the lens and turned coaxially with the lens axis, its lipophil properties remove every trace of grease. As this material is made from a fluid starting material, scratching particles cannot occur in any freshly broken surface. There should be no trace of xylol on the lens, as this dissolves the polystyrol.

3. Oil-immersion objectives should always be cleaned after use, even with

MAINTENANCE AND SMALL TECHNICAL DIFFICULTIES 135

use of non-hardening oils, as these also alter slowly with time. Preferably first use dry lens paper, which is mostly sufficient, or polystyrol (see at 2); finish when necessary with a piece of linen slightly moistened in xylene. When too much of a solvent is on the cloth, touch it with a dry part; the lens should never be soaked in any cleaning fluid. Some very old objectives have lens cements which can be weakened by xylene; cleaning of older objectives should preferably be done entirely dry.

4. Never attempt to take an objective lens to pieces for cleaning; even when everything has been brought back into place correctly, mutual distances can be changed. This does not hold true for eyepieces with their more simple construction; they can be unscrewed for cleaning without any danger. With most modern objectives unscrewing has become impossible because the mount is made in one piece. Condensers, on the other hand often have a front lens which can be unscrewed to lower the aperture of the illumination apparatus; even when this device is not used, dirt can accumulate between both lens systems and should be removed from time to time. Condensers with a swing-out front lens quickly become dusty on the uncovered top side of the large lens. When they cannot be cleaned sufficiently with an air current from a blower brush (a soft brush at the end of the spout of a rubber blower, also useful for cleaning the top cover of a built-in illumination) it should be taken out and cleaned in the manner already described.

Any dirt or other contamination of an objective (or any other lens or optical surface) will influence the optical path somehow; this does not mean, however, that a light absorbing dust particle will always manifest itself by anything like a sharp image. On the other hand, contaminations on surfaces in the optical path are most disturbing when they come into focus or nearly so.

Dirt of any kind on an objective will virtually always lead to the same effect, i.e. a reduction in the sharpness of the image. This holds true too for contamination of an objective front lens by completely transparent materials, such as Canada balsam or other resins or immersion oil. In the common situation where a 'dry' objective has got some immersion oil on the front lens, the covered part of the lens will form a hazy image (fig. 7.9A); The best way to detect such a contamination is to reflect light from the window on the front lens into the eye; any irregularity at the glass surface becomes manifest immediately in this way (upper left in fig. 7.9). It should be noted in passing that this inspection technique cannot be used as effectively when the front lens has a concave surface, as is often the case with modern ob-


jectives with plan-correction. Cleaning of the lens surface may have a dramatic effect on the image quality, even in the case of a contamination which might seem very slight (fig. 7.9B).

A

B

Fig. 7.9. A Dry objective (achromatic) with a front lens partly covered with a thin film of immersion oil, as shown with reflected light with at the right side a photomicrograph (90 x ) from a section of cat kidney made with this objective in this very condition; B the same objective after careful cleaning of the front lens, with at the right side a photomicrograph of the same area.

Apart from the front lens, the back lens of the objective has sometimes to be freed from accumulated dust and dirt. This can be avoided to a large extent by maintaining the tube as much as possible as a closed system; eyepiece and objective should therefore be left in the microscope and open places in a revolving nosepiece closed off by screwing a black cap into the


hole. It is clear that leaving out the eyepiece for some reason is very harmful: all dust particles falling into the tube will accumulate at the deepest level, i.e. on the back lens of the objectives and with a binocular tube at the surface of the dividing prism.

With regard to the eyepiece, dust or grease on one of its components causing spots and specks in the image may be troublesome. They can be located easily by rotating the entire eyepiece or the front lens alone after unscrewing the top. If the specks move only in the latter situation, the dirt must be located in the front lens.

Apart from the situations already discussed with objectives and eyepieces, the most important other locations of contaminations on surfaces in the optical path are the light source, or that which acts as such (ground glass screen, area in front of collector with a Kohler illumination and eventual filters) and the specimen itself, e.g. the cover glass. Dirt on the condenser or on the mirror generally is of lesser importance.

Frequently occurring minor technical troubles Even with the most correct use of a good microscope, a great variety of small technical derangements are bound to occur. Table IX lists the most common of these, with the corresponding measures to be taken. It should be pointed out that the microscope is assumed to be without major optical or mechanical faults; it is obvious that a hazy image can be caused not only by a trivial contamination but also e.g. by a loose front lens of the objective. Although the latter event is far from fictive, it is wise not to think too early in terms of faults of this kind and give the front lens a thorough cleaning first.

defect

coarse adjustment is too stiff

tube or stage sinks spontaneously under its own weight (image drifts out of focus)

TABLE IX (continued on page 138-139-140)

possible causes

mechanism has been faulty adjusted

dirt in rackwork

remedies

with many stands easy to adjust (often by moving the two control knobs in opposite directions)

clean and put on new grease

incorrect adjustment of as above rackwork and/or lubri-cation with too thin oil

faulty adjustment of focus control

as with first entry


defect

micrometer movement is blocked to one side

drift of focus with the slightest movement with the fine adjustment (especially with oil immersion objectives)

veiled, spotty image

sharply focused spots or specks in the image which change and disappear in moving the condenser up and down

hazy image, which cannot be brought sharply into focus

TABLE IX (continued)

possible causes

fine adjustment at the end of its travel

remedies

bring a 10 x objective into position with the revolving nosepiece; set the fine focus control at the middle of its range and then refocus with the coarse adjustment

a) objective insuffi- a) evident ciently screwed into the revolving nosepiece; b) surface of the b) avoid the use of coverslip stuck to the viscous immersion oil; objective by the layer clip specimen firmly of oil

dirt or grease on the cleaning where neceseyepiece (spots move sary when the eyepiece is rotated in the tube) or objective; contaminations on cover slip (spots move when specimen is shifted) or on any sur-face of the illumination apparatus

dirt near light source, as above; when the or a diffusing screen in contaminated surface front of it with critical cannot be reached, illumination, or at the change the focussing of cover plate of a built-in the condenser slightly illumination or a filter near to it with Kohler illumination

wrong immersion (oil use correct immersion; instead of air, air in- clean where necessary stead of oil, air bubble in oil), transparent con-tamination on objective front lens


defect

object field partially illuminated

object field unevenly illuminated

drift of a cloud across the field; after this, image out offocus (oil immersion)

sharply delineated bright spots in the image

possible causes

cover glass too thick, too thick layer of mounting medium

remedies

use of objective with correction collar, or (better) immersion objective

irregularly distributed clean with dry cloth or remnants of immersion paper tissue; beware of oil on the cover glass, xylene, as this may when using high-power weaken or dissolve the dry objective mounting medium

slide upside down on turn slide; make sure the stage (only with that label is not stuck high-power objectives) to the wrong side of

the slide

filter holder partially evident in light path

objective not clicked into position

condenser (or swingout lens) not in optical axis

mirror not correctly in position

condenser not centered (with critical illumination)

irregularity in light source and/or diffusing screen (with critical illumination)

move condenser slightly up and down; use ground glass in front of light source

air bubble in the im- wipe off the oil from mersion oil; the specimen and set oil in image space with up anew; a dry objective clean slide and objec

tive carefully

transversal reflections in the interior of the microscope (often sickle- or ring-shaped)

try another eyepiece, use correct Kohler illumination


defect

unsharp bright spots in the image

TABLE IX (continued)

possible causes remedies

longitudinal reflections use lenses with antiin the tube, causing reflection coatings, more round light spots change combination

objective-eyepiece

contaminations at a when the localization lens surface, upper- or of the contamination under side of the ob- cannot be traced, the ject, or air in immer- effect can be reduced sion oil of condenser often by opening the (differentiate as ex- condenser diaphragm plained before) somewhat more


H. Adam und G. Czihak: Arbeitsmethoden der makroskopischen und mikroskopischen Anatomie. Gustav Fischer Verlag, Stuttgart 1964.

R. Barer: Lecture notes on the use oj the microscope. Blackwell Scientific Publications, Oxford-Edinburgh 1968.

H. M. Carleton and R. A. B. Drury: Histological technique, 3rd ed. Oxford University Press, London 1967.

E. C. Clayden: Practical section cutting and staining, 5th ed. Churchill Livingstone, Edinburgh-London 1971.

B. D. Drisbey and J. H. Rack: Histological laboratory methods. E. & S. Livingstone, Edin-burgh-London 1970.

F. Flirst: Die 14 'schwarzen Punkte' beim Mikroskopisieren. Mikroskopie 18 (1963) 25-34. M. Gabe: Techniques histologiques. Masson, Paris 1968. D. J. Goldstein: Relation of effective thickness and refractive index to permeability of

tissue components in fixed sections. J. Roy. Micr. Soc. 84 (1965) 43-54. E. Gurr: Synthetic dyes in Biology, Medicine and Chemistry. Academic Press, London-New

York 1971. J. A. Heddle: Graphical conversions of mechanical stage readings for field finding in

different microscopes. Stain Technol. 42 (1967) 109-111. G. L. Humason: Animal tissue technique, 3rd ed. Freeman, San Francisco 1972. R. D. Lillie: H. J. Conn's Biological stains, 8th ed. Williams & Wilkins, Baltimore 1969. L. C. Martin and B. K. Johnson: Practical microscopy. Blackie, London 1958. R. McClung Jones: McClung's Handbook ojmicroscopical technique, 3rd ed. Hoeber, New

York 1964. H. Pabst: Neue Methode zur Reinigung von Objektiv-Frontlinsen. Leitz-Mitt. Wiss. u.

Techn. VI (1973) 67. B. Romeis: Mikroskopi~che Technik. Oldenburg, Mlinchen-Wien 1968. S. Ramaswamy: A numbered-grid locator slide for relocating microscopic fields. Stain

Technol. 45 (1970) 173-176. A. W. Wachtel, M. E. Gettner and L. Ornstein: Microtomy, in: Physical techniques in

biological research, vol. IlIA, ed. A. W. Pollister. Academic Press, New York-London 1966.

PART II

ADV ANCED TECHNIQUES OF MICROSCOPY

CHAPTER 8

SPECIAL TECHNIQUES OF ILLUMINATION

OBLIQUE ILL UMINA nON

With conventional illumination of a microscopic object the light cone thrown on the object will contain rays in different directions, from those parallel to the optical axis in the centre to oblique rays in the mantle of the illumination cone. As explained in chapter 6 and 7, the aperture of the illumination is adapted to that of the objective by means of the substage diaphragm; rays too oblique to enter the objective only give rise to glare and loss of contrast. The situation with this conventional illumination is drawn schematically in fig. 8.lA.

When the illumination cone is 'excavated' in such a way that only oblique pencils of light approach the objective, the situation of 8.lB is reached. As explained in chapter 5, theoretically some gain in resolving power may be expected under those conditions. This is, however, not the reason why this peculiar illumination is sometimes applied. With one-sided oblique illumination, with an inclination angle about equal to the aperture angle u of the objective (fig. 8.1), a curious plasticity of the image can be observed in

obI

cond

Fig. 8.1. A Schematic view of a conventional illumination with a cone with half top-angle u; B oblique illumination, in which the rays leave the condenser with an inclination equal to u with regard to the optical axis.

144 SPECIAL TECHNIQUES OF ILLUMINATION

Fig. 8.2. Pollen grains of Sonneratia ovata, 110 x. A photographed with conventional illumination (nearly closed condenser diaphragm); B with oblique illumination.

comparison with conventional illumination (fig. 8.2). For quite a long period this technique of oblique illumination has frequently been used as a means of enhancing the contrasts in objects difficult to stain. The question is in how far this is a real reproduction of the object and what creates this type of image.

In contrast to what is sometimes thought, this three-dimensional impression in transmitted light bears no relation to real shadow effects; this would be possible only with incident illumination. The effect is caused by an inclination of the image plane, occurring with oblique illumination. As the object plane naturally will remain perpendicular to the optical axis, small objects like the pollen grains of fig. 8.2 will be overfocussed at one border and underfocussed at the other border. These de-focussing effects cause interference phenomena, which make the object seem bright at one side and dark at the other and this again entails the plastic impression of the image. This is, however, for the greater part an optical artefact and does not necessarily bear a direct relation to the shape of the object. Apart from this effect, there is an enlargement of the resolving power in a single direction, as explained in chapter 6. This is usually not the reason why oblique illumination is applied, however.

Apart from one-sided transparent illumination with oblique rays discussed so far, an all-sided oblique illumination is also used, in which the illumination bundle has the shape of a conical surface. In principle, this is subject to the same criticism as a convex image plane is formed in this situation. The increase in contrast obtained with this type of illumination is less impressive than with one-sided oblique illumination as much glare is usually

OBLIQUE ILLUMINA TION 145

formed in the specimen. Oblique illumination was frequently applied in microscopy before phase contrast came into general use; in many cases it was the only method for enhancing the contrast of certain transparent refractile objects, such as diatoms. With all objections which can be raised against this type of illumination, it may sometimes render some service in the searching for small refractile objects in a specimen when no phase contrast is at hand, be it often at the expense of definition. It therefore still makes some sense to discuss briefly how an illumination of the type shown in fig. 8.1B can be obtained.

The classical illumination apparatus according to Abbe was provided with some devices enabling experimentation with oblique illumination (especially a joint enabling the mirror fork to be brought out of the optical axis and a substage diaphragm which could be moved sideways and rotated). With modern microscopes this type of illumination apparatus is as a rule no longer present. By means of a filter holder (with a piece of cardboard or a dark filter) a substantial part of the central opening of the condenser can be covered, so that the light can enter only from a border at the periphery of the condenser. The uptake of light can be controlled by taking out the eyepiece from the tube; only a sickle-shaped part of the backlens of the objective should be filled with light. Somewhat more advanced is the use of metal discs with a cut-out part in the filter holder for one-sided oblique illumination, or a ringshaped opening with a central stop for allsided oblique illumination. With a phase contrast condenser, such an illumination can be easily achieved with the phase-ring, in combination with an ordinary objective (see chapter 9). By displacing the phase-ring far out of the optical axis, one-sided oblique illumination can be attained when only a part of the phase-ring admits light to the objective. All this may give rise to intriguing images; in the long run, however, more benefit will be derived from using the phase contrast equipment for its original purpose.

DARK-FIELD ILLUMINATION

Oblique illumination, as discussed in the previous section, can be considered as a variant of normal transmitted illumination, in so far as the light bundle from the condenser can be accepted by the objective. When, with increasing inclination of the incoming rays, the conical surface of light reaching the objective in fig. 8.1 B comes to lie outside the entrance pupil, the light from the condenser can no longer take part directly in image formation. Even in the case where all the light falling within the aperture cone of the objective


has been suppressed (i.e. when a 'hollow cone' of light comes from the condenser), no total obscurity will occur in the image plane; the light, scattered in all directions as a consequence of refraction, reflection and diffraction in the specimen, will be partly captured by the objective. In principle the same phenomenon can be made to occur with incident illumination when directly reflected light is excluded from entering the objective (fig. 8.3).

\ \

""'7'""",,""""=-_J

/ /

1'1

Fig. 8.3. Schematic view of the dark-field principle with transmitted illumination (TI) and incident illumination (II).

As a consequence of the absence of direct light entering the objective in the situation of fig. 8.3, the image of the object will show nothing but darkness, except where light is scattered by refraction, diffraction etc. This phenomenon is called the dark-field principle, as opposed to bright-field, where direct light can reach the image plane1 . The parts of the object where light scattering takes place, e.g. small particles in a homogeneous medium, will function as small light sources against a dark background. A curious phenomenon is to be noted here. When the luminance of the diffraction spot of such a particle in the intermediary image is sufficiently high, such a particle will remain discernible in the microscopic image, even beyond the

1. The term bright-field is also frequently used - wrongly, in the writers opinion - for indicating conventional microscope objectives as opposed to e.g. phase contrast objectives. The fact that a considerable light absorption takes place in phase contrast objectives, and that phase contrast images have sometimes a dark back-ground, has nothing to do with the dark field principle.

DARK-FIELD ILLUMINATION 147

point where it can no longer be observed with bright-field illumination; a similar phenomenon is well-known with stars against a dark sky. Although much smaller particles than can be resolved with conventional illumination can be made visible in this way, it is clear that the brightness of such small light-emitting particles cannot be increased to an unlimited degree. In using powerful light sources, particles down to a diameter of 5 nm (0.005 fLm) have been observed; this is not only dependent on the light source and the degree of contrast obtained between particle and background, but also on the N.A. of the objective used.

The most extreme form of the dark-field principle is reached when the illuminating rays are projected perpendicularly to the optical axis. In colloid chemistry, particle analysis has in the past been performed quite often with a set-up, in which a slit-like light band is sent through a solution, with perpendicular observation of the light scattering particles: the classical ultramicroscope l of Siedentopf and Zsigmondy. The images which can be attained with the so-called Tyndall-effect in e.g. a colloidal solution of gold particles, can be compared with the well-known phenomenon of dust particles, clearly visible when a band of sunlight falls into a dark room, disappearing when the light is switched on.

The form of dark-field microscopy just described has found little application in biological-medical research; an important reason for this is that a prerequisite for particle-analysis with the Tyndall-effect is that the light scattering objects should be in a perfectly homogeneous medium; this can be met in colloid chemistry and crystallography, but not with biological material. Dark-field microscopy is applied in biological and medical microscopy to visualize certain light-refracting objects but the 'ultramicroscopic' aspect is here relatively unimportant. The theory of ultramicroscopy, while not of any great importance in contemporary microscopy (no longer even in colloid chemistry), has been dealt with because it has given rise to misunderstanding. In contrast to what is sometimes thought, the use of darkfield illumination does not augment the resolving power as compared with bright-field illumination under otherwise identical circumstances. The extremely small particles which could be observed only with dark-field illumination, had been made visible because they gave rise to a diffraction disc, but they were not resolved. The intensity of the bright points observed

1. This term was formerly introduced because very small particles can be observed with this technique. In the present situation this term can give rise to misunderstanding, as ultramicroscopic is sometimes used as synonymous with electron-microscopic. In practice the confusion is not important, as electron-microscopic techniq ues have in fact superseded ultramicroscopy in its original sense, even in colloid chemistry. It is self-evident, however, that these principles of observation are essentially different.


can (under constant condition of material and illumination) form an indication of the size of the particle; there is no question, however, of the formation of a geometrical image of an object.

With the dark-field microscopy as applied in biology and medicine, the geometrical impression of the object is not always reliable, the image is built in fact mainly from scattered light, generated in interfaces where different refractive indices meet. This is one of the reasons why dark-field microscopy, in former days the observation technique of choice for certain objects stainable with difficulty (such as certain bacteria), has been replaced for a large part by phase contrast microscopy.

Although a black paper disc glued in the centre of a round glass which fits in the filter holder and other similar devices can approach the conditions of dark-field illumination, true dark-field microscopy is not possible without a special illumination system. The demands for such a system are 1) a sufficient inclination of the illuminating rays (without pencils of light which can penetrate directly into the objective) and 2) a powerful light source. A third requirement is a centering device for the condenser.

The oldest type of dark-field condenser for transmitted illumination is the so-called paraboloid condenser. Optically, the property of a hollow mirror with a surface in the form of a paraboloid is that parallel rays of light are reflected in a focal point, even those far from the optical axis. This condenser (fig. 8.4A), which is the oldest type of dark-field condenser, consists of a

A B

Fig. 8.4. Dark-field condensers: A paraboloid condenser, B cardioid condenser. To hold the ray diagram as simple as possible, refraction effects in changing media have not been taken into account.


solid mass of glass with a paraboloid surface, which functions as a mirror by total reflection. The paraboloid condenser has a few obvious advantages in its simple construction and comparatively long working distance; on the other hand, it is virtually impossible to correct for image errors. Moreover, 'leakage' of direct light which can penetrate the objective is possible, although this can be prevented for a large part with a central stop (fig. 8.4A). Paraboloid condensers are used mainly at lower apertures where the disadvantages just mentioned are less disturbing. Much more effective for obtaining dark-field illumination with high apertures are double mirror condensers, of which the so-called cardioid condenser (fig. 8.4B) is the most important representative. It has been shown by Siedentopf (1912) that a good correction for spherical aberration (important for concentrating the highly inclined light rays in a small focus) is possible when the first mirror surface has the shape of a cardioid, i.e. the rotation figure of a heart-shaped curve. As only a small region of the cardioid surface plays a role in the actual reflection, a spherical surface is often used, as this is easier to make; theoretically this is less efficient, however. As a consequence of the double reflection and the blackening of central regions in the first and second mirror combination, a purely hollow cone of light can be produced with a high aperture (fig. 8.4B). As the image of the light source is formed thus at a very short distance from the condenser surface, the thickness of the object slide is here of some importance.

It is self-evident that all that has been said about condenser apertures in chapter 6 will hold true for these mirror condensers. Consequently, numerical apertures of over 0.95 cannot be reached without immersion, as pencils of light with a greater inclination than corresponds with this aperture will exceed the critical angle of reflection and are thrown back into the condenser. Thus, an aperture of 1.2 or 1.4, as specified with a given dark-field condenser, can effectively be reached only with some kind of immersion between condenser and slide. In using dry objectives - which can only have an aperture of under 1 - direct light from the hollow illumination cone cannot enter the objective. The conditions are different with an immersion objective with a N.A. of 1.30; the dark-field effect would be totally lost when the direct light rays with an inclination corresponding to this aperture would enter the objective. It is necessary, therefore, to stop down the aperture of the objective just until no more direct light is passed into the objective. Special immersion objectives (often with a comparatively low magnification, 40-70 x) exist which are provided with a built-in graded iris-aperture diaphragm for darkfield observation; alternatively, a so-called Davies-shutter which contains an iris diaphragm can be screwed onto the base of the objective mount, and


used for the same purpose. This device lengthens the tube, however, and for other optical reasons also a built-in diaphragm is to be preferred.

A very special problem with the setting up of a dark-field illumination is the fact that the actual image of the light source cannot be observed, when no direct light passes into the objective (which is the ideal condition). The question is, therefore, how the conditions of fig. 8.4 can be achieved. The adjustment of the illumination can be made easily, using a low-power objective and a diffuse light scattering part of the object in the optical path. (Note: with no object or with the object slide alone no scattering occurs and nothing will be seen.) When the condenser is in focus, i.e. the image of the light source is in the image plane, a small disc of scattered light can be observed which can be brought into the optical axis with the centering screws. When a ring-shaped light effect is seen, the position of the condenser is probably too low or too high (fig. 8.4). The proper dark-field effect is only observed on the spot of the illuminated disc. With low power magnification this will only fill a part of the field of view with high N.A. condensers of the type offig. 8AB.

The cells shown in fig. 8.5 differ in refractive index with their surrounding; this is why their contours come out clearly in the dark-field image. In a similar way, the granules and nuclei in the cytoplast manifest themselves in their turn by the local deviation in refraction which they cause. The most striking parts of the objects are two air bubbles which scatter much light; this can also be the case with all other contaminations in the object which differ greatly in refractive properties with other parts of the object. With most stained preparations for transmitted illumination, where the refractive index of the medium matches as much as possible that of the tissues (cf. chapter 7), dark-field illumination will not produce striking results; as a consequence of the staining, the image will be often confusing. Unicellular organisms, cells in suspension and bacteria in aqueous media have always been the classical objects for studying with dark-field illumination. As previously stated, considerable loss of ground has occurred here to phase contrast microscopy. On the other hand, the possibilities of a combination of darkfield illumination with fluorescence microscopy should be mentioned as a new development; it will be dealt with briefly in the next section of this chapter.

The dark-field principle can be applied both with incident and transmitted illumination (fig. 8.3). It is not easy, however, to give a hollow cone of incident light a sufficient aperture angle. In any case this is not possible with an illumination via the objective itself, as applied with vertical illumin-


Fig. 8.5. Smear from oral epithelial cells in human saliva (300 x) with at right two air bubbles; upper image: conventional illumination, condenser aperture stopped down to a rather low value; lower image: dark-field illumination, as obtained with a cardioid condenser, N .A. 1.2, used with condenser-immersion.

ation (fig. 6.9). As a rule, use is made here of a spherical or parabolic hollow mirror surface, applied in ring-form around the objective as shown in fig. 8.6. Such systems are used in applied technology for detecting minimal irregularities in surfaces, e.g. of metals. In recent times incident dark-field illumination has also been applied in biological research, often in combination with fluorescence microscopy.

A special type of illumination which has some relation to the different types of illumination discussed so far is the so-called Rheinberg illumination. To obtain this, a two-coloured filter is mounted under the substage condenser (e.g. in the filter holder) in which the central zone has a complementary colour to the ring-shaped peripheral zone. Under favourable conditions and with adequate relation of the absorption in both zones of the Rheinberg-filter, a normal bright-field image is obtained in one colour and a kind


-IMP

L c RD

Fig. 8.6. Set-up for incident dark-field illumination. L light source, C condenser, RD ringshaped diaphragm, IMP intermediary image plane.

of oblique illumination image projected over the first in another colour. Although it is sometimes possible to bring out beautifully certain refractile elements in a microscopic specimen, this system can give rise to rather confusing results and has now fallen into disuse.

FLUORESCENCE MICROSCOPY

General principles Fluorescence can be defined as the phenomenon displayed when certain substances are struck by electromagnetic radiation, absorb a part of that radiation and re-emit it with greater wavelength. The process of emission is a very short one and takes only a few milliseconds, during which the atoms struck return to their ground state. In contrast to fluorescence, phosphorescence, depending on other types of transition, has a much longer emission period; this phenomenon is of no importance for microscopy.

In most cases, substances showing the phenomenon of fluorescence do

FLUORESCENCE MICROSCOPY 153

this optimally when they are radiated with light of a determined excitation wavelength, while emitting light with a certain emission wavelength; both wavelengths are related to the physical characteristics of the fluorescent substance. It should be noted that the fluorescence light is never monochromatic, even when the excitation light is monochromatic. Strictly, there is not such a thing as an emission wavelength: it is always an emission spectrum; also the excitation corresponds with a certain spectral pattern. In practice, however, there is in most cases one major peak to reckon with and seldom two. The phenomenon of fluorescence is a general characteristic of electromagnetic radiation, it is by no means limited to visible light, or even light. Fluorescence can also be produced by X-rays (fluorescent screen in X-ray apparatus); in electron-microscopy, the viewing and focussing of the image of the (invisible) electron rays is accomplished also by means of a fluorescent screen.

One generally understands by fluorescence microscopy the application of the phenomenon fluorescence in light microscopy, i.e. the study of light emitted specifically by fluorescence. In this connection, a clear difference should be made between a) primary fluorescence or autofluorescence, which occurs by natural properties of certain substances and b) secondary fluorescence, induced by substances with fluorescent properties which have been attached to certain components of the microscopic specimen. With most histological substances, primary fluorescence is very slight or absent and use is made exclusively of secondary fluorescence. The preparative side of fluorescence microscopy cannot be treated here; it should be pointed out only in passing that an excitation-wavelength is always a wavelength at which a fluorescing substance absorbs some light. Consequently it is not to be expected that a completely colourless material would show fluorescence phenomena of any importance, when excited with visible light. Visible fluorescence light can be obtained by irradiation with ultraviolet rays; on the other hand, chlorophyll occurring in green plants shows fluorescence phenomena when excited with visible light, but the fluorescence emitted lies mainly in the invisible infrared.

When the light emitted by fluorescence has to be observed, two different measures h~ve to be taken. In the first place, the light falling on the object should be as rich as possible in the wavelength at which optimal excitation is obtained, avoiding as much as possible light of other wavelengths which would not contribute to the fluorescence effect. This is attained most often with a filter which passes only light of a certain wavelength and bandwidth, the excitation filter; in principle, this can also be accomplished with a


monochromator giving off light of only a very limited wavelength range. In the second place, the excitation light which has not been transformed by the fluorescence effect should be filtered out by means of a so-called barrieror cut-off filter (German: Sperrfilter), so that only the emitted fluorescence light reaches the eye or the photographic material (fig. 8.7, 8.8).

BF

300 400

,-, I \ , \

, I , I , I , \ , I , I I \ , \ , \

500

\ \ \ \ \ \ , "

600 nm

Fig. 8.7. Excitation-spectrum with concomitant emission-spectrum (dashed line) with maxima EX and EM of a fictive fluorescent substance. The line indicated with BF gives a transmission curve for an ideal barrier filter for this particular situation.

The ideal properties of both filter systems, which are essential for the observation of fluorescence, are totally dependent on the properties of the fluorescing material and show considerable degrees of variation. Generally filter sets are used, with groups of filters which can be brought into the light path separately or in combination. Both the distance between the peaks and the form of the curves of excitation and emission spectrum are important; in fig. 8.7 a (fictive) case has been presented in which the conditions are very convenient, as the barrier filter has a limit of transmission which passes precisely between the peaks of the excitation- and emission spectrum. It should be noted that in most cases the spectra of excitation and emission (for the determination of which a spectrofluorimeter is needed) are not known as exactly as they are drawn in fig. 8.7 and that the circumstances with regard to distance and shape of the peaks will not be as favourable. It is clear that when excitation and emission peaks come closer to each other it will be more and more difficult to separate them.

Many biological substances and quite a few fiuorochromes, specially prepared chemical reagents such as acridine orange or rhodamine, are

FLUORESCE],;CE MICROSCOPY 155

===\iil'== SF

==~==o

EF

Fig. 8.B. Schematic representation of the functioning of a fluorescence microscope. The light source sends out a multitude of spectral lines, from which the excitation filter (EF) selects those within a certain range (e.g. those from the proximal UV-region). To this excitation light, light of other wavelengths is added in the object 0 by the phenomenon of fluorescence. The barrier filter BF takes away the remaining excitation light, so that only the light formed by fluorescence remains.

stimulated in the blue region of the spectrum and the proximal ultraviolet, i.e. in the wavelength region 350-430 nm. When the excitation peak is near or not too far from the 400 nm region (e.g. 366 nm), these rays can still pass glass, which has many advantages, and can be separated rather easily from emitted light, which is then always further in the visible region. It is much more difficult to separate excitation- and emission peaks in the middle of the visible region, especially when the peaks are only some tens of nm apart. This is the case with the fluorochrome fluorescein isothiocyanate


(FITC), as often used in immuno-fluorescence, with an excitation wavelength and fluorescence wavelength of 495 and 520 nm, respectively; this asks e.g. for an excitation filter with a sharp cut-off at the upper limit of the excitation spectrum.

For the set-up of a fluorescence microscope as a whole, it is clear that the excitation filter should be localized somewhere before the object is passed by the illuminating bundle and the barrier filter behind it. A common situation is shown in fig. 8.8; the excitation filters are then mostly attached to the lamp housing, while the barrier filters are often mounted in the tube in a slide or a rotable disc, but their position varies with different makes.

Optical arrangements with afluorescence microscope The application of fluorescence phenomena was introduced into microscopy by A. Kohler at the beginning of this century; the first good instrument was built in 1911, and fluorescence photomicrographs were published as early as 1913.

Generally, fluorescence phenomena have been studied mainly with conventional transmitted illumination, although in recent times a tendency more often exists for using incident illumination. Apart from the use of excitation and barrier filters, the general conditions are optically comparable to those with conventional light microscopy, although the image formation is quite peculiar. As a rule, the condenser aperture should be as high as possible to concentrate a maximum of light flux in the specimen under observation. Matching of the numerical aperture of the condenser to that of the objective is pointless, as the fluorescent parts of the object can theoretically be considered as self-luminous objects (cf. chapter 5). For the same reason, the image quality of the condenser is virtually of no importance in fluorescence microscopy and a simple condenser of the Abbe-type can give quite good results. In bright-field fluorescence with transmitted illumination a Kohler illumination is to be recommended. Curiously enough - in contrast to conventional microscopy - the use of an achromatic condenser for this illumination is generally not to be recommended, unless it is specially developed for fluorescence microscopy: when excitation light in the proximal ultraviolet is used, often quite a proportion of the light is absorbed by the cementing material of the more highly corrected condensers. In using condenser-immersion (which is more often applied than with ordinary microscopy, in view of reaching as high an aperture as possible, stray light being virtually of no importance) special non-fluorescent oil with, moreover, a low absorption of ultraviolet should be applied. It should finally be noted that in focussing the field diaphragm with Kohler illumination, blue light


should be used when the excitation wavelength is in the blue or proximal ultraviolet region; there may be quite a difference in focus of the condenser when using 'white' light (e.g. from an auxiliary light source) and blue-violet light in the 400 nm region.

Much that has been said about the condenser holds true also for the objective; the correction degree does matter here, however, and ultraviolet absorption is in itself more of an advantage than a disadvantage. Unfortunately, many objectives of the better correction grades (especially when fluorite has been applied in the construction of the objective) not only absorb ultraviolet light, but show a considerable degree of fluorescence themselves. Special fluorescence-free objectives are available; their use is especially indicated when very feeble emission light has to be demonstrated. With a view to passing as much light as possible, such objectives have a comparatively high aperture for their focal length; in some cases, these objectives have been designed for oil-immersion down to the 10-30 X range. In using these objectives for incident light fluorescence (see next section), this high aperture is equally efficient for the illumination cone.

Apart from the presence of excitation filters, the illumination in fluorescence microscopy must meet very special demands, depending on the types of fluorochromes used. Generally, its light yield should be very high, in view of considerable losses at the passage of the different filters and the low percentage excitation light which is ultimately converted into fluorescent light. In most cases the ultimate yield in flux of fluorescent light is only of the order of 0.1%, or even less, of that emitted by the light source. As has been explained in chapter 6, all incandescent lamps have a very poor yield in the blue-violet region of the spectrum; as this precisely is the region where a large proportion of fluorochromes are excited, it may be stated that, apart from some special situations, incandescent lamps are less suited for application in fluorescence microscopy. Often gas-discharge lamps are used, particularly high pressure mercury lamps, which have powerful spectrallines in the proximal ultraviolet (especially at 366 nm) and in the visible blue-violet (near 405 and 435 nm): see fig. 6.10, page 110. Notwithstanding their need for complicated accessory equipment, their limited life and high operation costs, these lamps have gained an irreplaceable position in fluorescence microscopy. In the recent period there is a tendency, however, to use xenon-burners of medium power for fluorescence work. Even with these high-yield light sources, the image still may suffer from an inadequate brightness, so that details cannot be observed clearly and exposure times for photomicrographs become excessive. Barer (1968) has suggested a combination of transmitted and incident illumination in these cases.


The mirror also merits some special attention; this cannot be of conventional design, as light from the ultraviolet region is only partly reflected by silver. Use is made, therefore, of a specially designed mirror (aluminium, covered with a thin layer of silver) which gives a good reflecting surface for UV light; sometimes a totally reflecting prism of quartz is applied. In some situations with feeble UV fluorescence object slides of quartz are used, in order to allow as much UV light as possible to pass to the specimen. The coverslip can be of ordinary glass, of course, as only the fluorescence light in the visible range is of importance for the image.

As stated before, the excitation filters are generally localized in or near the lamp housing when transmitted illumination is used, whereas the barrier filters are mostly placed in a tube-slot disc. In former years, the barrier filter used to be applied as a cap-like device directly over the eyepiece. As all non-converted excitation light fulfills no useful function, the barrier filter should theoretically have a position as low as possible, i.e. in the cover glass of the specimen. Although special cover glasses with barrier filter function have been developed and ultraviolet absorption filters have been built-in even in the objective, these solutions have remained exceptions, as they cannot cope with variable circumstances. Under usual conditions, therefore, much excitation light (often ultraviolet) enters into the objective, together with the scanty amount of light converted by fluorescence. This is the reason why fluorescence in the immersion fluid and in the lens cement of the objective should be avoided as far as possible by using special objectives, as the fluorescence light from the specimen might easily be dominated by these concomitant fluorescence phenomena.

The excitation light must be filtered out as efficiently as possible by the barrier filter, so that non-fluorescent parts of the object will show virtually dark (fig. 8.11), bringing out the fluorescent image as clearly as possible. Autofluorescence, if present, showing a different excitation- and emissionspectrum may reduce the contrasts in black-and-white photomicrographs. The presence of excitation light beyond the eyepiece is also undesirable because remnants of ultraviolet excitation light can lead to fluorescence phenomena in the eye, lowering the image contrast. Moreover, such remnant excitation light can spoil photomicrographs completely, as photographic materials are much more sensitive to this energy-rich radiation than t~e retina of the eye. The images of fig. 8.9 show two photomicrographs, made with the same light source, excitation filter and optical equipment, differing only in the fact that picture B has been made with a less efficient barrier filter. When these photomicrographs were made, the differences were


Fig. 8.9. Section of cortical bone tissue (120 x ) with the fluorescent substance tetracycline incorporated; transparent excitation light in the 370-430 nm region. A photomicrograph made with an adequate barrier filter: some (primary) background fluorescence is present, the specific secondary fluorescence in the so-called Haversian systems around the blood vessels dominate the image, however. B the same area with the same excitation light, but photographed with a barrier filter which let pass a part of the energy-rich excitation light.

discernable but very slight; in the film emulsion, the remnants of excitation light have come to dominate over the fluorescence effect, which has all but disappeared. Safety reasons which are often mentioned in this respect are not primarily at stake, as the ultraviolet which has passed so many glass filters and lenses can be considered as relatively harmlessl .

Even under the most favourable circumstances with regard to the barrier filter, the background is often not totally dark with bright-field fluorescence microscopy. This is for a large part due to the fact that unwanted excitation light having the same wavelength as the fluorescence light is seldom completely eliminated, except in the case of very high quality barrier filters. Apart from this phenomenon, a variable degree of primary background fluorescence occurs which has another excitation wavelength. Careful experimentation with different combinations of excitation- and barrier-

1. Ultraviolet radiation can damage the cornea of the eye, but generally this does not form a great problem with the type of rays used in fluorescence microscopy (the situation is, however, quite different in ultraviolet microscopy, see chapter 11). A much more real danger in working with gas-discharge lamps is an excessive image brightness during observation with an unfiltered source threatening the retina. In fluorescence microscopy, this situation may occur sometimes when during observation filters are thoughtlessly changed so that at a given moment an unfiltered light bundle can reach the eye.


filters can often bring this down to a certain minimum. On the other hand, a light background fluorescence, especially when in different colour, enables a good over-all orientation of the specimen. When fluorescence microscopy is used for quantitative purposes, this is of course undesirable. With most modern stands for fluorescence microscopy, devices exist for a quick change between fluorescence set-up and conventional illumination with an incandescent lamp, e.g. by the facility of swinging out the base-plate of the mirror, so that a built-in illumination can be brought into action.

Bright-field fluorescence as discussed so far gives rise to a minimal light loss (still often over 99%!) and is, therefore, generally preferable for detecting very weak fluorescence when a good combination of excitation- and barrier filter can be found. When this is not possible for some reason, dark-field fluorescence should be tried. As discussed in a previous section of this chapter, no direct light can enter the objective with a correct set-up of the dark field; the requirements for the barrier filter are less stringent, therefore, under these conditions as long as the aperture angle of the illuminating rays is larger than that of the objective. Although this system thus has many advantages (as virtually no excitation light enters the objective and moreover fluorite and apochromatic objectives can be used) it can be applied only for rather strongly fluorescent specimens, because of severe loss in fluorescence light caused by the unavoidable reduction of the maximal N.A. which can effectively be applied in dark-field illumination. In some circumstances, it is possible to combine dark-field fluorescence with a second conventional illumination with aspecific light (e.g. using an excitation filter which transmits some light from the intermediate or red part of the spectrum, so that fluorescent and non-fluorescent parts of the specimen can be observed simultaneously in different colours.

For fluorescence microscopy with incident illumination a dark-field system can be used with a set-up as shown in fig. 8.6; with newer technical developments, however, bright-field is now often preferred. Incident illumination ('reflected light fluorescence') has long been used in the investigation of rocks and crystals in geology and mineralogy. In the last few years its application in biomedical microscopy has greatly increased, especially for investigation of fine details with high-power objectives, as in the study of chromosomes and in the case of very feeble concentrations of fluorochromes (cf. PI oem, 1971). In principle use is made of the system of normal incident illumination with a beam-splitting mirror of rather complicated construction; formerly semi-transparent plates were used for this purpose. As discussed in chapter 6, this system enables the use of the full aperture of


the objective both for illumination and observation of the reflected light, albeit with a high loss of light energy. The beam-splitting dichroic mirror on this so-called vertical fluorescence illuminator as developed by Ploem (1967) has an interference coating which has a high reflectance for the light passed by an excitation filter, but is transparent for the fluorescence light of a longer wavelength (as well as other light than that of the excitation wavelength). Consequently, this beam-splitting mirror also functions as a barrier filter, although a second 'real' barrier filter is necessary for full absorption of unwanted excitation light (fig. 8.10). As most of the excitation

t BF

, ,,<

;:;'-;:;'-;:;'-;:;-;:;-~-:':-:';""~-=--+=-";;;-:':-:';-::";-::'-::;-=--=-";;;-~-:';-::";-=-=Ilk-------->---_t:

i

Fig. 8.10. Schematic view of the course of the rays in a vertical fluorescence illuminator with dichroic mirror. OB object plane, BF barrier filter; at right a dark layer, absorbing unused excitation light, which has passed the dichroic mirror.

light that is reflected by the glass surfaces of objective and cover glass has been deflected by the dichroic mirror, this barrier filter can be of comparatively low absorption, whereas a complete suppression of untransformed excitation light can be reached with optimal instrumentation, so that a completely dark background enables the detection of minimal amounts of fluorescence light (fig. 8.11).

A dichroic mirror with matching excitation- and barrier filter can be constructed thus for a special type of fluorochrome, e.g. tetramethylrhodamine isothiocyanate (TRITe). As these complexes can be manufactured


as interchangeable units, it becomes very easy to change from the optimal circumstances for the observation of one kind of fluorochrome to those for another, the light source (a powerful gas discharge lamp) and all other parts of the microscope remaining the same. With this system it is even possible to combine incident fluorescence microscopy with conventional transmitted illumination, so that with a correct balancing of both illumination systems an over-all image of the specimen is seen simultaneously with the specific fluorescence image. This can be advantageous, e.g. in looking for certain details in the specimen which bleach quickly under the bundle of excitation light. Only when the desired parts of the specimen are in optimal position for observation or photomicrography, the incident excitation illumination is switched on. Sometimes, phase contrast is used for this additional image with transparent illumination. True phase contrast cannot be used with fluorescent images, on the basis of the fact that the fluorescent parts of the specimen behave like self-luminous objects so that direct and diffracted light cannot be separated. Polarized fluorescence microscopy, based on the fact that a variable degree of polarization exists in the emission light, is a highly specialized tool oflimited application.

Fluorescence microscopy has undergone a rapid development in latter years in different fields of application in biology and medicine. A very important and widely used technique is the immunofluorescence, enabling precise localization of proteins in biological specimens when treating them with a fluorochrome-labelled antibody, which is bound locally by antigenantibody interaction. Use is often made here of fluorescein-isothiocyanate

Fig. 8.11. Fluorescence photomicrograph of membrane-bound IgE antibody on the surface of a human basophilic granulocyte, as demonstrated with anti-IgE conjugated to a fluorescent dye (FITC). Incident bright-field illumination, 700 x ; note the complete absence of any background fluorescence of the elements in the suspension around the cell. (Photomicrograph kindly provided by Dr. Thea Feltkamp-Vroom.)


(FITC, cf. fig. 8.11). In other different fields of microscopy, such as chromosome analysis in cytogenetics, histochemistry of nervous tissue (in which by special treatment certain specific substances can be shown by fluorescence microscopy) and also in demonstrating proteins and nucleic acids, fluorescence microscopy has become an important tool. When it is possible to localize certain substances in cells or tissues with fluorescence microscopy, it is possible to detect minute quantities of it using fluorescent dyes, Rigler (1969) was able to demonstrate 10-15 g DNA in a single cell. Moreover, it has appeared that when an isolated detail in the object shows primary or secondary fluorescence, it is possible to measure the total amount of fluorescent material present, e.g. a specifically bound fluorochrome, such as acridine orange bound to DNA. With this technique, called microjfuorometry or cytojfuorometry involving only relatively simple instrumentation, a high degree of accuracy can be attained in measuring the fluorescence light given off by such a fluorescent object. This will be dealt with in some more detail in chapter 11. When the binding between the substance and the fluorochrome is specific, it is possible to measure with a high degree of accuracy cytochemical parameters such as the amount of nuclear DNA or the total protein content of a cell.


R. Barer: Maximum-intensity fluorescence microscopy. Nature 217 (1968) 672. M. Berek: Betrachtungen zur Darstellung des Abbildungsvorganges im Mikroskop und

zur Frage des Auflosungsvermogens im Hellfield und Dunkelfeld. Z. wiss. Mikr. 41 (1924) 1-15.

G. H. Bourne (ed.): In vivo techniques in histology. Williams and Wilkins, Baltimore 1967. G. G. Guibault: Practical fluorescence microscopy: theory, methods and techniques. Marcel

Dekker, New York 1973. W. Kraft: The technology of new fluorescence illumination systems. Mikroskopie 31

(1975) 129-146. R. T. Mayer and E. L. Thurston: An improved method for standardization of micro

spectrofluorometers. Stain Technol. 49 (1974) 61-64. J. S. Ploem: The use of a vertical illuminator with interchangeable dichroic mirrors for

fluorescence microscopy with incident light. Z. wiss. Mikr. 68 (1967/68) 129-142. J. S. Ploem: A study of filters and light sources in immunofluorescence microscopy.

Annals N. Y. Acad. Sci. 177 (1971)414-429. J. S. Ploem, J. A. de Sterke, J. Bonnet and H. Wasmund: A microspectrofluorometer with

epi-illumination operated under computer control. J. Histochem. Cytochem. 22 (1974) 668-677.

G. Prenna, S. Leiva and G. Mazzini: Quantitation of DNA by cytofluorometry of the conventional Feulgen reaction. Histochem. J. 6 (1974) 467-489.

J. Rienitz: Kritik des Positiv-Negativ-Verfahrens und der mikroskopischen Reliefverfahren (schiefe Beleuchtung, Schlieren-Mikroskopie und Shearing-Interferenzmikroskopie). Mikroskopie 22 (1967) 169-193.


R. Rigler: Fluorescent probes for analysis of nucleic acid structures and their interactions with proteins. Exp. Cell Res. 58 (1969) 460.

F. W. D. Rost and A. G. E. Pearse: Microfluorometry of primary and secondary fluorescence in biological tissue. Histochem. J. 6 (1974) 245-250

A. Sato: Fine structure of human nuclear chromatin in interphase as observed by polarized dark field oblique illumination. Acta Cytol. 30 (1969) 218-223.

H. Siedentopf: Dber bispharische Spiegelkondensoren ftir Ultramikroskopie. Ann. Phys. 39 (1912) 1175-1184.

A. A. Thaer and M. Sernetz: Fluorescence techniques in cell biology. Springer Verlag, BerlinHeidelberg-New York 1973.

CHAPTER 9

SPECIAL TECHNIQUES OF IMAGE FORMATION

PHASE CONTRAST MICROSCOPY

Basic principles The formation of an image of a microscopic specimen depends on the changes brought about in the image-forming agent by the specimen. This not only holds true for light microscopy, but also for microscopy with X-rays or electron rays (chapter 12).

In light microscopy the two most important changes occuring in the light passing the specimen are changes in amplitude and changes in phase; both with incident and transmitted illumination do these two effects occur simultaneously to a varying degree. As the differences between changes in amplitude and phase are fundamental for the understanding of the basic principles of phenomena dealt with in this chapter, they will be discussed first on the basis of the schema of fig. 9.1.

Fig. 9.1. Schematic view of the effect of amplitude- and phase-objects on a passing light wave. A control wave, passing without meeting any obstacle; B change brought about by a pure amplitude object; C effect due to a phase object, not affecting the amplitude.

a. Amplitude objects. With a classical object for conventional microscopy, e.g. a stained section, the contrasts in the object are brought about mainly by local differences in light absorption; as discussed in chapter 7, the conven-

166 SPECIAL TECHNIQUES OF IMAGE FORMATION

tional microscopic specimen is prepared so that contrast formation as a consequence of other phenomena (refraction, diffraction) is avoided as much as possible. In light-absorbing material with the same refractive index as the medium in which the wave was previously being propagated, only the amplitude of the wave will be changed, the other characteristics of the wave, the wavelength and the stand of the wave at a given position or time, the phase of the wave remaining the same. Changes in amplitude are visible to the eye and can also be made to produce contrasts on photographic material; image formation in conventional light microscopy of stained specimens is therefore mainly amplitude-contrast microscopy.

b. Phase objects. In the situation where a light wave passes a completely transparent (i.e. non-absorbing) medium with a different refractive index, quite another phenomenon will take place. The amplitude of the wave will remain unchanged, but the propagation velocity will change, as well as the wavelength. The refractive index is nothing but the relation between the propagation velocity in an absolute vacuum (c) and that in the material in

question (V): n = ~ ; in the case of fig. 9.lC the refractive index is V

higher, which means a decrease in velocity. On leaving the transparent medium with increased refractive index, wave c has kept its original amplitude and the wavelength returns to its original value. The wave has fallen 'out of step' with wave A and B, however: a so-called phase-difference has arisen. It should be noted in passing that a similar phenomenon arises when waves pass parts of the specimen with identical refractive index, but with different thickness. These changes are not visible to the eye, in contrast to amplitude changes. Band C differ both in amplitude and phase, the vibration frequency (which determines the colour impression) has remained the same in all three waves. The situation in fig. 9.1 is, of course, entirely schematical; virtually any amplitude object will also show some phase shift and vice versa.

When the formation of an image of an amplitude object is considered from a theoretical point of view, the Abbe theory formulated just over a century ago in 1873, states that the image is formed in two steps. The objective lens forms a primary interference pattern in the back focal plane of the objective, where this primary interference pattern is transformed into the intermediary image, as viewed through the eyepiece. The interference pattern obtained with any light-absorbing object is of course very complex; for theoretical considerations, it may be compared with an amplitude grating, a

PHASE CONTRAST MICROSCOPY 167

series of alternate opaque and transparent lines. With such a grating it can be shown physically that the primary interference pattern consists of series of arrays of maxima, with zeroth, first, second etc. maxima. The zeroth order maximum alone cannot produce an image. The more higher order maxima that can beformed (higher N.A. !), the more truthful the final image will bel. A very important point is that the interference maxima produced by such an amplitude grating all are in the same phase. A transparent object - e.g. an unstained animal cell - consisting of parts differing locally in refractive index and/or thickness or its abstract counterpart, a phase grating of alternating thicker and thinner strips of the same transparent material, would produce similarly a complicated primary diffraction pattern at the back focal plane of the objective. In the final image, however, the light intensities will be the same at all points of the image (which is really formed physically), leading to a totally invisible 'image'. Closer examination reveals, however, that some very fundamental differences can be observed between the primary interference pattern of a phase grating, as compared with an amplitude grating: 1) a phase difference of t A (90 0 )

exists between the zeroth order maximum (which arises by non-diffracted light) and the higher maxima; 2) the zeroth order maximum is much brighter in relation to higher order maxima than with an amplitude object when the phase retardations in the object are small (fig. 9.2 A and B). All this can be proved with theoretical arguments, requiring a thoroughly mathematical treatment, however (cf. Michel, 1964; Martin, 1966).

It has been the idea of the Dutch physicist Zernike2 to modify the primary diffraction pattern in such a way that a phase object might look like an amplitude object. Designed originally by its inventor for inspecting telescope mirrors, the principle was applied to microscopy as early as 1932 and even a photomicrograph of a diatom from that year has been published (cf. Zernike, 1955). At first, the optical industry did not show great enthusiasm for the idea which they regarded as only of limited value. Interrupted by the war of 1940-1945, effective development of phase-contrast microscopy lasted until 1946; the first commercially produced phase-contrast microscope (manufactured by the German industry) dates from 1942. At present, it forms an indispensable part of the equipment of any microscopist. In 1953, Zernike received the Nobel prize for his remarkable achievement.

Essentially, the principle developed by Zernike is, to a certain degree, a

1. Here lies in fact the real theoretical reason why the resolving power of an objective (and therefore that of a microscope) is determined by the lens system forming the primary image. 2. F. Zernike, 1888-1966, professor of theoretical physics at the university of Groningen.


t 1 r I t /\ //' " \ , \-,);: '" '" /' '" '" '"

A B C

Fig. 9.2. Simplified vectorial representation of the diffraction pattern of an amplitude grating (A), a phase grating (B) and at (C) the same grating with phase contrast, after t A retardation and reduction in amplitude of the zeroth order maximum (modified after Meyer-Arend ).

logical consequence of the theoretical knowledge about image formation just discussed. From fig. 9.2 it follows that all that would be needed to make a phase grating look like an amplitude grating is to change the relative phases and amplitudes of the zeroth and higher orders in the primary interference pattern so that they become like those of an amplitude object. This can be done 'by bringing about a separation between direct and diffracted light from the object and modifying the non-diffracted light in that the phase is retarded by t A (90°) and brought down in intensity, so that optimal circumstances for interference with amplitude effects occur (fig. 9.2C). What is achieved in this way, is the conversion of variations in thickness and/or refractive index into a variation in intensity.

Excellent accounts of the theory of phase contrast are given by Fran~on (1961), Martin (1966), Barer (1966) and Beyer (1973). From the viewpoint of the microscopist wanting to understand what he is doing in using phase contrast, the practical aspects of the phase contrast principle as explained above will be dealt with in the next section.

Practical realization o/the phase contrast principle The basic theory of the phase contrast, as explained in the previous section, calls for the following interventions in the light path, in order to achieve


the partial transformation of a phase interference pattern into an amplitude pattern in the primary image. 1. A separation should be reached between the light which has passed the

object directly and that which has been diffracted in the object. 2. The non-diffracted light should undergo a change in phase and ampli

tude, so that optimal conditions are created for interference between zeroth and higher order maxima in the primary image.

re 1. When a microscope has been focussed correctly on a stained specimen and the eyepiece is removed, the illuminated disc of the entrance pupil can be clearly seen. When the aperture diaphragm of the condenser is closed somewhat, it appears that there is no total darkness around this smaller bright disc, but that it is surrounded by a luminous circle fading away at the periphery. When the specimen is taken away, the luminous disc becomes sharply delineated and there is total darkness around it. The light observed around the border of the entrance pupil is light diffracted in the specimen; in the illuminating disc itself (the aperture of the objective) both direct and diffracted light occur. When a diaphragm is placed at the first focal plane of the condenser, an image of this diaphragm will be formed near the back focal plane of the objective. The entire aperture of the objective will be filled to a greater or lesser degree with diffuse diffracted light when an object is introduced in this light path; apart from the area of the image from the diaphragm, no overlap between direct and diffracted light occurs. It is clear that the smaller the diaphragm, the smaller the area of overlap.

With all modern phase contrast microscopes, a ring-shaped diaphragm or phase annulus is applied, usually in the form of a disc of opaque glass with a ring-shaped area of ordinary glass, which is imaged onto the phase plate in the objective (see below). It follows from the foregoing, however, that a ring-shaped illumination system is not essential for the phase contrast itself; the first phase contrast microscopes as constructed by Zernike had a central illumination diaphragm and corresponding phase plate (cf. Zernike, 1955). The annular diaphragm simply has been proven to give the best results in that it yields a better resolution than a small central opening. In theory, a very narrow annulus would enhance the separation of direct and diffracted light; this entails certain disadvantages, however, e.g. with regard to image brightness; most annuli have a width of7-lO% of the full objectiveaperture diameter.

re 2. For achieving the phase-shift between direct and diffracted light, a phase plate is inserted into the objective, near to (not necessarily in) the back focal plane of the objective, which should be covered by the phase

170 SPECIAL TECHNIQUES OF IMAGE FORMA nON

8 @

Fig. 9.3. Schematic view of the practical realization of the phase contrast principle; A phase annulus under the condenser, B the corresponding phase plate in the objective.

annulus to which it is conjugate (fig. 9.3, 9.5). This phase plate has two functions, a) influencing the phase of the undiffracted light (with, unavoidably, that of some diffracted light), and b) reduction of the intensity of the direct light (fig. 9.2).

In modern objectives the phase plate is made by evaporation of thin layers of magnesium or creolite in vacuo onto one of the back lenses of the objective, so that with proper adjustment of the layer thickness the desired change can be reached in the traversing direct rays. This can be both an advance of -1- II, giving rise to interference effects so that regions with higher thickness or refractive index will show up dark, or just a retardation of t II in phase of the passing direct rays, in which the reverse type of interference will occur and the regions with higher phase retardation will appear bright


against a dark background. These effects are called positive and negative phase contrast, respectively. Both types of phase contrast are used and many manufacturers supply objectives for positive and for negative contrast (the phase annulus, of course, remaining the same). The positive contrast is most commonly used, but for certain small details the negative contrast is often recommended; the choice can, however, be considered as mainly dependent on individual preference. Both types of phase plates are covered with an extremely thin light-absorbing layer which does not affect the phase displacement; a schematic view of both types of phase plate is given in fig. 9.4. It should be noted that positive or negative phase contrast effects

II

Fig. 9.4. Phase plates for positive (I) and negative (II) contrast with the same change in phase (+ and -, respectively) and amplitude.

are not bound to positive or negative phase plates respectively; due to the phenomenon of phase reversal the opposite may occur with certain objects, as will be dealt with in the next section.

The phase annulus should be imaged exactly over the phase plate near the back focal plane of the objective (fig. 9.5A). Consequently, phase annuli and phase plates should match each other; only objectives of identical geometrical characteristics can be used with the same phase annulus. Sometimes two or more objectives differing not too much in focal length can be made to share the same annulus, the phase plate having differences in size which correspond with the differences in magnification of the objectives. In most cases the variation in phase annulus is provided for by a revolving substage comprising rings of various size, so that they can be matched with the phase plates of different objectives. With the condenser according to Heine the size of the phase annulus can be varied by moving an optical system sliding within a tube, so that with one ring all objectives can be matched. Other systems also exist in which the dimensions of an imaged ring can be made to match the phase plate of the objective involved by a kind of pancratic systeQ1.

With most commercially produced phase contrast equipments, the phase plate gives a phase change of t A (90°; + or -). It can be shown that the exact values of the phase plate are not critical, virtually all values between 60° and 90° (i-t A) phase change giving good results; i A plates have been


manufactured, but they are infrequently used. Somewhat more variation exists for the absorption of the direct light in the phase plate. The reason for this is that with details of low retardation, most of the energy is in the direct (O-order) light so that a higher absorption of the non-diffracted light would in these cases create optimal circumstances for interference; with details of higher retardation, comparatively less energy is in the direct light, so that a high absorption would seem to be much less favourable. In practice most phase plates have a value of 70-75% absorption, the extremes may vary between 50 and 85% (i.e. transmissions from 50 to 15%). By choosing a very highly absorbent phase plate, one may reach an increased sensitivity for revealing object details of very low phase change, but such a plate might be quite useless for the observation of more refractile details in the same specimen; the reverse holds true for low-absorption plates. Further consequences of this situation will be discussed in the next section. The range of 70-75% absorption appears to give good results with a wide range of material. A few ingenious phase contrast systems with variable absorption have been designed, mostly making use of polaroid elements in both phase annulus and phase plate; by changing the angle between the axes of the polarizing material, the degree of tranf>mission can be adjusted infinitely (cf. Franc;on, 1961). These systems have, however, not come into general use.

The 'anoptral contrast' system for negative phase contrast makes use of a highly absorbing (90%) negative phase plate, which is non-reflecting and non-scattering, so that images with high contrast can be obtained with very thin objects (cf. Wilska, 1954). In contrast to other statements which have been made in this respect, this device is nothing more than a very sensitive negative phase contrast system.

From the foregoing it will be clear that for the setting-up of a phase contrast microscope the annular diaphragm should be imaged onto the phase plate in the objective in such a way that no 'leakage' of direct light which has not passed the phase plate occurs. This can be due to incorrect centering of the two screws permitting movement of the phase annulus holder beneath the condenser in two coordinates so that it fits onto the phase ring (fig. 9.5A and B). When the condenser is positioned too low, however, the image of the annulus is brought below that of the phase plate so that again much direct light which has not been changed in phase and tempered reaches the image plane (fig. 9.5C). All this entails a lowering of the contrasts and a somewhat distorted phase contrast effect.

The image of the annulus, as projected onto the phase plate can, of course, be observed by removing the eyepiece and looking down the tube; details


Fig. 9.5. Images (made via an Amici-Bertrand lens) of the ring-shaped dark phase plate in the objective back focal plane with the bright image of the phase annulus projected over it. A correct set-up, B insufficient centering of the phase annulus, C too low position of the condenser.

as shown in fig. 9.5 cannot be seen clearly enough, as the image of the entrance pupil seen in this way is too small. For this adjustment afocussing telescope is used which can be inserted in the tube when the eyepiece has been removed, or an Amici-Bertrand lens mounted in the tube which (as a swing-out lens, or built into a magnification changer) forms a telescopic system together with the eyepiece. These lens systems enable the formation of an enlarged image of the back focal plane of the objective, so that details as shown in fig. 9.5 can be clearly observed. A built-in Amici-Bertrand lens can also serve many other purposes, such as the precise adjustment of the condenser aperture (chapter 6), the setting-up of an oblique illumination, in polarization microscopy and in many other circumstances under which the filling of the aperture of the objective with light has to be controlled. The condenser of a phase contrast system can be used for setting up an oblique illumination (chapter 6); especially the Heine-condenser with variable annulus enables all kinds of experimentation with hollow illumination cones.

Phase contrast objectives can be recognized as such at a single glance when held with the front lens against the light, as the dark phase ring will then be clearly visible. It is not possible, of course, to tell whether the objective has been designed for positive or negative contrast; the presence of 'Ph' on the mount without further designation mostly indicates a positive contrast.

Objectives with the most commonly used + t A and 75% absorbing phase plates can be used for observation with conventional microscopy up to a N.A. of about 0.50 when demands are not high. Optimal image formation


with higher apertures is, however, seriously hampered by the phase plate in the region of the back focal plane.

Some further details about the phase contrast image with different objects Irrespective of the type of phase system used, the difference between a given point in the final image and its surroundings is related somehow to the phase change brought about by the object at the corresponding spot. This phase change, called also optical path difference or optical thickness (not to be confused with optical density, cf. chapter 11) is determined by the formula:

cp = (no - nm) t

in which no is the refractive index of the part of the object in question, Om the refractive index of the medium and t the thickness of the object.

With a thin object, a difference in refractive index of a few tenthousandths can be demonstrated with an average phase plate of e.g. between no = 1.5387 and nm = 1.5383, when the level of illumination is sufficient. This very high sensitivity determines the possibility of visualizing details in a transparent object with differences in optical thickness of only a few thousandths of wavelength.

It can be deduced from the formula just mentioned that if the difference between no and nm is at or very near to zero (in practice: smaller than

Fig. 9.6. Photomicrographs, made with positive phase contrast (+ 90°,75% absorption) of a suspension of human erythrocytes in solutions of bovine albumin (adjusted to an isotonic value with NaCl).

A Suspension in 26 If/O protein: all red cells show a positive phase effect; B suspension in 30 g% protein: both cells with positive and with negative phase effect can be seen and some in which the contrast is virtually zero; C suspension in 36 g% protein: all cells show a negative effect (from James and Dessens, 1962).


0.003-0.002) the optical thickness will be zero, independent of the geometrical thickness of the object. No phase retardation occurs and the object will be virtually invisible under the phase contrast microscope (fig. 9.6B). When no> nm, a positive phase contrast image will be obtained (in using a positive phase plate). When no < nm, a negative phase contrast image will occur, in which the regions appearing dark in the positive contrast show up bright against a darker background (fig. 9.6A and C). The relation between relative intensity in the image and phase retardation in the object is somewhat more complicated than would appear from the formula cp = (no - nm) t, which strictly only holds true for small values of cp as will be explained further on.

When the refractive index of the mounting medium of an object having a lower refractive index than its medium is enlarged in small steps, the following can be expected to occur. The object will first appear dark in ever decreasing grey-tones, become invisible when no, or virtually no, phase effect occurs and finally re-appear beyond the matching point in reversed phase contrast. In the situation that cp approaches zero, the refractive index of the medium can be considered to be identical with that of the part of the object which is in direct contact with the medium. This method for finding the sign of cp is ideally suited for statistical studies of large cell populations. It has been applied with erythrocytes suspended in albumin solutions of increasing concentrations, yielding a series of media with known shifts in refractive index. By counting the relative frequencies of dark and bright cells in different media from the series, the solution in which e.g. all cells are positive or negative and that in which 50% of the cells are dark and 50% bright (a total extinction will occur relatively seldom) can easily be determined (fig. 9.6). On the basis of such parameters, it is possible to detect very small differences in the refractive indices of cells in a population (cf. James and Dessens, 1962). As will be explained later on in this chapter, the refractive index of a cell can be taken as a direct measure for the concentration of solids in its cytoplasm. This measuring technique, essentially a sensItIve O-method, is called microrefractometry or phase-refractometry (cf. Barer, 1966).

For different reasons, phase contrast cannot be used for quantitative purposes other than with the microrefractometric null-method just described. The reasons for this are the following. The conclusion that nm > no when a negative phase effect is reached with a positive plate and vice versa, is correct only for smaller values of cpo It can be shown (and demonstrated theoretically) that the relative intensity in the image does not vary in direct proportion with the phase change occurring in the object. In using a positive phase plate and with no > nm, the decrease in intensity first shows a pro-


gressive decline with increasing phase retardation and then reaches a maximum. The object will subsequently appear progressively lighter, until at a given moment the same intensity is reached as with the first increase; ultimately, with ever increasing phase retardation, the relative intensity will again become identical to the background, i.e. the object becomes invisible. For values for cp exceeding this point, the relative image intensity will even become greater than that of the background, i.e. a negative phase contrast image will be formed (with a positive plate). This phenomenon is called phase reversal. The form of the V-shaped curve of image intensity plotted against cp is dependent on the degree of absorption of the phase plate: with a highly absorbing plate, the intensity shows a steep fall with increasing cp, reaching quickly its minimum and subsequently phase reversal. With a 90% absorption plate this (rather low) minimum is reached already at 5° (0.055 A) phase retardation, whereas with a more or less standard 75% absorbing plate this is reached only near a value for cp of 30° (0.33 A). Highly absorbing phase plates should be used therefore only to detect very small optical path differences, as they may give rise to confusing images when small and large phase shifts are generated in the same specimen.

For theoretical reasons which cannot be dealt with here, phase reversal is less likely to occur with negative phase contrast. This is why negative phase contrast can be preferable when the object contains a large amount of high phase details.

When a phase-retarding detail in the object appears dark in the image as a consequence of destructive interference of light, it would seem that light energy should be lost. This is, however, not the case; a phase contrast pattern only redistributes the light in the image plane. The light which appears to have disappeared in the dark regions reappears as a bright halo around darker objects. This halo is always opposite to the amplitude effect of the detail in the specimen; a bright halo can be seen around the dark erythrocytes in fig. 9.6 and a dark rim occurs around cells appearing in negative contrast. The darker the contrast in the phase changing detail, the brighter the halo and vice versa; with very low amplitude changes, the halo becomes almost imperceptible. If the halo images were 'added' to the proper image, the phase contrast effect would disappear completely; the light intensity would be the same at all points in the image plane and the 'image' completely invisible. It therefore follows that the halo is inherent to the phase contrast effect; the way it is formed is rather complicated and its explanation oflimited practical value. When the phase annulus is made narrower, the halo will become larger and more diffuse. This would make the halo less disturb-


ing, but on the other hand a very small phase annulus can give rise to difficulties with the light intensity and/or the adjustment of the annulus on the phase ring. As with the absorption percentage of the phase plate, a compromise must be reached, and usually the phase ring is made to fill between 7 and 10 percent of the aperture of the objective.

Another related phenomenon inherent to phase contrast is the effect called zone of action or shading-off. When a larger area of uniform phase retardation is observed with phase contrast, the contrast appears to decrease from the border of the area. There is thus a 'zone of action' over which the phase contrast operates optimally; the extent of this zone increases in inverse proportion to the relation between the width of the phase annulus and the objective aperture. Both halo and zone of action effect are a result of diffraction phenomena occurring at the boundary of regions with differences in optical path. These phenomena can be very disturbing in some cases and they are often described as 'artefacts' in the phase contrast image; as they concern nothing but direct consequences of the light distribution in the image plane, these effects are strictly no more an artefact than the phase contrast itself. They also contribute to the non-linear relation between the lighting intensity in the image and the phase change in the object, even in the first part of the V-shaped curve at low values for ([l.

In using the phase microscope for observation, halo and shading-off effect may sometimes enhance the contrast of edges, small granules in the interior of a cell etc., so that these effects are not always disturbing. On the other hand, in many instances the halo-effect in combination with phase reversal can distort the image in such a way that all details are lost. This occurs with rounded cells suspended in aqueous media with low refractive index (e.g. balanced salt solutions), which objects, moreover, show a lens effect as a consequence of their shape. The phase contrast image of such cells can be grossly distorted, so that it becomes inferior to that obtained with conventionallight microscopy; often a mere reduction of the factor no - nrn by adding some protein to the medium can then greatly improve the image.

With a given phase contrast equipment with fixed phase plates, the inclusion medium is the only means for obtaining a variation in the image contrast. The substage aperture diaphragm cannot fulfill any function here; it should be opened, as in closing this diaphragm the phase annulus might be covered partly or entirely. It should be noted in passing that the possibility of variation in the refractive index of the medium is often overlooked; the statement that a certain detail 'cannot be readily observed' with phase contrast strictly holds true only for a certain medium. By varying the medium, internal details in a cell which are not in direct contact with the medium,


can be demonstrated in different contrast. Media used for mounting stained sections or smears are not necessarily the best, as they are precisely intended to give rise to as small a refraction effect as possible (cf. chapter 7). On the other hand, too great disparity between no and nm should be avoided for the reasons just explained. For living cells, progressive concentrations of dried bovine albumin can be used with salts added to make an isotonic solution. Appendix I lists the most commonly used mounting media with increasing refractive index. It should be noted that the hardening mounting media show a change (generally an increase) in refractive index as the resins harden by evaporation, polymerization etc., which in some cases may take over a week.

The following remarks about stained preparations and the phase contrast microscope seem now to be pertinent. It will be clear from the foregoing that when a light-absorbing specimen is observed with phase contrast, effects brought about by amplitude changes will not be left undisturbed by the phase plate. The loss of contrast which this entails, is not necessarily compensated for by enhancement of the contrast in the final image on the basis of phase changes in the specimen. This will especially not be the case with more highly refractile parts of the specimen which will be distorted by halo and shading-off effects. In fig. 9.7 it is clear that the dense pigment granules come out much better in the conventional amplitude-contrast1 image than in that obtained with the phase microscope. The transparent cells of the surrounding liver tissue, on the other hand, which virtually show no absorption, form an ideal object for the phase contrast system. Generally speaking, it may be stated that in studying with the phase microscope routine specimens stained with the classical techniques, the results obtained may be confusing and not seldom the over-all image quality deteriorates in comparison with the conventional amplitude-contrast image. When it can be applied, the images represent neither a true phase contrast image, nor a conventional absorption pattern. It should be noted that the situation is generally much more favourable in this respect with differential interference contrast (page 186).

Even in unstained condition, routine sections of a thickness of 6-8 fLm are not suitable objects for phase microscopy. This is due not only to the fact that the phase changes occurring exceed the values for which the phase

1. There is no uniformity in terminology with regard to the conventional image formation, as opposed to phase contrast. In the view of the author, amplitude contrast is the only logical counterpart of phase contrast (although it is sometimes used to denote a special use of differential interference contrast, see last section of this chapter). Sometimes the term bright-field (German: Hellfeld) is used in this connection, but this is confusing as the only logical use of the term bright-field is as a counterpart of dark-field.


Fig. 9.7. Unstained liver tissue of a salamander with at the right hand side a group of pigmented cells; A conventional microscopy with fairly closed condenser diaphragm, B positive phase contrast image of the same area showing appearance of much more contrast in the transparent cells, whereas the contrast has become much less in the highly absorbing pigment cells.

plate has been designed, but also because the superimposition of phase changes in different layers in the specimen results in a blurred interference pattern. Thin specimens (such as 'semi-thin sections' of 0.5-1 [Lm as used in electron microscopy) are therefore ideally suited for study with the phase contrast microscope.

Different systems have been worked out to produce coloured phase contrast images from unstained specimens; although very ingenious, they have not found their way to frequent practical application. The combination of phase contrast with fluorescence microscopy may have a better future.

So far, phase contrast has been discussed only in connection with microscopy with transmitted light. Although this is the situation in which phase microscopy is mostly used in biology and medicine, the same principles can be applied in microscopy with incident illumination. The very high sensitivity of the phase contrast for minimal phase shifts is applied in technology in tracing small inequalities in the surfaces of non-transparent materials, such as polished metal surfaces. It is possible in this way to demonstrate level differences of a few tenths of a nanometer, far under the axial resolving


power of the conventional microscope. In using a positive phase plate, areas with a higher level will show brighter (shorter light path) in reflection and areas of a lower level will seem darker than the remaining surface. As absorption effects can also influence the image, sometimes an alternation of positive and negative phase contrast is applied (e.g. with an interchangeable phase plate) in order to separate phase and amplitude effects. When, in changing from positive to negative phase contrast a bright area in the image becomes dark or vice versa, it can be assumed that differences in level are responsible.

INTERFERENCE MICROSCOPY

Basic principles; the meaning of a refractive index The sharp distinction which is often made between phase contrast- and interference-microscopy is not justified to a certain extent from a purely theoretical point of view: both are based on interference effects as caused by phase changes in the specimen. An essential difference, however, exists in the way in which the interference pattern comes about. In interference microscopy, the separation between the two beams which are made to interfere does not occur by diffraction, as with phase microscopy, but by other means, so that interference effects can be controlled more closely. Interference microscopes convert phase changes into amplitude changes by interference between light which has passed through the object and another beam which passes outside of the object to be studied. The way in which this is reached will be dealt with later in this section.

With interference contrast a second image, the so-called ghost image, is formed as a consequence of the fact that - as with phase microscopy - only a redistribution of light in the image plane takes place: what has been 'substracted' somewhere, should be 'added' elsewhere (fig. 9.8). This ghost image, which is always opposite and conjugate to the primary image (lightdark and vice versa) is separated from it by a certain distance. If measurements are to be made the separation between both images should be complete, i.e. the image of the specimen must not be overlapped by the ghost image (neither, of course, by another part of the same specimen). By optical means, the ghost image can be moved, enlarged so that it becomes vague, etc. The absence of a halo in the immediate vicinity of a phase-retarding object in interference microscopy is essential for the measurement of optical path differences, but it is not necessarily of advantage for observational work. As discussed in the previous section, the halo formation with phase contrast often contributes considerably to the sensitivity of the system; an interference contrast image therefore sometimes seems to suffer from a

INTERFERENCE MICROSCOPY 181

Fig. 9.8. Squamous epithelial cells from an oral mucosal smear, photographed with an interference microscope according to Smith; at left cells in (negative) interference contrast, at right the fuzzy ghost image in opposite contrast. (Photomicrograph kindly provided by Dr D. J. Goldstein, University of Sheffield.)

low contrast, as compared with the corresponding phase image. As the ghost image is perfectly homologous with the halo in phase contrast, the statement that interference contrast images are free from halo is strictly somewhat misleading.

The optical arrangements of different types of interference microscopes will be dealt with in the next section; here the question of the possibilities and limitations of micro-interferometry will be first considered.

When a microscopic object, e.g. an isolated cell nucleus, is considered as a spherical bag with a homogeneous content (which is an oversimplification, and we shall have to consider this later on), it can be stated on physicochemical grounds that the refractive index no of such an object can be expressed in the following formula

no = ns + Q(. c

in which ns is the refractive index in the solvent (in this case water) and c the concentration of the solute, expressed in grams per 100 ml solution. The factor Q(. or specific refractive increment is defined as the increase in refractive index of a solution for every gram per 100 ml in concentration of a solute. As the value for Q(. can be shown for most organic substances to be constant over a fairly wide range of concentrations, it follows that the refractive index of a solution increases linearly with concentration.

At first sight, it seems a hopeless task to find a value for Q(. which would


make any sense in biological material, as one is always confronted with a variable mixture of widely different macromolecular substances (proteins, fats, carbohydrates and conjugated compounds) which contribute to the ultimate refractive index. It has appeared, however, that the value of IX for virtually all proteins occurring in nature lies within rather narrow limits: 0.00180 and 0.00185; nucleoproteins and lipoproteins do not fall far from that value. Some carbohydrates have a lower value, but taking into account that non-protein constituents usually contribute little to the solid content of protoplasm, fixing the mean refraction increment of 0.00180 for animal protoplasm seems not far from reality. It is thus possible to express the refractive index in terms of concentration of total solids; from this other conclusions can be reached such as the concentration of water (the specific volume of protoplasm being taken as 0.75), total wet mass and total dry mass via an integration of the product of cp and the projection area. Interference microscopes enable the measurement of the phase shift or optical path difference (o.p.d.), also called optical thickness, between specimen and background: cp = (no-nrn)t, the same factor which has been considered dealing with phase contrast in the previous section. On the basis of what has been just explained about the specific refraction increment and the conclusions to be drawn from the refractive index, the meaning of such a measurement becomes evident. It follows from the formula for cp that it is not possible to obtain by a single measurement of cp separate values for the thickness t and refractive index of the object no; the refractive index of the medium nrn is of course known or can easily be measured with a refractometer. The optical thickness can be used as a measure for the amount of solute present per unit area and standard depth, as has just been discussed; by measuring cp at a number of points in the specimen, an average value can be obtained per measured area, from which a value for the total dry mass of a specimen can be determined. If independent values for t and no are desired, separate measurements of cp can sometimes be made in two different media of different refractive index. By solving a pair of simultaneous equations, both unknown factors can be found. Details of the actual measuring procedure which is very much dependent on the type of instrument used cannot be dealt with in this general text in which only the outlines of methodology can be given (for details see Barer, 1966; Chayen, 1967; Ross, 1967; Beyer, 1973).

The data obtained with interference microscopy can be extremely useful, especially as they can be applied to living cells, or unfixed parts of isolated cells or tissues. The rather uniform value of IX for the most different biochemical substances which has made these interpretations possible, forms at


the same time both the strength and the weakness of the micro-interferometric technique. For it is not possible to say anything of the nature of substances which increase or decrease the total dry mass when this is observed under certain conditions. In some instances the causes of these changes can be made clear, but in other situations a combination of interference microscopy with qualitative or quantitative histochemical methods is necessary to establish more clearly the biological events involved.

Interferometric measuring systems As explained previously, micro-interferometry is based on the principle that two coherent beams of light of which one has passed through the object and the other outside of it are made to interfere; from the resulting interference pattern conclusions can be drawn which yield a value for the phase change ~ in a certain area of the object.

Unlike the situation with the phase contrast microscope, which has essentially been the discovery of a single man, the different types of interference microscopes have been more gradually developed from interferometers as they have been used in the nineteenth century in physics and astronomy, notably the lamin-interferometer from 1858 and that according to the MachZehnder principle from 1891. Details of these physical instruments are not relevant here, but it is important to note that with these instruments, as with the interference microscope, interference can only be made to take place between two waves which are coherent. One of the basic things in the optical construction of an interference microscope should therefore be that the object beam or measuring beam passing through the object to be measured and the beam passing through a clear area of field or the reference beam should be kept coherent.

A system based on the Mach-Zehnder principle which is the most simple from a theoretical point of view is drawn schematically in fig. 9.9; a splitting of the illuminating beam in two components is achieved by a semi-reflecting beam splitter (ensuring coherence of the measuring and comparison beam), whereas both beams are re-united via a similar semi-reflecting surface. Such an instrument would be ideal from a theoretical point of view in that object beam and comparison beam can be varied totally independently from each other, so that optimal circumstances for interference can be created. In practice, such a complete double-beam micro-interferometric system as devised by Horn poses very complex problems of adjustment, alignment of condenser and objective etc. and is very time-consuming to set up with a proper dummy specimen or a substituted adjustable rotating glass wedge.


Fig. 9.9. Scheme ofa micro-interferometric system according to Horn. P, and p. beamsplitting and re-uniting prisms; C compensation segment, with adjustable compensation wedge in the right hand beam; 0 objectives, over the measuring- and comparison-object.

Much more biological work has been done with the older interference microscope according to Dyson, one of the first commercially available interference microscopes. This microscope produces interfering beams by a complicated system of reflecting and semi-reflecting surfaces which split the beam into two halves, one of each passes through the specimen, whereas the comparison beam passes outside in a 'clear field' (fig. 9.9). There is only one condenser and objective, which makes the system more easy to handle but there is a considerable light loss and it is difficult to make measurements in the middle of a somewhat larger specimen, due to the limited distance between both beams. The interference microscope of Smith in its original version, which can be considered as a micro-interferometer based on the


P2

Pl

c

Fig. 9.10. Course of measuring- and comparison-beam in the interferometric system according to Dyson. C condenser, PI and P2 semi-reflecting mirror surfaces for splitting and re-uniting of the beam.

principle of lamin-Lebedeff, makes use of birefringent material to split the beam in a measuring and a control beam, which are separated by a distance of a fraction of a mm; they are united again with birefringent material after passage of one of the beams through the object.

In the introduction to this section it has been stated that when e.g. an isolated nucleus is considered as a bag with a homogeneous content, this is not correct, as from place to place the phase change will be different and this holds true for most biological objects. In determining the total dry mass of such a non-homogeneous object, it would be necessary to build up an average value for 'P from a great number of separate readings. In recent times, so-called integrating micro-interferometers have been developed which automatically measure a total integrated phase change due to the specimen to be measured and therefore give a value for the dry mass directly. The very elegant instrument designed by Smith (1972) avoids the difficulty of measuring the optical path differences at separate areas by a flying spot system, by means of which a great number of measurements made in a small area are integrated automatically. The accuracy and precision of this instrument seems to be quite promising (cf. Goldstein and HartmannGoldstein, 1974).

Differential interference contrast In the period 1950-1970, the phase microscope which is relatively inexpensive and easy to handle, has found gradually a widespread use for observation and photography in purely quantitative work, whereas the more


complicated interference microscope has found application on a much smaller scale as primarily a measuring instrument. The various colour effects on the basis of phase differences which can be achieved with interference microscopy (by destructive interference of light of mixed wavelength the complementary colour is brought out) can be beautiful to look at, but it is doubtful whether it contributes essentially to the observation and whether a skilful handling of e.g. a variable phase contrast system cannot give comparable results.

The position of the interference microscope as primarily a measuring instrument has changed somewhat in the recent period with the development of a special type of interference contrast microscope which has its main application in observational work. As such it can be considered as an alternative for (or a supplement to) the phase microscope. The basic principle of this so-called differential interference contrast (DIC) system as developed by Nomarski and variants of the same principle have in common with the conventional interferometers that measuring and comparison beam systems are made to interfere with each other, the difference being that the comparison beam also passes through the object. The distance between both beam systems is always very small, of the order of 1 fJ.m and is sometimes deliberately chosen below the minimum resolvable distance of the optical combination concerned. Consequently, the rays passing a given point of the object are made to interfere with rays which have traversed the object in its immediate vicinity. From this it clearly follows that the differential interference contrast cannot be used for measuring purposes, as the 'comparison beam' is to be considered as a heterogeneous complex passing through the entire object, instead of being a constant reading of the background. Only local differences in optical thickness come to expression, so that gradients of optical path differences are made visible in the object: hence the name differential interference contrast. The theoretical background of all this is treated by Allen, David and Nomarski (1969) and Rienitz (1969)

The interference pattern with this system is realized by splitting the oncoming rays of illumination into coherent halves with a polarizer, in combination with a so-called Wollaston-prism. The latter consists of two wedges of double-refracting material (calcite or quartz) with an angle of about 90° between their main axes (the Wollaston-prisms as applied here are slightly modified from the original type). Generally, this beam-splitting prism W I is localized just beneath the condenser (fig. 9.11); the reunion of the split beams is achieved with a second similar prism W II, somewhere between objective and eyepiece, in combination with an analyzer. The situation is

EP

o

08


Fig. 9.11. Scheme of the course of interfering rays in differential interference contrast. W I and W II Wollastonprisms, C condenser, OB object, 0 objective, EP eyepiece. An polarizer in front of W I and analyzer behind W II are not shown.

essentially different, therefore, to that in e.g. the Dyson microscope (fig. 9.10) where interference is made to occur with a constant comparison beam outside of the object. As the split beamcomplexes approach the specimen with a clear orientation towards the principal sections of polarizer and W I prism, the rotary position of the specimen can sometimes be of importance with objects having a substructure of parallel units.

The optical principle as explained in the foregoing has been developed into a workable instrumentation in 1955 by Nomarski for a microscope with incident illumination; it has been successfully applied later on for transmitted illumination and is manufactured commercially by different microscope firms since 1968-1970. As no more complicated changes in the light

path are entailed than a special condenser with a Wollaston prism and a second (adjustable) Wollaston prism (W II in fig. 9.11) somewhere behind the objective lens, both in combination with polarizing filters, differential interference contrast can be manufactured as a separate equipment to be adapted, in the same way as phase contrast, to an ordinary microscope. For different reasons, the objectives have to meet certain specifications (e.g. no double-refracting material); with some systems, the second Wollaston prism is mounted in the objective.

As stated earlier, the differential contrast is primarily an optical device for observation, no measurements of optical path differences being possible. It is often stated in booklets from the optical industry, that the Nomarskicontrast and similar systems have an image which is free from halo and at first glance this seems true (fig. 9.12). To a certain degree this is somewhat misleading, however, as is the statement that an interferometric image has no halo (cf. fig. 9.8). With differential interference contrast, the dark areas

188 SPECIAL TECHNIQUES OF IMAGE FORMA TION

in the image which arise by destructive interference are always accompanied by light areas in the immediate vicinity and vice versa, giving rise to certain effects which are physicalIy comparable with a ghost image pattern in the image itself. As is the case with phase and interferometric contast, the total light energy 'substracted' and 'added' in the image plane also remains zero with differential interference contrast. The occurring dark-bright effects give rise to a certain plasticity in the image as a consequence of shadow impressions: e.g. regions with greater optical thickness (showing up dark in such a way that they seem to be elevated from the background on the base ofthe ghost image pattern (fig. 9.12, left). When compared with a matching image with a conventional phase contrast (fig. 9.12, right) it appears thatthe

Fig. 9.12. Superficial cells in an oral mucosal smear, 360 x. Left: differential interference contrast, right: phase contrast (+ 90°,70% absorption).

information in both images is comparable, but that certain fine details in nucleus and cytoplasm come out more sharply and distinctly in the phase contrast image. This only holds true, however, for this very flat and thin object, showing only rather small optical path differences; in many occasions where the object is thicker and/or the optical path differences are greater, the situation can be quite the opposite. In thicker objects, the extreme sensitivity of the phase contrast system can itself destroy certain contrasts by the superimposition of other phase changes generated in parts


Fig. 9.13. Ground section of human cortical bone embedded in methacrylate; 200 x. A conventional microscopy, B differential interference contrast, C phase contrast.

of the object lying over or under the detail of interest, to which distorsion by halo and other effects are added. Fig. 9.13 gives an example of a specimen in which the fine canaliculi of osteocytes in a thick ground section of bone tissue can easily be demonstrated with differential interference contrast, while phase contrast fails so completely, that it even has no advantage over conventional microscopy. The reason for this is partly to be found in the fact that interference contrast makes use of a rather high aperture of the illumination, which is always reduced by the ring-shaped illumination of phase contrast. As a consequence, the depth of field with differential interference contrast is rather small and as this system is moreover less sensitive to extreme small optical path differences, there is less chance that a certain interference effect will be annihilated by a summation of minute other phaseretarding details. In the third place, the difference in which the interfering rays are generated is of importance for the formation of the image.

Apart from its use as a supplementary technique (and alternative) for the phase contrast observation system, the differential contrast can be used to advantage with stained preparations for the following reasons. As, unlike the situation with phase contrast microscopy, no separation in direct and diffracted light is made to occur, contrasts formed on the basis of amplitude changes do not need to be destroyed or changed. Differential interference contrast can be used therefore to enhance the contrasts in feebly stained preparations so that a certain addition of absorption- and interference-effects can be brought about. This can be applied with some more feebly absorbing stains, or old preparations which have bleached somewhat


Fig. 9.14. Cell in cover-glass culture of human fibroblasts stained w.th aniline blue and nuclear fast red; 900 x. Upper image: conventional microscopy; lower image: differential interference contrast.

The (somewhat faded) stain produces a rather low absorption contrast in the cytoplasm, whereas some nuclear details still come out quite clearly. In the differential interference contrast much more detail can be shown, without interfering with the absorption contrast present in the upper image.


with time (fig. 9.14). Under certain conditions, it is possible with similar equipment, by rotating one of the polarizing prisms until gradients in amplitude become compensated, to enforce only those contrasts which arise by local differences in absorption. This has been called 'amplitude contrast microscopy' (cf. David and Williamson, 1971). It has been applied to the study of feebly stained histochemical preparations resulting in a very high resolution in the image.

The plasticity of the image obtained with differential interference contrast may be rather impressive. As explained previously, the suggested heights and depths only represent local differences in the gradient of optical path differences through the cell. Although a certain detail in the object which gives the impression of an elevation may correspond indeed with a real relief-structure such as the cells of fig. 9.12, it is by no means sure (or even probable) that the nuclei would protrude in such a way as is suggested in this image. They have a relatively great phase-retarding effect due to their rather dense structure. It is simply not possible to say in how far these optical path differences are due to a longer path or a higher refractive index or a combination of both. That all this has nothing to do with a stereoscopic image can be shown moreover by the fact that the relief pattern observed with a certain detail can be varied and even reversed by changing the refractive index of the medium, the position of the adjustable Wollaston prism, and even the focus of the microscope.

As long as these limitations are well understood (when this technique was introduced, some descriptions have been published in which this was not taken into account; see Padawer, 1968, for a critical review), a very useful addition to the arsenal of the microscopist remains. It seems that the system has yet not gained the popularity which the phase contrast microscope had obtained shortly after its introduction. The reasons for this -apart from the costs - might be that the setting-up and adjusting in the first commercial versions were somewhat more complicated than with a phase contrast microscope. The newest systems are, however, very simple to operate; the equipment remains rather expensive, however.

It is worth mentioning finally that the image can be disturbed in a way which cannot be predicted when specimens are investigated with birefringent components. This is due, of course, to the fact that the beam-splitting is based entirely on polarization effects; this is again a phenomenon which plays no role in the image formation with phase contrast.

With phase contrast, interferometric contrast and differential interference contrast as more or less generally applied techniques, a new imaging system,


the modulation contrast microscope, is in an experimental stage (Hoffman and Gross, 1975). This system, of which time will show the practical value, makes visible phase gradients, optical gradients and surface slopes by means of a modulator behind the objective, conjugate with an aperture slit in front of the condenser. The resulting image leads to an illusion of threedimensionality, corresponding with gradients in optical density. The modulator does not cause phase changes and only affects light amplitude.

POLARIZATION MICROSCOPY

Basic principles of birefringence According to present ideas of wave optics, a beam of light can be considered as being composed of a succession of innumerable separate wave trains or quanta following each other at intervals of the order of 10-8 sec. Each of these wave packets can be regarded as an oscillation which can be resolved into vectors. Generally, these extremely rapid transverse vibrations take place in all directions perpendicular to the direction of propagation, in such a way that the amplitudes of the components in all directions are equal. In this most commonly occurring situation, the light is said to be unpolarized, whereas polarized light can be defined as light in which limitations have been put to the planes of vibration so that the oscillations are non-random. If all the oscillating components are removed save those vibrating in a single plane (the direction of propagation included), such a beam is said to be linearly polarized. The plane through the beam axis perpendicular to the direction of oscillation is called the plane of polarization. When the tip of the electric vector no longer oscillates back and forth, but remains constant in magnitude, the vector describes a circular motion (clockwise or counter-clockwise). Strictly, this motion is not circular at all, but helical; when viewed end-on, however, this model would show a circular motion of the endpoint of the light vector; this situation is called circular polarization of light. Elliptical polarization stands between linear and circular polarization; the vector can be considered here to oscillate perpendicularly to the propagation direction of the light, the orientation of the vector changing with a continuous angular velocity, so that the endpoint of the vector describes an ellipse (again, strictly an elliptical-helical movement). Linear and circular polarization can be considered as two extremes of elliptical polarization. Elliptical polarization is a very general type of polarization which commonly occurs. It should not be confused with partially polarized light, which is linearly polarized light to which some unpolarized light is added.

POLARIZATION MICROSCOPY 193

In optical rotation or optical activity, the plane of vibration of plane polarized (or elliptically polarized) light is rotated as a function of path length. This phenomenon is widely applied in physical chemistry and crystallography, but is of little importance in biological microscopy.

As previously discussed, the refractive index of a material n is nothing but the quotient of the propagation velocity oflight in a vacuum (c) and that in

the material ( n = ~), so that in glass with a refractive index of 1.515

the propagation velocity would be 30% lower than in a vacuum. As dealt with at the beginning of this chapter, the frequency or number of vibrations per time unit remains constant with this change in the nature of the medium. When the distribution of atoms and molecules is strictly equal in three perpendicular directions, the refractive index will be the same for light beams passing the material in any direction. These materials are called optically isotropic; many anorganic or organic objects such as crystals or natural fibres in animals, however, show to a greater or lesser degree the property that the refractive index is not the same in all directions. Materials showing this phenomenon are called anisotr(Jpic or birefringent. The latter denomination of this phenomenon refers to the fact that light passing such a material is split into two components polarized into mutually perpendicular planes. One component, the ordinary ray or O-ray obeys the laws of refraction in the usual way, whereas the other component, the extraordinary or E-ray has a propagation velocity that is different from that of the ordinary ray and therefore has another refractive index (fig. 9.15B). The situation is even more complicated in that the extraordinary ray has a velocity which varies with the direction of the rays through the birefringent material. With any anisotropic material, at least one direction can be found in which the ordinary and extraordinary ray are propagated with equal velocity. This direction is called the optical axis: it should be noted that this is a direction and not a single line. A section through a birefringent material containing the optical axis is called a principal section. It has appeared that the differences between the refractive index of the extraordinary ray (ne) and that of the ordinary ray (no) vary with the angle between the incoming ray and the optical axis, the maximal value for ne-no, the so-called birefringence, being a property of the anisotropic material concerned. Generally, the maximal deviation between ne and no is reached with incoming rays in a direction perpendicular to the optical axis. It should be noted that the sign of the birefringence can be both positive and negative, as the extra-


EO E

A B c

Fig. 9.15. A a light ray passing through a rhombohedron of isotropic material; B a light ray passing a rhombohedron of anisotropic material (e.g. calcite) is split into an extraordinary ray (E) and an ordinary ray (0); C Nicol prism: the ordinary ray is removed by total reflection at the balsam layer.

ordinary ray may show an increase or a decrease in velocity when it is deviating from the direction of the optical axis. In the case of the strongly birefringent Iceland spar or calcite, which is found as rhombohedric crystals, the refractive index of the ordinary ray is 1.66 and that of the extraordinary ray perpendicular to the optical axis l.49. This birefringence of - 0.17 is an extremely high value; for quartz the positive birefringence is no more than 0.01. In most biological materials these values are much lower and seldom exceed a few thousandths.

The high degree of birefringence of calcite can be applied to produce plane polarized light by removing one of the two beams into which an incoming beam is split. In the prism of Nicol the extraordinary rays are removed by total reflection (and subsequent absorption to the blackened side of the prism) at a boundary between two diagonally cut calcite rhombohedra, which have been cemented together with canada balsam. This cementing material has a refractive index just intermediate between Do and ne, so that at the balsam layer the E-ray is transmitted, whereas the a-ray is reflected and removed by absorption at the blackened side of the prism (fig. 9.15C); this will occur only for a certain angular field of the entering light beam. Other types of polarizing prisms such as the Ahrens- and the Thompsonprism exist which have certain advantages which cannot be dealt with here. They are likewise made of calcite.

When two Nicol prisms are placed in series with their principal sections

POLARIZA TION MICROSCOPY 195

parallel, the beam of polarized light coming from the first prism will pass through the second prism unaffected, apart from light losses due to absorption and reflection. When the principal planes are perpendicular to each other, however, the extraordinary ray from the first prism comes into the vibration plane of the ordinary ray and will be reflected. The result in this situation with so-called crossed prisms is that no light passes the second prism. It should be noted that the amount of light passed by two Nicols in series is therefore a function of the angle S between their principal planes; when this is rotated from 0° to 90°, the amplitude of the light vibrations which are transmitted by the second Nicol is proportional to cos S.

A variety of molecular orientations may give rise to the phenomenon of anisotropy; most liquids and non-crystalline solids are isotropic by virtue of a perfectly random orientation of their chemical bonds. The following types of orientation are generally discerned: a given medium may show only a single type of orientation, or a combination of more than one type.

1. Intrinsic or crystalline birefringence. This is essentially anisotropy resulting from an asymmetrical alignment of chemical bonds. It characterizes many crystals, but is also found in certain biological objects such as collagen fibres, tonofibrils, cellulose fibres and certain parts of myofibrils in muscle. It is independent of the refractive index of the medium in which the object is immersed.

2. Form birefringence. This is caused by the presence of asymmetrical particles (rod lets, platelets) of a given refractive index which have a certain orientation within a medium of different refractive index and of which at least one dimension is smalI in relation to the wavelength of light. This situation can be differentiated from intrinsic birefringence by the fact that the form birefringence is dependent on the refractive index of a medium which is made to penetrate between the oriented asymmetrical particles.

3. Strain birefringence. When a distorting force acts on bonds within a substance, a preferential orientation in the configuration of molecules may occur which leads to anisotropy. A well-known example is the fact that stressed glass shows birefringence, whereas unstressed glass is isotropic; stretched elastic fibres are more or less anisotropic, whereas unstretched elastin is isotropic.

4. Flow birefringence. Certain liquids which are anisotropic under normal conditions may become birefringent when subject to a shearing stress, due to a change in the orientation of solved particles. Good examples are formed by elongated particles suspended in a liquid.


Apart from the difference between ordinary and extraordinary rays with regard to the planes of vibration, some birefringent materials also show different absorptions for the 0- and E-rays, an effect called dichroism. This phenomenon is found with some naturally occurring substances such as the mineral tourmaline. Similarly to the situation with propagation velocity, this phenomenon is variable with the orientation of the object to the incoming light beam. With crystals showing more than one optical axis, the phenomenon may become very complex (pleochromism). In biological objects, outspoken dichroism is not common; it can sometimes be induced in certain tissue components by appropriate staining methods. This induced dichroism should be clearly distinguished from intrinsic dichroism, which is again a property of the molecular configuration in the material concerned.

In analogy to form birefringence of propagation velocity, form dichroism is also known. The phenomenon of dichroism is also applied in obtaining plane polarized light in sheets of polyvinyl alcohol impregnated with a dichroic material. Similarly to the situation with two more or less crossed Nicol prisms, a continuous variation of light intensity can be obtained by rotating the position of two film polarizers with respect to each other. This is applied in different fields of microscopy where the possibility of a continuous variation in light absorbance is desired. such as variable phase contrast devices and drawing prisms.

Finally it should be mentioned in this connection that polarized light can be produced not only by refraction (as in a Nicol prism) or absorption (in a dichroic material), but also by reflection and by scattering. When light is reflected at certain angles from a surface of a refracting medium such as glass, light with a plane of vibration parallel to the plane of the refracting surface tends to be preferentially reflected. This phenomenon can be held responsible for a good deal of the depolarization seen sometimes in the border parts of an object between crossed prisms. Scattering may be of some importance as it is responsible for the visibility between crossed prisms of small, highly absorbing particles such as heavily stained nuclei or grains of carbon.

The polarization microscope Polarization microscopy is essentially the application in microscopy of the technique of polarimetry such as is used in physicaly chemistry for the detection of rotation of azimuth, retardation etc. in birefringent media. Quantitative polarization microscopy is an extremely delicate tool which is used in mineralogy and petrography for the identification of minerals of


which the polarization-optical properties are known in the finest detail. In biology and medicine these possibilities are seldom exploited in full, partly because in biological specimens anisotropy is often less outspoken and of a simpler type than in inorganic specimens; e.g. materials with more than one optical axis are seldom found in biological material, whereas they are commonly met with in inorganic nature. In a similar manner to interference phenomena, polarization effects can also be applied for purely qualitative observational purposes. Both qualitative and quantitative polarization microscopy demand a microscope provided with special equipment, which should, however, be of a considerably more specialized construction if it is to be used for quantitative analysis. This will be explained in some more detail in the next pages.

A polarization microscope in its most simple form consists of a microscope provided with a device - usually situated somewhere under the condenser - for producing an illumination with linearly polarized light, the polarizer, and a second optical device with a similar effect, the analyzer, usually located somewhere in the microscope tube (tube analyzer) or just above the eyepiece (cap analyzer). In a binocular stand, the analyzer should always be placed just above the objective, as reflections in the prism of the binocular head prevent adequate crossing of the polarizer and analyzer. Polarizer and analyzer should both be readily removable and at least one of these devices should be rotatable. In the simple form of a polarization microscope such as is commonly applied in qualitative biological work, this is usually the polarizer whereas the analyzer mostly is inserted with a slot in the tube. In a specially designed polarization microscope, the polarizer is always in a rotatable mount graduated in degrees to enable the reading of rotation angles.

Until about 1950, polarizer and analyzer consisted of a Nicol or an Ahrens or Thompson prism, which are rather large costly, have a limited angular field, and, moreover, displace the emergent ray laterally. Sheet polarizers are in general use now for producing plane polarized light. As discussed before, their effect is based on the presence of dichroic material, absorbing the O-ray and transmitting the E-ray, but unfortunately neither of the effects is complete; the phenomenon of dichroism is somewhat wavelength-dependent, moreover. Thick films ofthis material in its newest version compare favourably with Nicol prisms. They transmit about 40% of the unpolarized incident light when parallel and less than 0.01% when crossed. In the more advanced types of polarization microscopes especially those used for quantitative work, polarizing prisms, however, are still often preferred.

With just a polarizer and analyzer (e.g. using inexpensive pOlaroid filters)

198 SPECIAL TECHNIQUES OF IMAGE FORMA TlON

a simple polarization microscope is obtained from an ordinary microscope and much qualitative work can already be performed with this equipment. When one looks down the tube of a microscope with the polarizer in a fixed position rotating the analyzer (or vice versa), the field becomes maximally bright when the principal planes of polariser and analyzer are parallel. When in this situation of 'parallel prisms' the analyzer is rotated, enlarging the angle e to 90°, the field becomes entirely dark. In this position with crossed polarizer and analyzer ('crossed prisms') no light should come through. When, however, a birefringent object is introduced in the light path between the crossed prism, i.e. in practice between condenser and objective, a variable amount of light passes at the site of this object. The image brightness of the object will vary four times from maximum to zero when the specimen is rotated on the stage. This is typical for any anisotropic object, so long as it is not studied parallel to the optical axis, in which case it will behave as an isotropic object.

This can be explained as follows (fig. 9.16). When the light from the

PO PO PO PO

~ N

I 45°

Fig. 9.16. Appearance of an anisotropic object between crossed polarizer and analyzer (with axes PO and AN) in different rotatory position with regard to the polarizer axis. It has been supposed that each time the object has been rotated clockwise through 45°.

polarizer passes isotropic material in the specimen, it will cause no change in the plane of vibration of the passing light rays, so that they will be totally reflected or absorbed in the analyzer. With anisotropic material, the same will hold true when the light passes in the direction of the optical axis; as explained before, in this situation the material shows no difference in propagation velocity of the extraordinary and the ordinary ray, so that it does not behave as a birefringent material. In all other cases, the polarized band of light arriving at an anisotropic object will be resolved into two components, swinging into the two privileged directions, corresponding to the O-ray and the E-ray. Both components pass the anisotropic object with different velocities; they are combined when leaving the object. No interference will


occur between these generally elliptically polarized beams, as they have different long axes of their vectors. When this light beam reaches the analyzer, however, both components are projected into the plane of vibration of the analyzer. Interference may now occur, as they come to vibrate in a common single plane.

Moreover, when 'white' light is used which has a mixture of vibration frequences, colour effects occur as a consequence of the extinction of light with a particular wavelength, leaving light with the complementary colour for the observer. This effect, sometimes known as chromatic polarization, can yield important data with regard to the nature of the birefringent object which will be dealt with later on.

When the azimuth, defined as the angle between the plane of vibration of the light leaving the polarizer and both (perpendicular) privileged directions in the object, is 45°, maximal light intensity will be found in the image; when the azimuth is 0°, total darkness occurs (fig. 9.16). This variation of polarization effects with the axis of a birefringent object is the cause of different well-known phenomena in polarization microscopy. A classical example is the occurrence of dark polarization crosses (Maltese crosses) when Haversian systems in bone tissue with their helical orientation of concentric layers of birefringent collagenous fibres are studied in transverse section between crossed polarizer and analyzer.

A microscopic specimen containing anisotropic elements which have different orientations will show a varying aspect when it is rotated on the stage, as the azimuth will change from 0° to 45° in all of these elements consecutively. In the anisotropic protein fibers of fig. 9.17, composed of longitudinally orientated tropocollagen molecules running in different directions, but with a main orientation, a totally different aspect can be produced with various rotatory positions.

When a fiber as in fig. 9.17 can be observed at all between crossed polarizer and analyzer, it means that an ordinary and an extraordinary ray have travelled it with different velocities. When emerging from the fiber, both rays are recombined, but one will be retarded with regard to the other. This phase retardation or phase difference of an anisotropic object, generally expressed with the symbol r, is related to the birefringence (ne-no) which has occurred with the azimuth in that particular position and the thickness t, according to the following formula:

r = (ne - no) t.

Usually, r is expressed as a fraction of a wavelength, or it may be given in


Fig. 9.17. Varying aspect of the meshwork of collagenous fibers in a spread-out whole mount of rat mesentery in two azimuth positions differing 65 0 ; 160 x . In the centre three mast cells, slightly stained, enabling an orientation. The minute bright spots are small contaminations.

nanometers. With a fully equiped polarization microscope, it is possible to measure these phase differences with great accuracy.

As stated before, the interference occurring in the plane of vibration of the analyzer causes interference colours to be formed. This phenomenon is most easily observed with light of mixed frequences (,white light'), while the wavelengths of the light passed by the crossed analyzer are related to the r which has occured in the birefringent object, in relation to the wavelength pattern from the light source. This chromatic polarization can be applied therefore to measure the phase retardation which has arisen in the object. When the colours formed are analyzed in those positions that maximal light intensity is observed (in four positions, when the object is rotated over 360°, cf. fig. 9.16), far-reaching conclusions can be drawn about some physical characteristics of the object. Unfortunately, phase retardations in biological specimens usually are too small for phenomena of chromatic polarization to occur.

Interference colours become perceptible to the eye with phase retardations of about 100 nm. Up to about 550 nm, each increase with 20-30 nm brings about a characteristic shift in interference colour. At a r of over 550 nm a new series of interference colours recurs (second order) and at 2 x 550 or 1100 nm a third order repetition can be observed. With increasing order the colours become less varied, as the chances for extinction of more than one colour increase; from the seventh order, 'white of higher order' occurs. All these complicated changes can be analyzed with the colour table of Michel-Levy, as reproduced in most treatises on polarization microscopy. From this, the value for the birefringence (ne-no) can be read for a given colour with an accuracy of about 25 nm, when the thickness of the object

POLARIZA nON MICROSCOPY 201

is known. As ne-no is known for most crystalline materials, this can be used for the identification of a given material in an object. As the interference colours have a periodic character, it is often not possible, however, to say (e.g. when the colour observed is reddish-orange) whether this corresponds with a r of e.g. 500, 1000 or 1500 nm. Moreover, with low degrees of retardation the colour effects can be rather vague and with most of the sections of a thickness of 5-10 [J-m the birefringence is too low to produce interference colours. In the situation of fig. 9.17 not the slightest trace of chromatic polarization could be observed.

Only under special circumstances in biological material, e.g. with the statoconia in the organ of equilibrium consisting of highly birefringent calcite (fig. 9.18), a clear chromatic polarization is found. In all circumstances where interference colours cannot be used or do not yield sufficient information, compensators should be used. Essentially, a compensator is a device to enhance or to reduce the phase retardation brought about by the object, usually brought into the light path with a tube-slide. Depending on the type of compensator the phase change introduced may be fixed or variable. A simple type of compensator often used is a so-called quarter wave plate. This is a mica or quartz plate, introducing into the light path a birefringence of t A (140-150 nm); it will consequently bring the interference colours on a level about 150 nm higher. Another compensator with a fixed effect is the first-order red plate, giving a retardation of 551 nm. Variable compensators are crystal plates of variable thickness (e.g. quartz wedges) which can be moved in the light path, or devices like the Ehringhaus compensator in which a combination of two anisotropic plates can be tilted with respect to the optical axis, the tilting angle corresponding with a certain phase difference. With all these devices the possibilities for analysis and identification can go rather far: apart from the effect on chromatic polarization, other conclusions, which cannot be discussed here, can also be drawn from the effect by the introduction of a known phase retardation. With optimal instrumentation, phase differences as small as a few tenths of a nm can be detected.

Variable compensators can also be used for purely qualitative work, e.g. the finding of an optimal contrast between objects with feeble birefringence and the background or ascertaining the sign (+ or - ) of the birefringence occurring e.g. in a protein fibre such as collagen. With material showing only weak anisotropic properties, the variable degree of strain birefringence in the glass used for making lenses can be disturbing. For more advanced polarization microscopy, special strain-free objectives have to be used. These are mostly of the achromatic type, as some of the optical materials


used in the manufacture of f1uorite- and apochromatic objectives are birefringent. These objectives are not usually of special design; manufacturers simply select ordinary achromatic objectives to find those which happen to have least strain.

As stated before, the most exact quantitative application of polarization microscopy lies outside biology and medicine. Precise determination of optical axes, and identification of crystals on the basis of their birefringence is every-day praxis in petrography and mineralogy. For this analysis, a three-dimensional tilting stage is sometimes used, for determination of the relationship between optical axes. In the field of biology in its broadest sense, where the accent is more often on qualitative work, an adapted ordinary microscope with sheet polarizers as analyzer and polarizer is generally used with just a rotable stage. Only where more feeble birefringence must be demonstrated and with quantitative work do such sophisticated devices as strainfree objectives, prisms instead of sheet polarizers, compensators and an extra powerful light source become a necessity.

The polarization microscope fitted with polarizer, analyzer and the other devices just mentioned, functions like any other microscope. In polarization microscopy of crystals, this is called the orthoscopic use of the microscope. With so-called conoscopy or indirect observation, an auxiliary lens of Amici-Bertrand is used between objective and eyepiece. With this telescopic system, interference patterns in the back focal planes of the objective can be observed e.g. in the analysis of crystalline material. With virtually all biological applications of polarization microscopy, conoscopic observation is not used. Most modern research microscopes have in any case an Amici-Bertrand system in view of phase contrast adjustment, control of the filling of the back lens of the objective etc. It is somewhat exaggerated, therefore, to use the term conoscopic microscope as if it were a special type of microscope.

Some applications of polarization microscopy in biological research When sections are to be used for polarization microscopy, the following general rules should be kept in mind. Sections should never exceed 10 [Lm in thickness in view of the overlapping of birefringence phenomena. When possible, a tissue block should be cut in three perpendicular planes. For the mounting of sections no albumin-glycerin should be used, whereas it should be kept in mind that usual routine methods for the deparaffinizing of sections in xylol are insufficient to remove small remnants of paraffin which can give rise to an extremely disturbing birefringence. The paraffin should, therefore, be removed rigorously by treatment of the sections in methanol-

POLARIZATION MICROSCOPY 203

Fig. 9.18. Statoconia ('hearing stones') in the utriculus of the organ of equilibrium of a mouse embryo; 5 f.l.m section submitted to micro-incineration (heating to 450 0 C) so that all organic material was burned, increasing the birefringent properties of these slightly ovoid structures. Upper image: parallel polarizator and analyzator, lower image: crossed prisms; 120 x.

chloroform at 50 0 C for 24 hours, which also removes remnants of plastic material often mixed with paraffin in modem embedding media. With ordinary paraffin wax, bringing back the sections through alcohol to xylene an extra time usually is sufficient (cf. Goldstein, 1965). Also other contaminations than paraffin show clearly in the polarization microscope (fig. 9.17).

Different fibrous proteins, such as collagen, elastin (under certain circumstances), to!J.ofibrils, and myofibrils in muscles show a clear anisotropy; they all have a positive form-birefringence with respect to their long axis. In the past, these properties have enabled investigators to reach very detailed conclusions regarding the molecular arrangements in these fibers, long before this could be detected by other techniques. Similarly, the peculiar

204 SPECIAL TECHNIQUES OF IMAG E FORMA nON

structure of myelin sheaths around nerves consisting of alternative layers of lipids and proteins, well-known from modern work with electron microscopy, has been predicted quite accurately by early investigators on the basis of analysis with the polarization microscope. It is also possible to show the course of myelinated fibers by using (even partially) crossed prisms on a stained preparation (cf. fig. 9.19).

For a variety of biological material, such as amyloid, qualitative polarization microscopy plays a role in the recognition of this material (Missmahl and Hartwig, 1953). Also in the detection oflipids (e.g. cholesterol), polarization microscopy is often used in histochemistry, as well as in the demonstration of quartz crystals in silicose etc. A review of these applications is given by Scheuner and Hutschenreiter (1972).

As investigation with polarization microscopy does not need to entail any special intervention with the specimen, it is possible to investigate living animal- or vegetable cells with the polarizing microscope. Anisotropic elements are not often found in cells which can be studied in the living state, however. Recently, the (rather feeble) birefringence of the mitotic spindle (due to the orientation of protein molecules in the submicroscopic microtubuli of the spindle) has been applied on a large scale for studying qualitative and quantitative changes in this structure during mitotic division in different cells (cf. Bajer and Mole-Bajer, 1972). Even cine-recordings have been made of changes of the spindle during the division process, using polarizing prisms with a high extinction factor l and a very powerful light source, or even a combination of the luminous flux from two light sources.

In those cases where very feeble anisotropy has to be detected, prisms instead of sheet polarizers have to be used and strain-free objectives; also reflex-poor optical glass surfaces and other devices correcting the loss of contrast due to reflections, as well as the use of special fluorescence free immersion oil become necessary to reduce the amount of background light which obscures faint birefringence phenomena. More easily detectable anisotropy as that of the statoconia of the inner ear which are composed of calcite (fig. 9.18), or cholesterol crystal in the adrenal cortex do not call for more equipment than just a pair of simple sheet-polarizer filters. In some situations, even a total crossing of analyzer and polarizer is not necessary to detect anisotropy. This can be applied sometimes as an easy way to locate birefringent structures in a stained specimen without totally extinguishing

1. The extinction factor is the ratio of the intensity of the light transmitted with a parallel position of analyzer and polarizer to that transmitted with analyzer and polarizer in a crossed position. This factor may vary with different types of equipment between 7000 and over 100.000.

POL ARIZA TION MICROSCOPY 205

A B

Fig. 9.19. Nerve cells in a haematoxylin-phloxin stained section of human medulla oblongata; 180 x. A Conventional microscopy, B photomicrograph made with partially crossed polarizer and analyzer: birefringent myelin sheaths of nerve fibres between the cells become apparent.

the illumination of the isotropic parts of the object, thus enabling a better over-all view of the specimen (fig. 9.19).


R. D. Allen, G. B. David and G. Nomarski: The Zeiss-Nomarski differential interference equipment for transmitted-light microscopy. Z. wiss. Mikr. 69 (1969) 193-221.

A. S. Bajer and J. Mole-Bajer: Spindle dynamics and chromosome movements. Academic Press, New York-London 1972. Int. Rev. of Cytology, suppl. 3.

R. Barer: Phase contrast and interference microscopy in cytology, in: Physical techniques in biological research, vol. III, part A, ed. A. W. Pollister. Academic Press, New YorkLondon 1966.

H. S. Bennett: The microscopical investigation of biological materials with polarized light, in: McClung's Handbook 0/ microscopical technique, 3rd ed., ed. R. McClung Jones. Hoeber, New York 1964.

H. Beyer: Theorie und Praxis der Inter/erenzmikroskopie. Akad. Verlagsgesellschaft Geest & Portig, Leipzig 1973.

J. Chayen: Interference microscopy, in: In vivo techniques in histology, ed. G. H. Bourne. Williams & Wilkins, Baltimore 1967.

G. C. Crossmon: Mounting media for phase microscope specimens. Stain Technol. 24 (1969) 241-247.


G. B. David and Brenda S. Williamson: Amplitude-contrast microscopy in histochemistry. Histochemie 27 (1971) 1-20.

M. Fran~on: Progress in microscopy. Pergamon Press, Oxford-London-New York 1961. D. J. Goldstein: Relation of effective thickness and refractive index to permeability of

tissue components in fixed sections. J. Roy. Micr. Soc. 84 (1965) 43-54. D. J. Goldstein: Detection of dichroism with the microscope. J. Microscopy 89 (1969)

19-36. D. J. Gold~tein and I. J. Hartmann-Goldstein: Accuracy and precision of a scanning and

integrating microinterferometer. J. of Microsc. 102 (1974) 143-164. H. Gundlach and H. H. Heunert: Zur Anwendung der Interferenzkontrast-Mikroskopie

in der Biologie. Microscopica Acta 76 (1975) 305-315. N. H. Hartshorne and A. Stuart: Crystals and the polarizing microscope, 4th ed. Edward

Arnold, London 1970. R. Hoffman and L. Gross: Reflected-light differential-interference microscopy: principles,

use and image interpretation. J. of Microsc. 91 (1970) 149-172. R. Hoffman and L. Gross: The modulation contrast microscope. Nature 254 (1975) 586-588. P. Huber: Zur Untersuchung ungeriirbter histologischer Schnitte mit Phasenkontrast.

Mikroskopie 21 (1966) 1-24. J. James and H. Dessens: Immersion-refractometric observations on the solid concentra

tion of erythrocytes. J. Cell. Compo Physiol. 60 (1962) 235-241. L. C. Martin: Theory of the microscope. Blackie, Glasgow and London 1966. K. Michel: Die Grundlagen der Theorie des Mikroskops. Wiss. Verlagsgesellschaft, Stutt

gart 1964. H. P. Missmahl und M. Hartwig: Polarisations-optische Untersuchungen an der Amyloid

substanz. Virch. Arch. 324 (1953/54) 489-508. R. Miiller: Zur Verbesserung der Phasenkontrast-Mikroskopie durch Verwendung von

Medien optimaler Brechungsindices. Mikroskopie 11 (1956) 36-46. G. Oster: Birefringence and dichroism, in: Physical techniques in biological research, vol. I,

eds. G. Oster and A. W. Pollister. Academic Press, New York-London 1955. J. Padawer: The Nomarski interference microscope; an experimental basis for image inter

pretation. J. Roy. Micr. Soc. 88 (1968) 305-349. J. Rienitz: Der Bildcharakter beim differentiellen Interferenzkontrast. Mikroskopie, 24

(1969) 206-228. K. F. A. Ross: Phase contrast and interference microscopy for cell biologists. Arnold,

London 1967. F. Ruch: Birefringence and dichroism of cells and tissues, in: Physical techniques in biological

research, vol. III, part A, ed. A. W. Pollister, Academic Press, New York-London 1966. G. Scheuner und J. Hutschenreiter: Polarisationsmikroskopie in der Histophysik. Thieme

Verlag, Leipzig 1972. F. H. Smith: A Laser-illuminated Scanning Microinterferometer for determining the dry

mass ofiiving cells. The Microscope 20 (1972) 153-160. M. M. Swann und J. M. Mitchison: Refinements in polarized light microscopy. J. Exp.

Bioi. 27 (1950) 226-237. A. E. J. Vickers: The polarizing microscope in organic chemistry and biology, in: Modern

methods of microscopy, ed. A. E. J. Vickers. Butterworth, London 1956. A. Wilska: Observations with the anoptral microscope. Mikroskopie 9 (1954) 1-80. M. Wolman: Polarized light microscopy as a tool of diagnostic pathology. J. Histochem.

Cytochem.23 (1975) 21-50. F. Zernike: How I discovered phase contrast. Science 121 (1955) 345-349.

CHAPTER 10

RECORDING AND REPRODUCTION OF MICROSCOPIC IMAGES

PHOTOMICROGRAPHY

General principles Photomicrography may be defined as the reproduction of minute objects, magnified by a compound microscope; the result is called a photomicrograph. On the other hand, a microphotograph is a minute photograph of a larger object, in which a compound microscope or possibly a simple lens system may have been used. Microphotography in the latter sense, as used e.g. for document reproduction, falls out of the scope of this book, although a compound microscope in some form may be used for this particular purpose in the inverted direction. There is some confusion, however, as the term microphotography is used incidentally instead of photomicrography. In many European languages, moreover, the correct equivalent of photomicrography is something like Mikrophotographie (German) or microphotographie (French); due to the totally different fields of application this state of affairs seldom leads to confusion.

Photomacrography is again a rather unclear term; it can tentatively be defined in this connection as photography in which the image-forming system consists of only one lens or lens-complex, and the image is larger than the object. As with a photographic camera lens (which can have quite an advanced degree of correction), a certain magnification can be reached, there is some overlap between the domain of photomicrography and photomacrography in the range from 5 X to 20 x. Using one of the modern I x objectives, a direct magnification down to 10 x can be reached with the compound microscope, whereas with so-called macro-lenses (see chapter 3) which can be considered as highly corrected simple microscopes, photomacrography is possible in the same range. These macro-lenses enable the photographic recording of large object fields; even in combination with a wide-field eyepiece, the 1 x microscope objectives have an object field with a diameter of no more than about 10 mm; this is easily surpassed by macrolenses, luminars or whatever these lenses for use without an eyepiece may be called. The problems with these low-power images do not lie so much

208 RECORDING OF MICROSCOPIC I MAGES

with resolving power or chromatic aberration as with homogeneous illumination ofthe object field, curvature offield, etc.

The making of a photomicrograph with any type of objective or microscope always has a purely optical aspect - the projection of a real image onto the photographic material - and a photographic aspect, pertaining to the properties of the light-sensitive emulsion on which the image is projected and its further treatment. This duality has for long been a source of conflict -especially when microscopy and photography are represented by two persons or two different departments - as both aspects, which cannot be clearly separated, playa role in the ultimate result. As a rule such conflict situations arise from a difference in attitude. It often occurs that the microscopist is too optimistic about the possibilities of producing good prints from a negative which is bad in the eyes of the photographer. On the other hand, a photographer with no experience in photomicrography often tries to retain a few basic ideas from general photography, which do not hold true in photomicrography, in which enlarged images are always recorded, often at the border of the resolving power of the imaging lens system.

Between the use of a microscope for visual observation and projection microscopy only a small - but essential - difference exists optically. With visual observation, the virtual image is formed at a certain distance in the direction of the foot of the microscope. When, using the same eyepiece, a real image has to be formed on a screen, viz. the photographic material, the intermediary image should be located past the second focal plane of the eyepiece lens. This can be reached in two ways, as explained with the drawings of fig. 10.1. In the first place, the tube-length can be increased; when the microscope lacks a draw-tube, this can be achieved by pulling out the eyepiece somewhat and fixing it with adhesive tape or by the use of a projection- or photographic eyepiece with an adjustable front lens. The second possibility consists of reducing the image distance of the objective lens with the fine adjustment so that the intermediary image comes to lie within focal distance of the eyepiece (fig. 1O.1C). As all these displacements are very small (apart from very short projection distances) the focussing is mostly made by the most easily performed method i.e. adjustment of the intermediary image although theoretical objections could be raised against this. Consequently, with photomicrography with a simple attachment camera, ordinary eyepieces (which are not designed for projection) are often used. Some of the existing special photo- or projection-eyepieces cannot be used for visual observation, because of a very small eye clearance or even a negative pupil height (chapter 4). It follows clearly from the fore-

PHOTOMICROGRAPHY 209

A

B

c

F,g. 10.1. Ray diagram In projecting an image by means of an eyepiece: A intermediary image in focal plane of eyepiece front lens, B projection on a short distance by enlargement of tube length, C similar effect as with B, but reached by changing the projection-distance.

going that a microscope that has been correctly focussed for observation will not automatically be in focus for projection and that, in changing the projection distance, small adjustments of the fine focussing control are necessary; these fundamental facts are of practical importance.

Photomicrographic equipment Cameras which can be used for photomicrography with a standard microscope can be classified as follows: 1. conventional 2. attachment 3. bellows type 4. photomicroscopic stands (camera microscopes). In the first three categories camera and microscope can be separated; in the fourth microscope and camera are built as a single unit.

210 RECORDING OF MICROSCOPIC IMAGES

The way in which the image is focussed on the light-sensitive emulsion and its control depends on the type of camera used. The same holds for the other main photographic aspect of photomicrography, the determination of the correct exposure time. More specific points with regard to this aspect will be dealt with, therefore, in treating the different types of photomicrographic cameras. The selection of a camera is most often governed by the amount of photography and the demands for the quality of the photomicrographs. It should be kept in mind, however, that the camera in itself is of little importance for the quality of the image, as the image-forming system is in first instance the microscope.

1. The most simple way to make a photomicrograph is by the use of an ordinary camera, fixing it with a vertical stand constructed out of wood or metal (e.g. an enlarger) or with microscope and camera in vertical position where the stand enables this. If the lens cannot be removed from the camera, as will often be the case with most conventional cameras, the following rules should be kept: a. The camera should be put into such a position with regard to the eyepiece that the exit pupil (eye point) is at the front surface of the camera lens; this can be verified with a piece of white paper or smoke (the exit pupil is at the smallest diameter of the outcoming light bundles, see fig. 4.4 on page 65). The penetration of other light than that from the microscope should be avoided with a sleeve. b. Focus the microscope with a relaxed eye, i.e. without accommodating (first throw a glance out of the window); when the camera distance setting control is put at infinity, the image will be in focus on the film plane, when the requirement a) has been fulfilled. c. The aperture setting of the camera lens should be as large as possible (f-number small); with most fixed-focus cameras the aperture behind the lens will reduce the field size. Anyhow, the aperture setting only reduces the illuminated field on the film plane; it exerts no influence on image brightness.

With a single-lens reflex camera, the situation is simpler in that the microscopic image can be focussed with the view-finder of the camera. Often the camera lens can be removed (it does not fulfill any function and only reduces image quality). With some makes special microscope adapters exist which can be screwed into the lens holder. A disadvantage with this system is that the focussing has to be made on the ground glass of the view finder; this rather coarse ground glass can interfere with details of the microscopic image. When a strip or disc of clear glass is present in the center of


the ground glass, the image can be focussed with a hand magnifier1• When the image does not move in relation to a fixed point on the clear section of the ground glass when the eye is moved before the magnifier, it is sharply focussed in the plane of the screen.

2. Attachment cameras differ from conventional cameras in that they have been specially designed for photomicrography. They have no (photographic) objective and can be mounted directly on the microscope, usually by fastening into the tube, sometimes with a more sophisticated intermediate piece. As a rule, they have a focussing telescope with a rectangular frame in conjugate position on the film plane; the viewing prism can be rotated so that the focussed beam falls on the photographic material (fig. 10.2). Use

A B

Fig. 10.2. Schematic view of a type of equipment for photomicrography by means of an attachment camera with focussing eyepiece (A) and with a bellows extension camera with ground glass screen attached to a vertical bar (B).

1. Such a clear area on a ground glass can also be prepared by placing a drop of Canada balsam in the centre of the rough side of the ground glass and covering it with a round cover-glass.


is often made of smaller frame sizes (24 x 36, or 65 X 90 mm). A special form of attaching a camera is with a so-called trinocular tube, in which a binocular tube head is used for visual work and a third upright tube is present into which the image can be projected for photography by tilting the beam-deflecting prism. Visual and photomicrographic optical systems are often parafocally adjusted, i.e. an image focussed on a frame in the eyepiece will come exactly in focus on the emulsion when the beam is deflected towards the upright tube, so that a separate focussing telescope is not always necessary.

3. Bellows extension cameras. In former years, this type of camera was often used in a horizontal position in some sort of optical bench arrangement. Modern microscope stands cannot be brought easily into a horizontal position (in contrast to the hinged stand as shown in fig. 2.5 on page 27). The vertical assembly as depicted in fig. 1O.2B and in which the camera is entirely separated from the microscope, needs a heavy base and a metal bar or rail to which the camera is secured. The image is focussed on a ground glass screen via a mirror located at an angle of 45° which can be removed from the optical path when the exposure is made. Although this type of equipment is rather heavy and may look somewhat primitive when compared with the more sophisticated types of attachment or automatic camera microscopes, it is still widely used for 9 X 12 cm or 4 X 5 inch sheet film. Different advantages exist with this type of equipment: the large format causes minimal loss to occur in resolution and minimal disturbance by small scratches or spots in the negative, the sheet film enables individual treatment of the negatives, whereas the bellows makes a continuous change of magnification possible. On the other hand, the illuminance or lighting intensity which varies with the surface area of the image (cf. chapter 1) will be rather low with such a large format of the negative. As the illuminance determines the exposure time, it is important to realize that the surface area of a 24 X 36 mm frame of a 35 mm roll film and a 90 x 120 negative differ by a factor of over 12.5. A powerful light source is necessary, therefore, with this large format equipment and when a somewhat long exposure time might be of lesser importance in many cases, the use of these large negatives may be impossible for the registration of certain low-intensity images, such as occur in fluorescence microscopy.

4. Photomicroscopes. Specially designed large microscope stands incorporating a camera with automatic exposure control and film movement have come now into general use. Focussing is mostly by a circle, graticule or


frame which can be observed through the microscope eyepiece and which should be brought into sharp focus first before focussing the image proper. By its fully automatic functioning, the making of a photomicrograph can become really a part of the observation, without any special measures to take but pushing a knob for making an exposure. Their simple operation makes them very suitable for all kinds of routine registration and documentation. They are, however, by no means the answer to all kinds of photomicrographic problems. These camera microscopes are mostly limited to 35 mm roll film in which any scratch or spot in the negative always has serious consequences, because of the small image area; moreover, no possibility exists for making (on purpose) double exposures or give an individual treatment to special negatives. The use of 90 x 120 mm sheet film has been undoubtedly greatly reduced by the development of camera microscopes which all are designed for the use of 35 mm roll film, but for special purposes in a non-routine situation the individual exposure of larger format sheet film (or plates) can be essential. Some newly developed large research and camera microscopes enable the alternative use of different formats (fig. 2.8 on page 30) but these large research microscopes are a type on their own. Another new device which has come on the market, is a fully automatic attachment camera with built-in electric transport and other features of a camera microscope.

With all these newer technical developments, it should be kept in mind that - quite apart from the object - the quality of the negative is determined primarily by the properties of the projected image and this again by the adjustment of the illumination and the quality of the lenses of the microscope. When the most simple type of achromatic objective is used, any type of microscope or camera will produce images which suffer from remnants of chromatic and spherical aberration. The types of microscopic objectives and their field of application for visual work and/or photomicrography have been treated in chapter 3.

The photomicrographic exposure Exposure is essentially the light energy administered by the projected image on the sensitive emulsion: it could be defined as lighting intensity on the projection area X exposure time. While rather large deviations from the correct exposure are tolerated with black and white photomicrography, colour materials are rather critical. There is a definite tendency nowadays towards automatic exposure control. Apart from the fully automatic camera microscopes just dealt with, different more or less automatic systems


for use with attachment cameras or conventional cameras adapted for photomicrographic work exist. Exposure meters designed for general photography are usually less suited for exposure measurement in photomicrography. Special types of meters exist for photomicrography; they are often inserted into the microscope as a light-sensitive probe replacing the eyepiece and joined with a wire to a light meter. With fully automatic exposure control also a shutter is opened and closed; most meters give only some value which can be transferred somehow into an exposure time, e.g. with a table or a calculating factor. When the set-up of the microscope is fixed, it is not at all necessary to measure the lighting intensity directly at the film plane; by precise calibration it is possible to predict the exposure time from a reading elsewhere of the same image (e.g. from the position of an eyepiece or a beam-splitter).

It would be useless to sum up the different types of exposure meters applied in photomicrography. In principle they have a built-in photocell of the classic type or - with some of the modern types of camera microscopes -an electronic photomultiplier as detector. These photomultiplier systems have a very high sensitivity and a broad range of exposure determination; this enables automatic exposure control, e.g. with very low image brightness, where ordinary photocells fail completely. It is important, however, to realize that an automatic exposure control does not ensure a correctly exposed negative in all situations, even with faultless materials and equipment. Apart from special circumstances (dark-field, fluorescence) in which the image proper is formed by the most illuminated parts of the image for which most exposure control systems have not been designed, the following factors have to be taken into account when using automatic or semiautomatic exposure control.

1. Without exception, the luminous energy admitted to the light-sensitive material is regulated by the time in which light is allowed to fall on the material, i.e. the shutter time; a lens diaphragm as applied with a conventional camera can of course not be used. As the shutter time will have a certain minimum period, e.g. 1/50 sec, it is very well possible that with low-power magnification and an intense light source even this minimal period is too long, so that an overexposed negative is obtained in spite of the fully automatic control. Certain systems have a safety device (such as a warning light) for this common situation, but even then it is of no use to aim at short exposure times in photomicrography of non-moving objects. The lighting intensity of the image can best be adapted in such a way (by enlarging the resistance on the lamp tension, or using a neutral density


filter) that the exposure time is of the order of a few seconds. The duration of the exposure is mostly audible as a click for opening and shutting and sometimes can be seen from a control lamp. As stated before, very sensitive photomultiplicators and other electronic light sensors have come into use for automatic exposure control, which enable very short exposure times (down to 1/1000 sec), but apart from moving or quickly changing objects there is no point in using such exposures under reasonable conditions of stability of the apparatus.

2. When a part of the object with a certain detail which is the real subject of the photomicrograph, e.g. a few isolated cells, forms only a small fraction of the entire field, most automatic systems tend to chose such an exposure that the background comes on the negative in a medium tone. Certain darker details may get lost in this way, because there is strictly an underexposure of the negative over a particular important fraction of the field. In some cases this can be overcome, as it is possible to regulate the exposure time also on the basis of the absorption measured in a small field which can be seen in focussing the image and on which the detail in question may be placed. This is called spot measurement, as opposed to integral measurement over the whole image area. When only integral exposure determination is possible, the only remedy in the situation with isolated cells just mentioned is to set the control of the film speed to a lower value.

3. Reciprocity failure (also called Schwarzschild effect). When exposure times have to be used which are extremely long due to low lighting intensity of the image, as compared with calibration exposures made previously with an automatic exposure control, it seems that the film speed becomes lower than it was. This phenomenon is named after the German astronomer Schwarzschild, who discovered it in making photometric observations on stars with low radiant intensities. It is difficult to give exact rules, as also the properties of the photographic material and its processing playa role, but when the lighting intensity on the film is e.g. 50 times lower than with a control exposure which proved to be correct, the optimal exposure time will not be 50 times as long, but something like a hundred times; with still lower intensity of image brightness, the difference may become more than a factor of two. This effect is principally of importance with colour photomicrography (see page 229), but is met with occasionally in black and white photography of feebly illuminated images. An automatic exposure control generally does not take this effect sufficiently into account, although sometimes some compensation mechanism exists. The remedy is rather obvious:


for extremely long exposure times, which cannot be avoided (e.g. by use of another light source) the film speed selector should be set for a slower film than that actually used.

Apart from the three situations dealt with in the foregoing, a number of other occasions exist in which an automatic exposure control is obviously an important aid in facilitating the finding of a correct exposure time, but often some experimentation remains necessary. When no automatic control or exposure meter is present or the use of such a device is limited, as in the case of dark-field or fluorescence microscopy, a correct exposure time has to be found on the basis of trial exposures, e.g. increasing with a factor of two each time from an exposure time which is certainly too short. This must be done by exposing a series of negatives with roll film or, in the case of sheet film in a cassette, by pushing the slide of the film holder for about two centimeters each time, so that the successive steps will have received exposures according to an arithmetic progression (2, 4, 6, 8, 10 etc. times the initial unit). Care should be taken to change nothing in the microscope setting or the illumination when taking such a series of exposures. With black and white photography a relatively large exposure latitude exists; it is useless, therefore, to make series of e.g. I, 2, 3 and 4 seconds, differing by less than a factor 2.

As the exposure time varies in inverse proportion to the lighting intensity (subjectively experienced as 'brightness'), any factor influencing this wiII change the exposure time; a weB-known pitfall in practice is when a correct exposure time has been measured with an exposure meter, while the microscopist finally somewhat re-adjusts the condenser diaphragm just before the exposure is made, which leads to an incorrect exposure of the emulsion. When the optical conditions from one microscope setting to another change more drastically, e.g. a change in objective or eyepiece, the illumination control remaining at the same level, the new exposure time can be estimated from the calculated change in lighting intensity. As the latter factor is proportional to the quotient of the squares of effective numerical aperture and total magnification, the second exposure time (ET II) follows from the first one (ET I) by the formula:

ET II = ( N.A.I )2 X (VII)2 ET I N.A.II VI

in which N.A.I and N.A.u and VI and VII are the effective numerical aperture and magnification of the first and the second exposure, respectively. The exposure time calculated in this way cannot be more than an approximative


one, as e.g. any change in the condenser aperture will influence the effectively used aperture of the new objective. As discussed in chapters 3-6, the N.A. of an objective as mentioned on the mounting is seldom fully exploited.

With a more or less primitive set-up without automatic shutter, a very practical technique is to use a piece of cardboard in the light path to produce an exposure time of e.g. a few seconds with an opened camera which has been focussed before. The shutter of the camera can be used to close the camera· after the exposure has been made (with the cardboard in the light path). This very simple method has the great advantage that vibration in the camera set-up which can be the cause of focus drift (often a problem with makeshift situations), can be avoided as much as possible.

Finally, a few words about the magnification factor in photomicrographic negatives. As a projection image is made of the negative, the size of which will depend on the distance from the exit pupil to the projection surface, it is clear that the magnification factor cannot simply be the product of objective and eyepiece magnification, as with direct observation. The distance in mm from the exit pupil of the microscope to the emulsion is sometimes called the camera length, K. It should be noted that when the exit pupil does not fall outside the front lens of the eyepiece or projective (negative eyepoint height, see chapter 4) this length cannot be measured easily. The total magnification at the film plane V m can be calculated from the formula :

K Vm = Vo X Ve x-

250

in which Vo and Ve are the magnification factor of objective and eyepiece, respectively. This means that only with a camera length of 250 mm is the magnification exactly that of the product of objective and eyepiece magnification, if no other factors change the course of the light rays. In most attachment cameras and photomicroscopes the projection distance is fixed, but with the bellows type of camera (fig. 10.2B) this factor can be varied infinitely within certain limits. In most cases, the precise value of the ultimate magnification of the negative will be of limited value as compared with the area of the image it contains. As a general rule, the only real limitation is that the final magnification in the print when studied at a reading distance of 250 mm should not exceed 750-1000 times the effectively used objective aperture, as discussed at length in chapter 5.

In some cases it may be necessary to know the exact final magnification on a print or a negative. As some uncertainties always exist with regard to


exact camera length and/or magnification of the total optical set-up between specimen and film plane, the only way of knowing the magnification exactly is by measuring it. This can be most easily accomplished by using an object micrometer (see chapter 11), photographed or focussed on the viewing screen (if the magnification is not modified here with regard to the film plane) and measured with a millimeter ruler. For a bellows camera, a chart for magnification change can be prepared at different extensions of the bellows.

Contrasts in the negative Emulsions used in photomicrography for obtaining black-and-white negatives consist essentially of silver halogen ide crystals suspended in gelatin on a support of cellulose film; glass plates as support have how virtually disappeared. Such an emulsion is not uniformily sensitive to light of all wavelengths, there is a certain colour sensitivity which is characteristic for the type of film. Apart from the colour sensitivity, there is a complex of mutually more or less interdependent properties of a film which are generally referred to under the main headings of grain size and resolution, contrast and speed; these properties will be dealt with briefly further on in this section.

The photographic process in itself in photomicrography is of course no different to that in general photography and use is made of the same emulsions, albeit mostly of a special type in photomicrography. The whole of the photographic process in connection with microscopy is dealt with extensively in some specialized monographs on this subject (Michel, 1962; Brain, 1969; Gander, 1974). A very brief outline of the factors which determine the characteristics of a negative emulsion, as used in photomicrography is, however, necessary to form a general background for facts and problems with regard to contrasts in the negative.

The properties of the films which are commercially available and can be used for photomicrography vary according to different criteria. The colour sensitivity of a film is definitively fixed with the so-called sensitation-process in the manufacture of a usable film from an untreated silver-salt emulsion. The other properties which determine the character of a film emulsion, grain size and resolution, contrast and speed, can be altered to a certain extent by processing, in which they will alter in relation to each other.

Colour sensitivity. Films used for black-and-white photomicrography may be classified as blue-sensitive, orthochromatic, panchromatic and, at the other extreme of the scale, infrared sensitive. Blue-sensitive films cannot


record absorptions in the green and red regions; they can be used for photomicrography with blue-violet and proximal ultraviolet light. Orthochromatic emulsions have extended sensitivity in the green, insufficiently rendering the red, however. Panchromatic emulsions are sensitive for all colours except deep red, and a special type of panchromatic emulsion exists which is highly red-sensitive. Although the materials of different commercially available emulsions may vary greatly, it can be stated in general that the sensitivity of a panchromatic emulsion still differs quite substantially from that of the human eye (fig. 10.3). Infrared emulsions are used ex-

100

50

25

400

BLUE GREEN YELLOW RED

/ / --,,"

I /

/

500

I

I I

I

600 700nm

Fig. 10.3. Spectral sensitivity curve of a panchromatic emulsion, plotted in percents of the maximum on the vertical axis, as compared with the human eye (dotted line).

elusively with infrared photomicrography, as will be dealt with briefly in chapter 12.

Grain size and resolution. Although there is some relation between resolution and grain size, it is a common misconception that these two are always directly related. Even a film with a rather coarse grain may be capable of comparatively high resolution, but a film with a fine grain always gives a better reproduction of the image. Graininess in itself is not so much related to the actual size of the silver halide partieles (varying between a few tenths of a fJ.m and over a fJ.m) but mainly to the regularity of their distribution in the emulsion layer, the type of developer used and some other factors of lesser importance. In general, films with a fine grain are rather slow; as it is generally of no advantage to have very short exposure times in photomicrography of non-moving objects, fine grain films are usually applied. The basis of everything, however, is the quality of the microscopic image. I t should be noted that grain size is not a fixed property of the film; both exposure (degree of over-exposure) and processing (developer, developing time) can influence the grain size in the ultimate negative.


Contrast as a property of the film material (N.B. not to be confused with contrasts in the image). When a photographic emulsion is irradiated with light of a wavelength for which it is sensitive, a certain relation between the exposure (light flux x exposure time) and the amount of metallic silver in the developed negative can of course be expected. A measure for this amount of silver is the density, which is defined as the logarithm to base 10 of the reciprocal of the fraction of the incident light transmitted (cf. chapter 11). When this density D (as measured with a densitometer) is plotted against the logarithm of the exposure, it appears that this relation is not a simple linear one (fig. 10.4). Only in the part p - q does a linear relationship

D 4

3

2

-2

p

-1 o

q

0<

2 3 4 LOG EXP

Fig. 10.4. Density D in the image of a negative, as plotted against the logarithm of the exposure; the angle ex.

exist; after this, a further increase in exposure even leads to a decrease in blackening, a phenomenon (virtually absent with modern emulsions) caIled solarization. The linear part p-q has a definite slope which indicates the so-called gradation or contrast of the film; more precisely, it is expressed as tan oc (fig. 10.4) and called the gamma of the emulsion. When oc = 45°, the gamma is 1; when oc > 45°, a smaIl change in exposure will lead quickly to significant changes in blackening; the contrast is high and the film is called hard; when oc < 45° there is much more tolerance in reproducing variations in exposure, but when the object has very weak contrasts it will be reproduced rather poorly with this soft film. It will be clear from fig. 10.4 that the projection of p-q on the horizontal axis will give some indication of the exposure latitude of the film, and this factor is thus clearly connected with film contrast.


As the absorption differences between the components of most microscopic objects met in biology and medicine are seldom high, the contrast of the combination film/developer should certainly not be too low. Extremely high contrast is found with the type of film used in document reproduction but this is mostly too hard an emulsion for use in photomicrography.

Speed. Although speed is certainly not unconnected with the other variable factors of an emulsion dealt with so far, it should be considered briefly under a separate heading. In many instances the speed of a film is not of primary importance with non-moving objects; apart from situations involving quickly bleaching specimens or extremely feeble image brightness (fluorescence microscopy) there is no need to aim at the shortest exposure time possible, as mentioned before.

Speed, as measured in a branch of photographic technology called sensitometry, is expressed in Europe with the so-called DIN (Deutsche Industrie Norm) number, whereas the ASA (American Standards Association) system is used also outside of the U.S.A. The DIN value increases with three points at each doubling of speed, whereas the ASA number doubles in geometric progression. Some corresponding values of both numbers (with doubling of the relative speed each time) are given below:

DIN 9 12 15 18 21 24 27 30 33 36

ASA 6 12 25 50 100 200 400 800 1600 3200

As stated earlier, the variable characteristics of a film are connected with each other, and can be influenced to a certain extent by variation in the processing. Generally, it can be stated that the finer the grain, the steeper the gradation and the slower the speed. In reducing the development time, the angle IX. from fig. 10.4 will become smaller, so that the film contrasts will be lowered; the other factors will change then accordingly. The possibilities of making good eventual shortcomings in exposure and/or choice of the type of film by this means are, however, very limited. Generally films with fine grain, comparatively high resolution (up to 120 lines/mm) and moderate contrast are selected for photomicrography; in all other aspects they are not different from films used in ordinary photography.

As stated before, a photomicrograph can of course never reveal details not present in the original image. In order to obtain as good a negative as possible, both sharpness and contrast of the image should be optimal.


Sharpness of an image is, under given optical conditions, merely a question of correct focussing of the image on the film plane. Although this can be made in widely varying ways, depending on the type of equipment, a few general remarks in this respect should be made here. With many lowpower objectives, the focus is just correct, or not. As with high-power objectives the depth of field is often only a fraction of the specimen thickness (see chapter 5), focussing is no more a purely optical question~ the observer wanting to make a photomicrograph should make a choice here. If the investigator leaves the actual making of the photomicrograph to another, photographer or not, it is unfair to reproach him or her afterwards that the photograph is 'not right', unless the operator who does the focussing has been properly instructed. This is a very common (and totally unnecessary) ground of conflict. Often it is advisable to use a ground glass screen for focussing as depicted with the apparatus offig. 10.2, thus enabling discussion between two persons about the desired plane of focussing. It is much more difficult for two persons with the focussing telescope of an attachment camera to agree on .a certain image. Focussing with such devices is mostly made with a rectangular frame (the prospective image field), sometimes provided with fine double diagonal lines which should be seen as separate entities. In order to have as little accommodation as possible, it is advisable to focus the frame after having thrown a glance out of the window; the lines should be immediately sharp on looking down the telescope (or the eyepiece with frame in a photomicroscope). When bringing the image plane to that of the frame lines, these lines should not be fixed with the eyes; with a microscopic image the tendency to accommodate has appeared to be much less than with these thin lines. Any discussion about the plane of focus of the image can be held only between observers both seeing the frame lines sharply. Even small variations in vision will lead to sensible differences in the fixing of the image plane, especially when making highpower photomicrographs.

Quite another common problem in focussing with any equipment, including a ground glass screen, is the fact that with certain light filters, the plane of focus of the image may undergo a minimal change. It is advisable, therefore, to do the final focussing of a high-power image with the filter to be used for the photomicrograph in the light beam. With a 9 x 12 cm frame ground glass screen this may, however, again give rise to difficulties, when the filter takes away much light.

Contrast in the image, i.e. the standing out of a certain part of the object against its surroundings, is often confused in practice with sharpness or


focus. Under certain conditions, a lowering of the contrast may give a certain impression of unsharpness, the image being somewhat out of focus. Fig. 10.5 shows two photomicrographs, of which the image B would be called 'sharper' than A on first impression. In fact they have been focussed in the same way using the same optical equipment, except that the contrast is greater in fig. 10.5B, due to the use of a filter. In this particular pair of

Fig. 10.5. Effect of a contrast filter; photomicrograph of spread chromosomes of a metaphase of human fibroblasts stained with the Feulgen-technique. A exposure taken with unfiltered light from a low voltage lamp, B exposure taken with the same focus level of the microscope but with a narrow-band interference filter (). max. 546 nm) in front of the light source.

images, the greater contrast of B with regard to A could be considered as an advantage for studying e.g. size and form of these chromosomes; generally, however, the optimum for the contrast in a photomicrographic image is dependent on the purpose of the image. Contrast in a microscopic image should therefore certainly not always be as high as possible: a too high contrast level may lead to the disappearance of gradations within that object. On the other hand, the sharpness is generally only optimal in a photomicrograph when it has reached its highest value. Sharpness is used here in its sense of 'in focus'; unfortunately, the term sharpness bears also some relation to contrast, so that it is not in all respects a clear term.


The technical operations for enhancing contrasts in the object as described in chapter 6 and 7, such as adjusting the aperture diaphragm of the condenser and other measures for the control of glare, are at least as important in photomicrography as they are in visual microscopical observation. In making black-and-white photomicrographs, still another very important means exists for influencing contrast which is applied here on a larger scale than in visual observation, the use of contrast filters. These light filters can be subdivided, on the basis of the way they function, into absorption filters in which light of certain wavelength regions is removed by absorption and interference filters, in which light of a limited part of the spectrum is selectively transmitted by semitransparent layers which reflect light of other wavelengths. The part of the spectrum separated from the light from the source can be controlled more precisely with interference filters than with absorption filters. Generally, the transmission characteristics of a filter can best be given in the form of a curve plotted over the spectrum. The bandwidth of a filter is defined as the distance between the two slopes of the curve at exactly half of the maximal transmission, the transmission as the percentage of the light passed at the maximally transmitted wavelength. With a typical interference filter the bandwidth will be of the order of 10-15 nm with a peak-transmission of around 40%; socalled precision line-interference filters have a bandwidth of about 5 nm with a transmission of no more than around 10% at the peak. The latter type of filter has too narrow a transmission range as a rule for use as a contrast filter; these types of filter are used e.g. in microphotometry (see next section) or fluorescence microscopy. In the present situation, only glass filters are used in photomicrography; liquidfilters, e~sentially consisting of troughs of suitable solutions of certain chemicals absorbing light of a given wavelength range, are rather cumbersome to handle and they have practically disappeared now, although they theoretically permit infinite variation. Gelatin absorption filters consisting of a dyed gelatin film sandwiched in between glass or plastic are still occassionally used; they are not heat-resistent, however.

In selecting a filter which transmits preferentially light which is specifically absorbed by one of the dyes bound in the specimen, a stronger blackening will occur in the positive of the photographic print in the regions where this dye is localized. The effect will be maximal when the filter has such a narrow width of its transmission curve that only light falling within the absorption curve of the dye is passed by the filter. With such a high contrast all nuances in concentration of dye will get lost; on the other hand, an object illuminated preferentially with light of the same colour as its own, will have a low


over-all contrast but reveal much more shades of its colour. The use of contrast filters is essentially a question of complementary colours. A simple aid in this is a colour circle as shown in fig. 10.6, in which complementary colours are arranged opposite each other.

Fig. 10.6. Simplified colour circle.

When a specimen is stained with two dyes, it is e.g. possible to emphasize the absorption of one of the dyes by chosing a filter having a colour with a position more or less opposite to that of the dye concerned. When the film type and other factors remain constant, the grey-values in the negative can be adjusted in this way at will.

Generally, it may be stated that filters and dyes are characterized only partially by their colour, i.e. their hue in transmitted light; a more accurate description can be given with their transmission curves. To a certain extent, the colour impression is a subjective one, in which factors like the saturation (purity) and lightness of a colour, as well as the spectral characteristics of the light in which they are observed playa role. A general impression of the distribution of the transmission hue of the most important colours over the spectrum is given in table X. These values are no more than an orientation, but they enable to place e.g. a yellow dyestuff somewhere in the spectrum, or to say roughly which colour is to be expected from a filter with a transmission top of 546 nm and a bandwidth of 20 nm.

The transmission curves of most contrast filters in use are provided by the manufacturers; they are limited to three or four main types. For special purposes, a continuous running interference filter may be used, enabling the selection of a pencil of light from a desired region of the visible spectrum with a relatively narrow bandwidth (10-20 nm with a not too large slit).


TABLE X. WAVELENGTHS AND COLOURS (transmitted hue).

Violet 380-450nm Blue 450-500 Blue-green 500-520 Green 520-550 Green-yellow 550-570 Yellow 570-600 Orange 600-630 Red 630-780

This device, sometimes called an interference filter monochromator, is a longitudinally banded filter which can be moved in a sledge across the light path in front of the light source or in a special diaphragm assembly on a lamp-house, or on the foot-plate of a microscope with built-in illumination. This longitudinal filter is made in such a way that a linear relation exists between the wavelength transmitted and the position on the sledge, e.g. 6 mm shift of the filter corresponding with about 10 nm shift in the maximum. When a broad band of light is necessary to fill the object field with light with low magnifications, differences in colour may become obvious in the field of view, however, when this device is used. This variable filter can be useful when rather uncommon stains are applied or stains absorbing near the extremes of the visible spectrum. It should be kept in mind, however, that most ordinary objectives have not been designed for the latter situation so that apochromatic lenses should be used. True monochromators, in which light from the desired wavelength is selected from a source with a continuous spectrum by means of a prism (see chapter 11) are seldom used in photomicrography. The absorption characteristics of most stains are known from the dye industry and can be found in books like Lillie (1969), Harms (1965) and others. It should be noted, however, that many of the dyes used in animal or botanical microtechnique do not have the same absorption curve in a solution as when bound in situ to certain parts of a specimen. Only a microspectrophotometrically made curve (see chapter 11) in a small area of a microscopic specimen gives an impression of the real absorption characteristics of such a stain. In every-day practice the data given in fig. 10.6 and table X will do for a sensible choice of a filter; only in special cases will it be necessary to fall back on the objective physical properties of stains and filters.


Between the formation of the latent image, and the real negative image established after development, an interplay takes place between the characteristics of the emulsion and the spectral composition of the light from the light source, as modulated by the stained specimen. Even with panchromatic emulsions which are sensitive to all colours of the visible spectrum, the sensitivity curve has clearly another distribution than that of the human eye (fig. 10.3). Consequently, when looking at an image before taking a photomicrograph the importance of violet and blue components in the latent image is often underestimated, whereas the eye is slightly more sensitive in the red part of the spectrum. An extreme example of the consequences of this has been encountered in fig. 8.9 on page 159 in which an image was totally dominated by short wavelength excitation light, which impressed only as a vague blue hue when looking down the eyepiece. On the other hand, objects which transmit much light of longer wavelengths sometimes call for a greater exposure than one would estimate visually. Orthochromatic emulsions should of course never be used for photographing objects with many red components. They have some advantages, such as a steep gradation; they are often used with unstained material, e.g. in metallurgy.

It is not possible and not necessary to balance the spectral characteristics of light source, object, contrast filter and photographic material minutely in all cases of black-and-white photomicrography. The following basic rules can be given which hold true for most routine cases; they are illustrated by fig. 10.7. 1. An incandescent lamp emits (even with overtension) mainly red and

infrared radiation, to which the average film material is relatively insensitive. An unstained or stained specimen photographed without any filter, will therefore show up in the negative as if it were photographed with a spectrally perfectly homogeneous light source with a broad-band red filter. Unless major parts of the object transmit mainly light in the blue and green region, the contrast will always be on the low side.

2. Although most stains in use in microtechnique will differ widely in their absorption characteristics, they generally show quite a deal of absorption in the middle region of the spectrum. In using a yellow-green interference filter with a broad transmission curve, very good intermediate contrasts can be obtained in the negative (fig. iO.7C). Not only are both the film and the eye then about equally sensitive, but achromatic objectives also give their best performance with light from this middle region of the spectrum. This does not mean, by the way, that achromatic

A

B

c

o


objectives are always suited for black-and-white photography (see chapter 3).

3. For giving a higher contrast with stains absorbing in the more extreme parts of the spectrum, it is to be recommended to have a blue and a redorange filter at one's disposal (fig. 1O.7B and D). With these three (including the yellow-green filter already mentioned) contrast filters, in combination with a neutral density filter to temper eventual image brightness, virtually all routine circumstances in black-and-white photomicrography can be handled. It needs quite a deal of experience, however, before satisfactory results can be expected routinely with a widely varying series of objects and magnifications. Buying expensive equipment can never compensate for shortcomings in this respect.

Colour photomicrography Contrary to the situation with monochrome photography, colour exposures are mostly made on reversal film, which yields a transparent positive after developing. Negative films which can be printed into ordinary positives or material yielding directly a positive print (polaroid) are less often used. This preference is no longer primarily for economical reasons, because the price of a printed colour negative is no more as high as it used to be, but it is difficult to get really well-balanced prints from a commercial laboratory; the making of colour paper prints is too complicated an affair to perform in a photographic department of medium size. The use of reversal film, however, is easier in that with well-balanced exposures a satisfactory result may be expected from a commercial laboratory. On the other hand, virtually any experienced microscopist has come to know that colour slides are

Fig. 10.7. Section of the epithelium of the human trachea, stained by the Masson-Goldner technique; photomicrograph on panchromatic emulsion, magnification 400 x . This staining is composed of three dyestuffs, one of which shows a rather diffuse absorption throughout the visible spectrum (Weigert-haematoxylin), whereas the other two com,:onents, lightgreen and orange-G, have a maximal absorption near 630 nm (in the red) and in the blue region between 450 and 500 nm. A Exposure made without filter, which comes down to an illumination with more red light: a rather high contrast exists in the green stained basement membrane beneath the epithelium. B Photomicrograph made with a contrast filter with maximal transmittance near 480 nm (blue): very high contrasts exist in the epithelial cells with their cilia, stained mainly by orange-G; the basement membrane has been filtered out. C Exposure with a broad-band interference filter with maximal transmittance at 550 nm; well-balanced contrasts both in the epithelium and in the connective tissue beneath. D Exposure made with a red interference contrast filter (maximal transmittance at 630 nm); maximal absorption in basement membrane and fiber pattern in the connective tissue, virtual absence of contrast formation in the epithelium in which only the cell nuclei, as stained with Weigert-haematoxylin, can be discerned.


fine for demonstrations and lectures, whereas black-and-white prints have to be used for documentation and publication. In the publishing world printed colour photomicrographs of high quality are still very expensive; in many cases a first-class monochrome print with well-balanced grey tones will be much better than a second-rate colour print. For some special purposes when colour seems absolutely necessary, two separate black-andwhite prints, taken with different contrast filters, will demonstrate quite a deal.

Different extra problems arise in making a colour exposure in comparison with monochrome photomicrography. In the first place, this concerns the optics of the microscope. In the previous section, it has been explained that the chromatic aberration and spherical aberration of an achromatic objective can be held at a minimum by using a yellow-green filter (see also chapter 3). As this is impossible in colour photography, use has consequently to be made of fiuorite- or apochromatic objectives with compensation eyepieces. In many cases a condenser of a somewhat better correction grade is desirable, in combination with a Kohler-illumination.

The photographic-technical aspects of the colour film and the way in which colours are reproduced by an interplay of the three layers which are sensitive for light of different wavelength cannot be dealt with here (cf. Habermalz, 1975). A few specific problems which are directly related to the forming of the latent image in the multi-layered emulsion are of great practical importance, however. The impossibility of using contrast filters has been mentioned already in passing; in colour photomicrography the contrasts can be made to arise only by 'natural' differences in light absorption in the specimen; the spectral composition of the illuminating beam is therefore of great importance. A second important difference between monochrome and colour photomicrography exists in the fact that the exposure latitude is much smaller in colour work, and without an accurate exposure meter virtually no results can be expected. In the third place, the reciprocity failure (Schwarzschild effect) plays a much more dominating role in exposure control than with monochrome photomicrography. The situation is even more complex as this phenomenon has different effects in the three layers of the emulsion, entailing a shift in colour balance which cannot be counteracted simply by adjustment in exposure time only (Jenny, 1970). As the Schwarzschild effect as a whole is difficult to calculate exactly (a rough estimate is to double the exposure time with a lO-fold increase over a trial exposure and to take it fourfold with a lOO-fold increase) it is often advisable to avoid very long exposure times as much as possible by using a powerful light source and/or a faster film.


The spectral distribution of the light given off by a source with a continuous spectrum (see chapter 6), e.g. an incandescent lamp, depends on the temperature of the glowing filament. When this temperature increases, the maximum of the light emitted will shift to the blue side of the spectrum, whereas with a decrease in temperature of the filament, the maximum will shift into the red and infrared. As a measure for this, the notion colour temperature has been introduced, which is defined as the temperature in degrees Kelvinl to which a completely black body should be heated to give off light of exactly the same spectral distribution as the light source which has to be characterized. In practice it appears that the temperature of the lamp-filament and colour temperature are about the same, although the filament is not a black body. Colour temperature provides a complete description of an illumination from this point of view and makes possible the comparison of different light sources with continuous spectrum. In the case oflight sources with a band spectrum, such as the high pressure mercury burner (see fig. 6.10 on page 110) it is not possible to give a description of the spectral properties with a precise value for colour temperature. This type of illumination cannot be used for general colour photomicrography anyway, as certain parts of the visible spectrum are not represented at all.

Daylight, originating from the sun, has a high colour temperature (5500-6000° K) and therefore contains much more violet and blue components than a low-voltage bulb (e.g. 12V, 50W) at full tension which has a value of about 3000° K. It is illustrative to note that in the latter case no more than about 10% of the radiation given off falls within the visible spectrum; the rest consists of (useless) infrared warmth radiation (fig. 10.8). When the lamp voltage is lowered, the colour temperature will be reduced so that a still smaller portion of the radiation emitted falls within the visible range.

It is clear that the sensitization of a colour film should be matched somehow to the colour temperature of the light source. Two main types of emulsions can be distinguished, a tungsten film balanced for a colour temperature around 3000° K, and the other type, 'daylight' film, which is indicated for use with a light source having a colour temperature of around 5500° K (xenon burner, flash and fluoresecence work). It is clear from the foregoing that a daylight-film, used with a low-voltage lamp will yield positives with a red cast, whereas in the reverse situation the image will be

1. Degrees Kelvin or degrees on the absolute temperature scale = temperature in degrees centigrade + 273.16°. Apart from the Kelvin scale, another unit for colour temperature has been introduced, the Mired (Micro Reciprocal Degrees) which has certain advantages. 200 degrees Mired correspond with 5000° K, 300 with 3300° K, 400 with 2500° K and 500 with 2000° K.


Radiant energy In arbitrary units

120

100

80

60

4000° K

40

20

100 300 400 nm Wavelength

Fig. 10.B. Spectral distribution of radiant energy emitted by a black body heated to different temperatures.

too 'cold', with a bluish cast. This can be compensated for with so-called conversion filters which, however, absorb much light. Smaller differences can be compensated for by colour balance filters, generally of the complementary colour to a certain cast. Such filters can sometimes be pre-tried effectively by projecting a diapositive suffering from colour distortion through a few correction filters. In some instances where the balance is critical, it may be necessary to keep the incandescent lamp at a certain voltage yielding a correct colour balance. When this has been found with trial-exposures with a high-power objective, it may be that the exposure time apparently becomes too short when low-power objectives are used. Lowering the setting of the lamp voltage will of course disturb the colour balance. The use of a neutral-density filter can bring down the image brightness, without affecting the colour quality of the illumination. These filters


absorb a fixed portion of the passing light, so that a rough calculation can be made of the effect to be expected. Generally, the amount oflight absorbed by these filters is not expressed as a percentage, but as the density, defined as the logarithm to base 10 of the reciprocal of the transmittance, the percentage of light transmitted. Mostly use is made of a set of filters of 0.30, 0.60 and 0.90 density, corresponding with transmittances of 50%, 25% and 12.5%, respectively. They can be used singly or in combination.

Different other circumstances exist which can be the cause of a colour imbalance, apart from obvious causes like faults in processing or exposure, leaving a contrast filter in the light beam, use of a wrong light source etc. The most important factors to think of when colour photomicrographs systematically show colour imbalance are: heat absorbing filters (built-in with some types of light sources) which can absorb quite a deal of red light, traces of ultraviolet radiation to which colour film is very sensitive, stained mounting medium of the microscopic specimen (all old preparations mounted with Canada balsam can show a light yellow cast, virtually unperceptible to the eye) and radiation exposure (X-rays, y-rays, e.g. when the films are stored in rooms where such radiation is present). Finally, colour shifts in using a condenser of low grade correction or chromatic objectives often are more obvious in a colour positive than when viewed through a microscope. This is the reason why, as stated before, the use of high-quality optics is indicated virtually without exception with colour work.

SOME SPECIAL TECHNIQUES IN PHOTOMICROGRAPHY

Microftash Essentially this is a light source with a typical position because it can only be used for photography. As a rule it is formed by a device which can be mounted under the condenser with a quartz or glass tube filled with a gas (e.g. xenon); by means of a loaded condensator a discharge can be made to take place in the tube, which is immediately followed by an intense light flash with a duration of (depending on the tension given off by the condensator) 1/500-1/5000 sec. As it is not possible - and even dangerous - to focus with the flashlightl the object has to be pre-focussed with some kind of regular microscope illumination, e.g. with a ring-shaped flash tube or with an auxiliary lamp incorporated in the flash unit. With the recently

1. In this field, van Leeuwenhoek was again a pioneer when he tried to observe the explosion of gun-powder under the microscope, reporting to the illustrator what he saw; he nearly lost an eye with these experiments.


developed flash steering device 'visuflash' the possibility exists to use the flash tube for focussing and/or visual observation at a low brightness level (van Maaren, 1974).

The flash-unit is used mainly in photomicrographic work with moving objects, in which the movement is so fast that the image becomes blurred even with the shortest setting of the shutter. The very short light flash can be considered to take the place of an extremely short shutter time with a powerful light source. As the duration and intensity of the light flash can be varied only in a very limited way (by the current load given off by the condensator), the correct exposure with a given optical combination can only be found by trial and error, i.e. experimenting with different neutral density filters and film materials. In fact, the situation comes down to the curious condition that all other circumstances have to be matched to a fixed 'shutter time'. It is evident that any type of automatic exposure control is of no value: the shutter should be open when the flash 'passes'. For colour photomicrography it is important to note that the colour temperature of such a flash generally lies around 6000° K, so that the use of daylight-type film is obligatory.

Stereophotomicrography To produce stereoscopic images, two different exposures have to be taken at a certain angle to each other; these two pictures have to be studied each with a separate eye, so that both images can be seen as one picture with depth through its structure. Both prints should be identical in tone; they can be viewed with a stereoscope as used in viewing macroscopic stereoscopic images, e.g. in some anatomical atlases. The pair of photomicrographs can be taken simultaneously with a so-called Drilner-camera, in combination with a stereo-microscope (cf. chapter 2). In this set-up, the image planes of both half-images enclose an angle of circa 1680 • It is also possible to keep both image planes in a parallel position and make the two exposures one after the other, moving the object in between from one side of the object field to the other, or shifting the camera over a corresponding distance. With a so-called stereo-ocular both pictures can be taken simultaneously, which avoids having to keep the tone of both images identical on the ultimate prints.

All techniques for stereophotomicrography are of limited practical value, however, as with increasing magnification the stereo-effect is lost due to a rapid reduction in depth of field (cf. chapter 5). Stereophotomicrography is only of value, therefore, with lower magnifications. To elucidate spatial relationships, it is sometimes possible to re-create the depth effect such as is

SOME SPECIAL TECHNIQUES IN PHOTOMICROGRAPHY 235

reached with direct microscopic observation with up-and-down movements of the fine adjustment by taking overlapping exposures at different depths in the same negative. The contrasts generally are rather low, however, in these cases, and it is more effective to make separate negatives at different depths, reconstructing the structure of the specimen by interposition of glass plates or sheets of plastic. This technique, sometimes known as photomicrosynthesis is very tedious and the results only seldom justify the time-consuming work. Quite another situation exists when a total reconstruction of a complex structure such as an embryonic heart is made from photomicrographs of serial sections, but this is far removed from stereophoto micrography proper. A single photomicrograph can never contain all the information of a truly stereoscopic image. This holds true also for the new types of microscopes which enable the photographing of a microscopic image which oversees a very deep field: the scanning electron microscope (see chapter 12) and its light-microscopic equivalent, the scanning photomicroscope of McLachlan (1964). In all these cases a so-called isometric image is obtained with full depth in the image but, not unlike viewing with one eye, it misses the really stereoscopic dimension of perspectivic observation.

Holography This new development in image recording has been invented by Gabor in 1948, and it has been linked from the beginning with microscopy. Although it still remains a promising technique, it is fair to say that it has not come up to its earlier expectations as a true revolution in microscopic observation. Essentially, holography comes down to a reconstruction of the wavefront emitted by a light-emitting or illuminated object rather than a recording of the image formed by that wavefront itself such as is made with conventional photomicrography.

The holographic microscope as it has been developed by Gabor and Goss (1966) is built essentially on the interferometer principle in which coherent light from a laser is split into a fraction passing through the object and the other (larger) part remaining outside of it. All of the information with regard to amplitude and phase is available and with suitable means amplitude contrast, phase contrast and even dark-field images can be reconstructed from the same hologram. The three-dimensional reconstructed image has a very large depth of field in comparison with a conventional two-dimensional image. Different technical problems reduce the actual value of this recording system, such as diffraction noise and dust particles. Holograms can be stored in photographic emulsions but also, curiously enough, in crystals oflithium niobate (LiNb03 ).


Cinemicrography Cinephotomicrography, or for short cinemicrography, is the application of cinematography in recording microscopic images. A similar confusion as pointed out on page 207 is prone to arise, as in most European languages the equivalents for cinemicrography are terms like microcinematographie (French) or Mikrokinematographie (German). As - in contrast to the notion microphotography - microcinematography is not used for minute cinerecording of a large object, there is less chance of confusion as with the terms photomicrography and microphotography. The term microfilm is used only in connection with microphotography of documents, for which the same frame and type of material is sometimes used as with cinemicrography.

Cinemicrography is applied in widely different fields of microscopy. It is often used to record dynamic phenomena of moving objects such as animal cells or bacteria; in chemical technology it is applied to record mechanisms of crystal growth, melting phenomena performed on a hot stage etc. Before applying cinemicrography for educational or research purposes, its pros and cons should be weighed against the use of television video-recording (see next section of this chapter). Undoubtedly, the application of film has lost some ground to TV-recording, but for different reasons, cinemicrography will keep its place as an aid in the recording of events under the microscope. In the first place, the quality of the image is clearly superior and the use of colour does not create many extra complications, apart from the cost. In the second place it is relatively easy to change the speed of a certain dynamic event in recording it . In technical microscopy, and sometimes in biological work, some processes taking place at high speed can be studied with slowmotion cinematography, which is defined as taking more frames per second with the camera than pass when the film is projected at normal speed. When e.g. the film is transported in the camera (which should be specially adapted for this) with 64 frames/sec and it is projected with 18 frames/sec, a slowing down of a fast process with a factor 3.5 is reached. On the other hand, with a so-called time-lapse recording, the number of frames per time unit is less than that when the film is projected. As no limitations exist with regard to film speed or minimum exposure as with slow-motion recording, it is possible to speed up the recording of certain processes at will. When an exposure is taken every 20 seconds and the film projected at a speed of 18 frames/sec, a process taking place in one hour (e.g. a mitotic cell division) can be overseen in 10 seconds. Special equipment is necessary to ensure that after a certain period one picture is taken and the camera moved one frame. With


very long intervals between the exposures, it is often advisable to cut off the illumination to the specimen in the interval period, also in view of damage to the specimen by the intense light. As this technique is widely used nowadays, this type of equipment can be obtained commercially as a complete set.

Direct recording of a dynamic event without slow motion or time-lapse, with subsequent showing of the film in a later stage with a projector (the film can also be studied image for image) occurs only in a minority of the situations in cinemicrography. As a rule, more or less adapted cameras for conventional cinematography are used, although specially constructed cine-microscopes have been built incorporating a camera with focussing telescope, microscope, light source, time-lapse adjusting equipment etc. Mostly, use is made of standard 16 mm cameras, preferably with electric drive, mounted on a heavy and very rigid support which should be separated from the microscope stand in view of the vibrations of the film camera.

Cinemicrography comes down to the taking of a large series of photomicrographs on a small image area, with a more or less limited amount of time for the exposure of each frame. Even in making a time-lapse film it should be taken into account that as a rule the object is slowly changing; therefore a great freedom in the exposure time in photomicrography does not exist here. Another consequence of an object which is moving is the fact that the optimum plane of focus is bound to change; even the object itself (e.g. a wandering cell) may move out of the field of view! Consequently the focus at the object plane should be controlled continuously during the cinematographic recording. Often a semi-transparent beam splitter is used, in combination with a focussing telescope as shown in fig. 10.9.

When the light coming from the eyepiece is divided equally over both directions, it is clear that a loss of no less than 50% of the light for the exposure of the film will occur. In view of the short time period available for the actual exposure, such a loss is often too great, so that partially silvered prisms with asymmetric light distribution (e.g. 70/30%) are used. In view of the usually high illuminance, even less than half of the illumination intensity will do for the focussing telescope. The light source used for cinemicrography mostly is a gas-discharge lamp (high pressure mercury or xenon); except for very low magnifications, no incandescent lamp has a sufficient light yield to ensure the necessary short exposure time under different conditions, especially with techniques such as phase contrast. With regard to exposure control, it should be noted that - not unlike the situation with micro-flash - the exposure time which is linked with the camera speed


Fig. 10.9. Focussing eyepiece for cinemicrography with image-splitting via semi-reflecting prism.

cannot be varied with the camera but within very narrow limits. With film material of different speed and neutral density filters, a correct exposure can always be reached, as long as there is sufficient 'reserve' in the illumination. The use of high-speed cameras in cinemicrography is limited as a consequence of the extremely short exposure time available; with 64 frames per second, 30 meters of film pass in a period of one minute, during which no less than approximately 4000 separate frames should be correctly exposed. Even with a normal speed of between 18 and 24 frames/sec (usual projection speed for silent or sound film, respectively) the exposure time cannot be more than of the order of 1/50 sec, as the time for the moving of one frame area to the next (during which the camera opening should be closed) has also to be taken into account.

The standard format for films used in scientific cinemicrography is the 16 mm film with an image size of 7.5 x lOA mm and 130 frames per meter. The cost of35 mm film ('normal-film') with only 52 frames per meter is often prohibitive. Moreover, its image area (16 x 22 mm) is 4.5 x as large as that of a 16 mm film, with all its consequences for the exposure time which is of course related to the lighting intensity at the image plane. Recently, the new format 'super-8', frequently used in amateur cinematography, with an image format of 4.2 x 5.7 mm has been introduced into cinemicrography. A great advantage of this is the relatively low cost of materials, as compared with 16 mm film. Although this format is not suited for demonstrations


for large audiences, it could become of great value in scientific work. The ordinary 8 mm format with an image size of only 3.6 X 4.8 mm is really too small for bringing over microscopic images with a sufficient amount of detail.

Although cinemicrography entails a series of complications which are non-existent with ordinary photomicrography, it is often possible to attain satisfactory results without large investments. The devices necessary for the continuous observation of cells within specially designed chambers, the maintenance of the specimen at a correct temperature with cells ofisothermic animals etc. essentially have nothing to do with cinematography as a technique for registration of dynamic microscopic events. In practice they are of great importance, but a more detailed description would fall outside the scope of this text (for reviews see Rose, 1967, Johnson and Wood, 1967 and Lawson, 1972).

OTHER TECHNIQUES FOR REGISTRATION AND REPRODUCTION OF MICROSCOPIC

IMAGES

Drawing devices In the past, drawing an image was virtually the only way for recording a microscopic image; the first photomicrographs were made as early as 1839 by J. B. Dancer in England in the same year as Daguerre made his first demonstrations of the photographic process. It is only after 1945 that photomicrographs have gradually started to dominate over drawings in the microscopic scientific literature. In the years between 1870 and 1939, virtually any larger laboratory working in the field of applied microscopy had its own specialized illustrator and many pioneers in microscopy were experienced drawers themselves. It should be kept in mind that in that period photomicrography was possible, but the equipment and light sources were rather primitive and the emulsions often unreliable. Then, as now, often only certain parts of a microscopic image were important for a given purpose and by skillful drawing it is possible to isolate somewhat the details concerned from a more complex image. A second important advantage of drawing is that problems of depth of field can easily be solved in making the drawing, so that a section can be recorded in its full depth at high power. This can be illustrated with the drawing of fig. 10.10, as made in 1934 by C. A. Vlassopoulos, illustrator at the Histological Laboratory of the University of Amsterdam; the connective tissue fibers shown here in their three-dimensional connection traverse the entire section thickness. Although


Fig. 10.10. Drawing with Indian ink of a 7 [Lm section from the papillary layer of human dermal connective tissue; silver-impregnation according to Laguesse, counterstained with aniline-blue and azocarmine. The meshwork of interwoven connective tissue fibers which traverse the entire section can be overseen in its totality, whereas in making a photomicrograph the depth offield would have been much smaller than the depth of the section at this level of magnification and aperture (cf. table VII on page 89). Drawing made by C. A. Vlassopoulos in 1934.

it is obvious that a drawing is not necessarily objective, it should not be forgotten that really first-class drawing artists had an experienced eye and often, curiously enough, did not know much about the subject; sometimes they did not even want to know about it. A drawing as shown in fig. 10.10 required up to several weeks of work with negligible material costs; it would be beyond price nowadays to use this just as an illustration material for a descriptive publication.

REGISTRA nON AND REPRODUCTION 241

The present situation is that only in selected cases the drawing technique is used and that first-class illustrators have become very rare. Unlike illustrators of the past, who often did not use any aid at all, it is advisable for the more simple type of drawing work to make use of devices which facilitate the transfer of a certain pattern from the microscopic image to the drawing paper. When a drawing is used for quantitative analysis of a microscopic image (see chapter 11) the use of such a device is even obligatory.

Drawing aids can be divided into two main groups: a) systems where by means of a mirror or prism a real image is projected onto paper, on which the outlines of the image can be traced, and b) optical devices enabling the projection of the drawing paper into the microscopic image. The latter system is known as camera lucida on the basis of the fact that it is not necessary to work in a darkened room, in contrast to the method a). As a third possibility it may be mentioned that sometimes the best technique is to make first a photomicrograph tracing certain details with drawing ink on the photomicrograph, after which the print is bleached with Farmer's liquid, or make the tracing on transparent paper over the photographic print. This principle, used sometimes to give an exact contour drawing accompanying a complicated photomicrograph, is in fact a combination of photography and drawing, which can be very useful, but is rather timeconsuming. With regard to the last-mentioned technique, it should be em-

NF

A B

Fig. 10.11. Drawing apparatus (camera lucida) according to Abbe (A) and with a semireflecting prism (B). NF neutral density filter.


Fig. 10.12. Use of a drawing apparatus with a semi-reflecting prism (fig. 1O.11.B) in determining cell areas.

phasized in passing that only chinese drawing ink can withstand treatment with Farmer's liquid which dissolves the silver from the emulsion of the print; ball-point or felt-tipped pen lines disappear with the silver in the print.

The principle of a camera lucida can be achieved in different ways. The classic drawing device of Abbe is based on total reflection at a prism interfase with a central opening, in combination with a mirror at some distance from the microscope tube (fig. lO.1IA). In a second device of later development, the beam-splitting is based on a semi-transparent prism, which produces a better image quality (fig. 10. llB). When the distance between the two parallel layers which reflect under an angle of 45° is sufficiently great, such as is the case with the larger types of drawing apparatus, the drawing paper can be placed at a comfortable distance from the microscope (fig. 10.12). The smaller types of drawing prism which can be put over the eyepiece are mostly based on the Abbe-principle, but often the paper must be brought very close to the foot of the microscope stand, and even then it is possible to trace only a part of the field of view. The neutral density filter NF in fig. 10.11 serves to balance the brightness of the image with that on the drawing paper; without such a device, one of the images would easily dominate over the other. This can be solved with a rotating disc with neutral density filters of different absorption grade, or a sheet polarizer, which can be rotated against a second polarizer (e.g. on top of the dividing

REGISTRATION AND REPRODUCTION 243

prism in the situation of fig. 10.11 B). When a correct balance has been reached between 1) image brightness in the microscope, 2) brightness on the drawing paper and 3) setting of the neutral density filter, lines drawn with a soft pencil will become clearly visible in the microscopic image (fig. 10.13).

Fig. 10.13. Isolated liver cells in a stained smear, as observed with a drawing apparatus (of the type shown in fig. IO.lI.B) with both the image of the specimen and that of the drawing paper in focus.

Microprojection Microprojection is essentially the projection of a microscopic image onto a screen by means of a simple or compound microscope. Although in different situations (such as photomicrography) use is made of microprojection, one generally understands by this term the situation in which the projected microscopic image itself forms the goal, e.g. for the demonstration of certain details in a specimen to a larger audience. Amidst newer technical means for 'microscopical communication', such as colour diapositives, cinematography and television microscopy, microprojection no longer plays a dominant role to-day. It has an important advantage, however, in its relatively simple type of equipment which does not exceed reasonable means and basic technical knowledge of an experienced microscopist. Of


the newer technical means, colour microtelevision (see next section) would be superior from a tutorial point of view, if it were not prohibitive because of the cost and the specialized skill demanded for its adjustment and maintenance.

The central problem with conventional microprojection is the image brightness; apart from short-distance projection, e.g. on a ground glass screen or for drawing purposes, the image brightness quickly falls below reasonable standards with a conventional illumination and a magnification of anything over the very low-power range. In the past use was often made of the sun as a light source, as mentioned in chapter 6. Even with high pressure mercury lamps and xenon burners (the carbon arc has now virtually disappeared as a light source for this purpose), the problems are not quite solved for large screen microprojection. Apart from losses by reflection and absorption, the total luminous flux remains the same when an image of an illuminated object is formed (see chapter 1); the illumination alters proportionally with the surface area ratio between object field and image. The lighting intensity undergoes a very drastic reduction, therefore; when the diameter of an object field is e.g. 500 fLm and the projected image 2 m, the lighting intensity will decrease in this situation with a factor of several millions. In order to keep a reasonable brightness on the projection screen, the light energy passing such a very small object field has to be so enormous that damage may be inflicted to the specimen by local warmth development (bleaching, softening of the mounting medium, formation of gas bubbles, etc.). This happens especially with darkly stained specimens which absorb a great deal of the light energy. A heat-absorbing filter (taking away a substantial portion of the infrared radiation) can reduce these effects to some extent, but problems tend to remain. Haematoxylinphloxin stained preparations (if not stained too heavily) tend to be rather light-fast. In general, it should be kept in mind that valuable specimens treated with certain light-sensitive stains can be spoilt immediately.

Even with the most powerful light sources, it is not possible to reach an acceptable image brightness at higher magnifications with a larger distance between projective and screen: the image brightness diminishes with the second power of the projection distance. With optimal equipment, using a gas discharge burner, it is possible to reach a projection distance of 8-10 m with an image diameter of up to 2 m, enabling (in a totally dark room) the use of high-aperture objectives up to 25 - 40 x, in combination with a special projective. Optical problems are virtually not met with in this situation, and when high-quality optics are used with a well-reflecting screen with fine structure, the image quality is excellent and all colours

REGISTRA TION AND REPRODUCTION 245

come over quite naturally. With this long projection distance, the intermediary image comes to lie virtually in the first focal plane of the projective, so that the interaction objective-projective is optimal. It is tutorially important for the audience to follow the adjustment of the image, searching for details and building-up the final high-power image as far as it goes. A well functioning nosepiece with parafocal objectives in combination with a special type of mechanical stage is essential for this.

Television-microscopy The brightness problems in microprojection dealt with in the preceding section can be entirely solved when the image is recorded over a closedcircuit television system. As the image is brought over electronically, it can be intensified virtually at wilP, and demonstration with the highest magnifications is possible with an unlimited number of monitors (receivers). TVmicroscopy even enables the direct demonstration of low-brightness images, such as those obtained with phase-contrast microscopy, to a large audience; this is unthinkable with any form of microprojection. A second practical advantage is that the room does not have to be darkened. With so-called projection-television a reconverted image is projected, not onto the fluorescent screen of a monitor, but directly onto a large convential projection screen by an ingenious transformation system. Problems of image brightness are re-encountered here, but they are no worse than with the projection of a motion picture film and the object is never in danger. This type of equipment has, however, remained prohibitively expensive.

Against all advantages it should be mentioned that some loss in resolving power (virtually nil with microprojection or cinemicrography) will inevitably occur. This does not need to be very dramatic, as long as it is taken into account that details to be shown should be magnified so that they do not interfere with the frame lines of the TV-system. In general it can be stated that it is advisable to take a larger magnification than would be necessary for direct observation. The rules for empty magnification etc. as dealt with in chapter 5 do not automatically hold true for television microscopy. It can be calculated that with a receiver with a 53 cm screen (diagonally) and 625 frame lines, the upper limit of the useful magnification at a viewing distance of 2.50 m would be something like 12.500 x for a 100 x objective with N.A. 1.25.

Quite another advantage of television microscopy is found in the possi-

1. Newer developments in television techniques, a.o. in ophthalmology with the so-called fundus camera, have made possible the recording of indeed very dimly illuminated images, but for television microscopy a normal camera will be sufficient.


bilityof using camera tubes with a special sensitivity for ultraviolet or infrared light; they can be applied with the same equipment (transforming unit, monitors) as used for visible-light television-microscopy.

For demonstration purposes, the image on the monitor(s) can be fed from two or more cameras, so that e.g. macroscopic drawings and schemes can be alternated with microscopic images. Moreover, with adequate equipment, it is relatively easy to make a video-recording of a certain program. Up to a certain degree, video-recorded television and cine-recordings have to compete with each other. They each have their own limitations and possibilities, such as can be seen from the summary listed below. It should be noted in passing that the value of a recorded programme, made without competent direction for educational purposes, is often overestimated both with film and television. A considerable degree of professional skill is necessary to know how to build up a coherent 'story' from independently recorded scenes, even under seemingly simple circumstances.

Television-microscopy recording

Loss in resolution of roughly 15 % (depending on the equipment), using oneinch tape, when the magnification is held high, so that details to be observed do not approach the distances between the frame lines

Colour-television microscopy (which can be recorded) possible, but calls for high technical skill for adjustment and maintenance; cost in many cases prohibitively high

No image-brightness problems, no speciallight source necessary

Relatively low material cost, once the equipment is present. No further processing of recorded tape; tape can be shown immediately and can be re-used

Immediate recording possible, up to 50 min continuously

Limited or no possibility for special techniques like slow motion and timelapse

cinemicrography

High quality image with all magnifications with neglegible loss in resolution on 16 mm film

Use of colour relatively easy, but a four-fold increase of material cost

Image brightness (and colour temperature in using colour material) a point of continuous concern

High cost of material, which has to be sent out for processing. No immediate inspection of results possible

30 m reels, which have to be exchanged

Slow motion and time-lapse possible

REGISTRATION AND REPRODUCTION 247

Only with a special type of recorder electronic mounting of independently taken shots into a programme possible; in all other cases the programme has to be recorded in a single sequence as mechanical mounting is not possible

In principle, the same equipment for recording and reproduction

Unlimited mechanical mounting of scenes into a recorded programme; this is very time-consuming work calling for professional skill

Camera and projector separate

When preparing a programme for educational or demonstrative purposes, these differences should be weighed against one another before the actual work is started. It should be noted that with a so-called tele-cine unit it is possible to mount film fragments into a television programme. All this is, of course, out of reach of the average microscopist, but use can often be made of the services of educational audiovisual departments. In itself, television microscopy does not necessitate any specialized equipment apart from that in common use for general educational closed-circuit television work, and a good microscopic equipment.


W. Belfield: The production of a composite drawing of microscopic material by means of photographs. Stain Technol.43 (1968) 339-340.

E. B. Brain: Techniques in photomicrography, 2nd ed. Oliver & Boyd, Edinburgh 1969. A. Castenholz: Kymographie mit dem Vitalmikroskop (Mikrokymographie). Microsc.

Acta 74 (1973) 89-109. D. Gabor and W. P. Goss: Interference microscope with total wavefront reconstruction.

J. Opt. Soc. Am. 56 (1966) 849-858. R. Gander: Mikrophotographie: Rezeptefilr Mediziner und Biologen, 2e Auflage. Urban &

Schwarzenberg, Munich-Berlin-Vienna 1974. F. Habermalz: Probleme der Belichtungsmessung in der Photographie und Mikrophoto

graphie. Microsc. Acta 71 (1971/72) 23-29. F. Habermalz: Die Korrektur des Farbgleichgewichts bei mikrophotographischen Auf

nahmen mit Umkehrfarbfilm. Microsc. Acta 76 (1975) 415-427. H. Harms: Handbuch der Farbstoffe filr die Mikroskopie. Staufen Verlag, Kamp-Lintfort

1965. L. Jenny: Schwarzschild-Faktoren vom Farbumkehrfilm. Z. wiss. Mikrosk. 70 (1970/71)

222-226. J. H. Johnson and S. Wood: Color cinephotomicroscopy of the living microvascular

system, in: In vivo techniques in histology, ed. G. H. Bourne. Williams & Wilkins, Baltimore 1967.

D. Lawson: Photomicrography. Academic Press, London-New York 1972. R. D. Lillie: H. J. Conn's Biological stains, 8th ed. Williams & Wilkins, Baltimore 1969. P. W. van Maaren: A new universal and variable flash steering device for visual examina

tion and photomicrography: VISUFLASH. Mikroskopie 30 (1974) 296-305.


D. McLachlan: Extreme focal depth in microscopy. Applied Optics 3 (1964) 1009-1013. K. Michel: Die mikrophotographie. Springer Verlag, Vienna-New York 1962. A. Niklitschek: Uber mikroskopisches Zeichnen. Mikroskopie 2 (1947) 195-216; 321-327. H. von Prosch: Vergroszern photographischer Aufnahmen unter gleichzeitigem Kontrast-

ausgleich. Mikroskopie 30 (1974) 321-326. 1. P. Rieb: Une installation de microcinematographie pour oeufs de Teleosteens, avec

prises de vues a intervalles de temps determines. Microsc. Acta 75 (1974) 338-345. G. Rose: Cinemicrography in cell biology. Academic Press, New York 1967. G. L. E. Turner: Microscopical communication. J. of Microsc. 100 (1974) 3-20. T. E. Wallis: Drawing from the microscope. J. Roy. Micr. Soc. 75 (1955) 77-87. E. Wenzel, W. Mirande and I. Weingartner: Fourier-Optik und Holographie. Springer

Verlag, Vienna-New York 1973.

CHAPTER 11

MEASUREMENTS WITH THE MICROSCOPE

GENERAL INTRODUCTION

In many cases a microscopic observation will not end with the observation itself, possibly supplemented with a photomicrograph or a drawing. For purposes of comparison with observations from other workers in the field, it may be desirable to know e.g. the exact dimensions of a particular detail observed in a microscopic specimen. Apart from linear dimensions, the only parameter which could be measured until the not too distant past, currently more complex factors can be measured such as the relative spatial relations between different elements of an object or the spectral absorption characteristics of an object part. In general, a definite tendency exists in microscopy to a change from descriptive phemenology to metrology (cf. lebsen-Marwerdel,1975).

In general, the methods used in measuring geometric dimensions (length, surface, etc.) of parts of an object are often called morphometric methods and when applied in measuring tissue components histometric methods. They can be performed with very simple means, but newer developments enable an automatic or semi-automatic analysis of these morphological parameters. Measurements which establish quantitative values of another nature are called by various names, such as photometry (in which light absorption is measured at a fixed wavelength) and microspectro(photo)metry in which absorption of light of an object part at different wavelengths is analysed. Other techniques for quantitative analysis of physical characteristics of tissue components are quantitative polarization microscopy and micro-interferometry which have already briefly been treated in chapter 10.

In the following sections different measurement techniques which are frequently used in biology and medicine will be reviewed from a practical point of view, with some emphasis on the measurements which can be made with relatively simple equipment. Some of the newer instrumental developments in measuring technique, however, can no longer be considered as belonging to the territory of the highly specialized worker; they will therefore be considered in their relation to microscopic observation. Quan-

250 MEASUREMENTS WITH THE MICROSCOPE

titative aspects of phase contrast, interference and polarization microscopy as dealt with in chapter 10, will not be treated again. Specific metallurgic (e.g. micro-hardness testing) or other measurements used in chemical physics, cristallography and different fields of technology, such as the application of warm and cold stages in determining melting points and other temperature-dependent phase changes will not be considered, as they fall outside the scope of this book.

MORPHOMETRIC ANALYSIS

Measurement of length in a focussing plane Measurements of distances perpendicular to the optical axis (with transverse settings) are often performed in practical microscopy. Distinction is usually made in this connection between relative and absolute units of measurement, the latter representing the value expressed in [.Lm, instead of in divisions of a measuring scale; relative units can easily be transformed into absolute ones by a conversion factor which can be obtained by a valuation procedure. When purely comparative measurements are made under standardized conditions, it is often completely useless to convert the readings obtained into absolute units, unless they have to be compared with data from other workers in the field.

Measurements of length are often made with a so-called eyepiece micrometer which consists of a scale with fine divisions etched onto glass, placed at the intermediary image plane. In a Huygens eyepiece such a scale, etched on a round glass, can easily be put over the field diaphragm; often a special type of measuring eyepiece is used with an adjustable front lens, so that the scale can be brought into sharp focus before bringing the intermediary image into the plane of the etched graticule. Usually, the divisions of an eyepiece micrometer are one tenth of a millimeter apart.

In measuring a certain distance in a specimen, e.g. the diameter of a cell nucleus, it is advisable to place this object with one border against a (not necessarily the first) ruling of the scale, so that one has to estimate the distance between divisions only at one side (fig. 11.1). In measuring with

o 10 20 30

I, , ,11' , , I.! I I I ••• , I, , 1 , 11 , , 1 I

Fig. 11.1. Measurement of an object in a microscopic specimen with an eyepiece micrometer so that estimation of distance between two divisions is only needed at one side.

MORPHOMETRIC ANALYSIS 251

ordinary high-power objectives, it is advisable to use only the central part of the scale, both in view of the performance of the objective and the curvature offield in the eyepiecel .

The reading of about 8.5 scale units for the longest diameter of the nucleus in the situation of fig. 11.1, can be brought on an absolute basis by means of a valuation, a determination of the value of the divisions of the eyepiece micrometer, as projected in the specimen. For this purpose use is made of a stage micrometer or object micrometer, which consists of a finely divided rule, etched photographically onto an object slide and mounted with a cover glass; the divisions are usually separated by distances of 10 [Lm. The microscope is focussed onto these lines, so that the image of the stage micrometer divisions falls over the lines of the eyepiece micrometer.

It is easy to see that the actual valuation comes down to determining the relation between the two scales. The following method for determining this relation can usually be applied successfully with higher magnifications. Difficulties are met especially under these circumstances, because of the fact that the divisions of the object micrometer scale, how fine they may be, will produce blurred images which are not sharply delineated at higher magnifications. By using the mechanical stage and rotating the eyepiece, the lines of the two micrometers are brought into a parallel position. Subsequently, two more or less distant lines of the stage micrometer are made to fall just beside two more or less distant lines of the object micrometer (fig. 11.2B); the micrometer-value, i.e. the real length of the spaces between

III IIII IIII IIII IIII IIII IIII III

A B Fig. 11.2. Valuation of an eyepiece micrometer with the image of a stage micrometer. A position of both scales in which no precise reading is possible; B correct position for a valuation reading: 13 divisions of the eyepiece micrometer correspond with 2 divisions of the object micrometer.

1. A rather extreme situation exists, of course, when the size of the distance to be measured exceeds the length of the micrometer scale. Unless a lower magnification can be used or a longer scale, there is no choice but to move the object with the mechanical stage over the scale, keeping in mind a characteristic spot which fell over a certain division and which is then again positioned at the beginning of the scale. When very large distances - the ends of which cannot be viewed simultaneously in '" single field - have to be measured, a mechanical stage with graduated divisions in combination with a cross-wire or eyepiece micrometer, can produce reasonable values. Here are the limits of what can be called measuring with a microscope.


the divisions of the eyepiece micrometer as projected in the specimen, can easily be calculated on the basis of the relation between the number of spaces included in each of the micrometer scales.

The system described which is illustrated in fig. 11.2B (under the conditions of fig. 11.2A only a rough estimate can be made) is generally superior to the method in which the beginnings of the two scales are placed over each other, although this can sometimes be used with lower magnifications when both scales can be overseen entirely. In the situation of fig. 11.2, the micrometer value is 2/13 x 10 fLm (= 1.54 fLm), when the divisions of the stage micrometer are 10 fLm apart as is usually the case; this would correspond to a magnification of the objective of 100/1.54, or 65 x. It should be noted that the total magnification of the microscope cannot be found in this way; when necessary, it should be determined by projecting the image of a stage micrometer on a distance of 250 mm from the exit pupil (see chapter 10). When used for microprojection or photomicrography, the total magnification can be determined likewise by projecting or photographing a stage micrometer under the conditions used.

The valuation of an eyepiece micrometer under the optical circumstances (objective, eyepiece, tube length) under which measurements have to be made does not form a problem in itself. Although it should be performed in duplicate or triplicate, it has to be made only once for a given optical combination. The real practical problems are more in the measuring procedure itself. In order to produce measurements as accurate as possible, a large final magnification has to be selected, often nearer to the limit of empty magnification than would be necessary for visual observation. The counterpart of this extension of the image over the micrometer scale would be to make the distances between the division lines of the eyepiece micrometer smaller. A closer apposition of the division lines of the eyepiece micrometer than about 0.1 mm, however, appears to entail a diminished visibility of the scale as a whole, due to diffraction phenomena.

The unavoidable estimating of distances between the division lines in the measuring procedure with a conventional micrometer is not only a tedious and fatiguing work, it often leads to results which are less accurate than would have been permitted by the optical limits. Even with all kinds of variants of the classical eyepiece micrometer, such as the contrastmicrometer in which the scale consists of black squares linked together diagonally (fig. 11.3) and other micrometer-variants such as the stepmicrometer, the problems of estimation remain unavoidable. These special types of micrometers have only the advantage that they are less easily distorted by diffraction than ordinary line scales; in using a contrast micro-


2 3

··1··········',··· Fig. 11.3. Contrast micrometer scale, as compared with a conventional micrometer scale.

meter, the borders of the object remain free and the actual measuring lines have no thickness. For accurate measurements, other types of eyepiece micrometer provided with mechanical devices to avoid guess-work are used. A screw-micrometer eyepiece (or filar eyepiece micrometer) consists of a scale with a rather coarse division at the plane of the intermediary image. A thin thread can be moved over this scale by means of a built-in mechanical screw device. The drum by which this movement is controlled has a division engraved on its circumference (fig. 11.4). As a rule, one entire rotation of the drum corresponds exactly with the movement of the thin wire from one line of the fixed division scale to the next, so that the esti-

A B

Fig. 11.4. A Screw-micrometer eyepiece as seen from the upper side, the dashed circle indicating the field of view. B The image as seen when viewing through the eyepiece; the object in focus has a length of 585 divisions on the drum.


mation between the fixed division lines is replaced by a reading on a finer scale. Similar to the situation with a conventional eyepiece micrometer, it is to be recommended that the object to be measured is made to 'lean' against a fixed line, so that a reading has to be taken with the drum only at one side (fig. 11.4). As a rotation over 3600 generally corresponds with exactly 100 divisions of the drum, the reading in the circumstances of fig. 11.4B would be 500 + 85 units. Also with objects smaller than one large scale division, it is often to be recommended to use this system of 'clamping in' of the object; the mere touching of the drum by the hand causes some bending to occur in the tube entailing small changes in the position of the image, so that it is not a good system to traverse an object to be mea~ured with the wire and take two readings. When the distance between two points or sharply delineated borders (e.g. the border line of a stained cell nucleus) has to be determined, it is often easier to fix the measuring points in a slit between two wires. Screw-micrometer eyepieces therefore are sometimes provided with both a single and a double wire, so that one of both can be used for measurement (fig. 11.4). The valuation of such a micrometer is very simple, as non-coinciding lines can be transformed into fractions of the divisions of the fixed lines of the eyepiece micrometer by means of the drum. The micrometer value of one division on the drum (1/100 from a division of the fixed scale) will fall below the minimum resolvable distance at even comparatively low magnification. It should be kept in mind that the seemingly precise readings which can be obtained with a screw-micrometer eyepiece are for a large part illusory; its accuracy is certainly not much greater, if any, than that of a conventional eyepiece micrometer with visual estimation of intervals. Some microscopists, such as Loquin (1956), even advise in general against the use of a screw-micrometer eyepiece, as the precision of the reading on the drum largely exceeds the errors introduced by the bending of the tube when the drum is touched with the hand.

In using any type of eyepiece micrometer, the measurement of lengths perpendicular to the optical axis entails two transverse settings. The accuracy of these settings is theoretically rather high: in most situations even under the minimum resolvable distance (cf. Fran90n, 1961). It should be pointed out, however, that setting accuracy is seldom the limiting factor in the measuring accuracy in these circumstances, as other factors such as contrasts in the object, distorsion of the object by the objective and diffraction phenomena are often more important. With so many variables, it is virtually impossible to give precise general rules for the accuracy which can be


reached with these length measurements. It is a general rule in metrology that the measuring instrument should have a precision which is ten times as high as the tolerances admitted with the object which has to be measured. This means that for measuring an object of 5 [Lm with some accuracy (i.e. close to the 'true' value), the precision (= reproducibility) of the measuring device should be within 0.5 [Lm. With a theoretical minimum resolvable distance of e.g. 0.3 [Lm and taking into account distorsion of the image, diffraction phenomena and errors in the eyepiece micrometer, this is about what can be reached. With lengths under 5 [Lm, the error may well be around 20%, whereas over 10 [Lm an accuracy of 5-lO% could be reached under favourable circumstances (use of high-quality plan-objectives, etc.). All these measurements should be considered more as approximations than as accurate determinations; the specification of a length measured as 5.74 [Lm can mean no more than that this length should be somewhere between 5.00 and 6.50 [Lm.

Finally, mention should be made of a relatively new type of length-measuring device, the image-shearing measuring eyepiece. This rather large device is inserted in the tube; it contains beam-splitting prisms and a moving mirror

A(!)

Fig. 11.5. Effects of an image-shearing measuring eyepiece on a schematic image of a cell with a nucleus. A complete overlap of both daughter-images, B both images moved apart over a distance corresponding with the nuclear width so that the latter can be measured, C position of both images so that the width of the entire cell can be read, D both images in free position.


or a rotatable prism which can be moved by means of a drum. The distance of the shear between the two images (appearing sometimes in different colours) can be read in arbitrary divisions of the drum. This type of mea suring device enables the reading of certain distances in isolated objects quite easily, in a way shown in fig. 11.5; its use is much less strenuous than reading the fine line scales of eyepiece micrometers. A valuation of the shearing distances as read on the drum is performed with an object micrometer. It seems that a rather high degree of accuracy can be reached with these systems (cf. Humphries, 1969, for details). It should be taken into account that in using these image-splitting devices in objects such as tissue sections with a complex pattern of details of all kinds, the patterns obtained with sheared images may become very confusing.

Measurement of distances along the optical axis The so-called depth measurements can be performed with any microscope provided with a graduated fine focussing control. In most cases, the manufacturer can specify how much !Lm tube- or stage-displacement corresponds with one division of the graduation (if this is not given on the control). A few special complications exist with depth-measurements, e.g. in the measurement of section thickness, which are often overlooked but should be kept in mind in performing any measurement along the optical axis.

In the first place any axial setting should be considered as a sharpness setting. The instrumental depth of field, to which both axial resolving power and geometrical depth of field contribute (see chapter 5) brings about a much larger degree of uncertainty in fixing the depth of a point in the object than with transverse settings. Axial settings are also influenced by the depth of accommodation of the observer's eye. All these factors are reduced dramatically with use of higher magnifications and high apertures; it is advisable, therefore, to keep the depth of field as small as possible by using high-power objectives. Nevertheless, the accuracy of the settings is always much lower than in the case of a single focussing plane. A second complication in depth-measurements is caused by the occurrence of refraction phenomena in the object space. When the upper and lower settings are made, the differences between both readings only correspond with the real difference in depth when the refractive index in the object space is completely homogeneous. When an oil-immersion objective is used (to be recommended in view of the high aperture), this condition is virtually fulfilled when - as usually will be the case - the object and its mounting medium have a refractive index in the same order as glass and immersion oil. When an object has been mounted in water or in air, a correction has


to be made for refraction effects. This important effect, which is often overlooked in practice, can be explained with the help of fig. 11.6.

When an object is in a medium with a higher refractive index than the medium between it and the objective, the depth of such an object will

A B

Fig. 11.6. Effects of light refraction on depth measurement. In A it has been assumed that in the upper part of the object space a refractive index n. prevails, which is lower than that of the medium n l which surrounds the object to be measured: the distance read on the graduations of the fine adjustment will be too short. In B the cIrcumstances are reversed: n. > no and the value read will be too large. Situation A occurs with any routine mounting medium and a dry objective, and situation B in using an oil-immersion objective with a specimen mounted in an aqueous medium.

seem to be smaller than it really is!. On the contrary, the depth will be overestimated (and consequently also measured as too large on the basis of object-objective displacement) when the object is in a medium with a lower refractive index than elsewhere in the image space, which entails refraction towards the optical axis. The correction which should be made is proportional to the relation between the refractive indices concerned; under the circumstances of fig. 11.6 it will, therefore, apply that the real depth P (equal in both situations) and the depth read on the divisions of the fine adjustment P A and PB would be:

P = PAX ~ in the situation A, and P = PB X ~ in the situation B. n2 n4

The presence of a cover glass (not drawn in the figure) does not make any change in the basic situation.

l. The same phenomenon is the cause of the well-known failure to catch a fish in an aquarium when looking from above.


Practically speaking, this effect is only of minor importance in the common situation where oil-immersion is used with an object in one of the conventional mounting mediums with a refractive index of around 1.5 (see appendix I) and immersion oil with a refractive index of 1.515. In using an oil immersion objective and an object mounted in water, the situation of fig. 11.6B arises, whereas the situation A will apply in the case of an object mounted in Canada balsam (n = 1.53) and focussed with a dry or waterimmersion objective. In measuring an object with a dry objective and with incident illumination, however, no correction is necessary and the reading on the fine adjustment can again be taken directly as the depth in the object.

In contrast to the situation with measurements perpendicular to the optical axis, a calibration for transforming relative values into absolute ones is not necessary, unless the divisions on the fine adjustment have an unknown value or one wishes to verify the values specified by the manufacturerl .

The best technique for this calibration is the use of a special cover glass, the thickness of which has been measured with a technical precision-micrometer. Both surfaces are semi-aluminized or treated with glass ink and subsequently scratched, so that the scratches can be used as reference marks to focus the surfaces of the coverslip. When p divisions of the fine-adjustment screw correspond with thickness of the cover glass of q [Lm and n is the refractive index of the cover glass, one division shift of the screw would

then correspond with a n x p [Lm travel when a dry objective has been q

used and ~ when the reading would have been taken with an oil-immersion q

objective. In most cases the divisions on the micrometer screw correspond with 2 [Lm; a much higher accuracy than of about this range cannot be reached in most cases due to the uncertainty in the axial setting and the refraction effects which can never be calculated accurately. For special purposes (e.g. in metallurgy) precision microscopes are made with a large micrometer screw attached to the objective with an extended scale. These stands which are generally made for use with incident illumination are not often used for biological work. Measuring of the thickness of a section is also possible by using quite another approach, i.e. by interference micro-

1. Like the situation with length measurements in a single plane of focussing, it is often unnecessary to calculate exactly the absolute values of the travel corresponding with one division of the micrometer screw, as long as the readings are taken under standardized conditions.


scopy. As has been dealt with in chapter 9, the optical path difference (phase retardation) 'P which occurs when a band of light passes an object with a thickness and/or refractive index differing from that of its surroundings can be described with the formula

'P = (no - nm ) t

in which no and nm are the refractive indices of object and medium, respectively, and t the thickness of the object. When the object is a section in its totality, its thickness can be found without knowing no by mounting the section subsequently in two mounting media with different refractive indices and measuring the phase retardation each time with an interference microscope. The pair of equations thus obtained can be solved so that t becomes known (Barer, 1966).

Measurement of length oblique to the optical axis When the two ends of a length to be measured are not in the same focussing plane, measurement remains possible, but entails a few complications. The two transverse settings as read on the micrometer eyepiece yield a distance t which is a projection of the oblique length onto a plane perpendicular to the optical axis. When both ends differ by an axial distance a as measured with the fine adjustment, the real length will equate (t2 + a2), as this is simply the third side of a rectangular triangle with t and a as the two other sides. When a dry objective has been used and the distances have been measured in a medium with refractive index n, a has again to be multiplied by n, as explained in the preceding section. The measurement of these oblique distances suffers from several sources of error which accumulate, so that a very accurate result cannot be expected.

MEASUREMENT OF AREAS AND VOLUMES

Measurement of areas Generally, in using the term measurement of areas one refers to areas perpendicular to the optical axis, as areas parallel or oblique to the optical axis cannot be measured directly with a reasonable degree of accuracy. The area to be estimated will often be some kind of projection of a threedimensional structure into the object plane, as a completely two-dimensional surface (or a structure optically behaving as such) will occur only in the case of a section which is very thin in relation to the object. Under some circumstances a relatively thick section from a three-dimensional complex


may lead to projection areas which overlap each other, the so-called Holmes effect (fig. 11.7). This is important in stereology, the application of geometricstatistical measuring procedures used for obtaining quantitative information about three-dimensional structures from two-dimensional images. In this complex of techniques, which will be dealt with in the next section, use is often made of surface-area measurements of components of a section, in which the Holmes effect can introduce an important systematic error. No further comment on this effect will be given in this connection, except the general remark that a reduction in section thickness will considerably reduce the amount of overlap, as can easily be seen from fig. 11.7.

A B

Fig.n.7. The Holmes effect: the projected areas A and B ofa comparatively thick section of two adjacent three-dimensional structures can overlap each other.

In particle size analysis, use is often made of rather simple technical aids, such as comparing directly the projection areas of the particles to be measured with circles or areas with other geometrical forms in an eyepiece graticule or as a projected image on white paper on which circles etc. have be drawn. The difficulties met in defining the size of the projection area of an irregular particle are often underestimated; generally one measures what is described mathematically as the 'maximum inscribed figure', but this is by no means the only approach. When large numbers of particles have to be measured systematically, all the methods just mentioned are cumbersome and application of some form of automatic image analysis (page 270) has to be considered.

For measuring somewhat larger areas, use is often made of a grid in the eyepiece at the plane of the intermediary image (fig. 11.8). The area can be estimated by simply counting the number of squares occupied by the area to be measured. It is necessary, of course, to estimate fractions of a square

MEASUREMENTS OF AREAS AND VOLUMES 261

or make a home-rule to count e.g. no partly covered squares at the left and upper sides counting in full all partly covered squares at the right and bottom sides of the grid. Calibration of such a grid can again be made with a stage micrometer. An analogous procedure is to use a camera lucida for drawing the contours of the area to be measured on squared paper. Calibration can again be made with a stage micrometer, projected onto the squared paper.

Fig. 11.8. Measurement of an area with an eyepiece grid.

The surface of a drawn circumference of an object part, e.g. cells in a smear, can also be determined by use of a planimeter. Another technique used is that of cutting out the image on paper selected for constant thickness and weighing the cut-out pieces. Instead of paper thin metal foil can also be used; the technique is rather time consuming, but highly accurate and reproducible (cf. Sillevis Smitt et aI., 1969 and van Mens et aI., 1975).

When interdigiting patterns of surface areas have to be measured systematically, as is often the case in stereology, all techniques dealt with so far fail on practical and other grounds. In order to obtain precise information about the relative areas occupied by different components in a section, quite another approach has to be made which is now generally known as the point-counting method. Essentially. this method consists of superimposing a regular point lattice over the image and counting the points falling over the surface to be measured. This principle can be applied by bringing a graticule with regularly distributed points at the level of the intermediary image of a special eyepiece (fig. 11.9). Such a device, sometimes called an integration eyepiece, is usually provided with an adjustable front lens, so that the graticule can be focussed separately and thus observed in the same plane as the image. The same principle can also be applied by bringing a


lattice printed on transparent film material over a photomicrograph. The latter principle is applied especially in electron microscopy, where direct counting in the microscope is virtually impossible.

Fig. 11.9. Measurement of a surface in a microscopic specimen with an integration eyepiece; the area shown covers 10 of the 25 counting points.

Provided an optimal relation exists between areas to be measured and spacing of the counting points, the point-counting system is the most reliable way for determining the areas of irregularly dispersed surfaces, expecially in the way used for stereo logical analysis (see next section). It has also been shown to be superior and easier to handle than the so-called linear analysis (derived, like the point-counting system, from geological measuring techniques). Essentially, this is very similar to the point-counting system, but instead of the number of points falIing over an object, the total length of linear probes falling over an area to be measured is counted. The analogy of both systems can be verified with fig. 11.9. Although the system of linear integration, as used in the 19th century in other fields, has certain advantages and can be applied with the help of special integrating stages or specially designed integrating eyepieces, the point-counting system has been shown to be the most efficient.

When using an integration eyepiece, the spacing of the counting points with regard to the object can be varied only by changing the objective magnification; with a printed lattice as in fig. 11.10 it is possible, of course, to make transparent prints with any desired size of squares, so that the spacing between the test points may be varied at will. A double lattice system as shown in fig. 11.10 allows simultaneous estimation of structures which are widely different in their surface area; larger structures may be counted with the coarse grid using the fine grid for delineating of the periphery, obviating the necessity of counting all points of the fine grid over


Fig. 11.10. Counting grid, as used for point-counting, e.g. in transparent prints placed over photomicrographs or electron micrographs. The thick lines enclose nine smaller squares enabling a quicker counting of larger areas, using the individual small squares for precise counting at the borders and for integral countmg of small areas.

larger areas (cf. Weibel, 1969, 1972). The optimal spacing of the counting points depends on many factors, e.g. the relative percentage of the object field filled by the average area to be measured. A too close spacing should be avoided; this condition follows from statistical considerations (each point sample should be independent of the other) and the accuracy of the sampling does not increase linearly with point density (cf. Sitte in Weibel and Elias, 1967). Whereas unconsidered zeal does not pay under these circumstances, the density of sample points should be great enough so that not too many features are missed in an individual counting. It is sometimes mentioned that the spacing of the counting points should be 20-25% of the mean size of the average area to be measured, but it should always be kept in mind that the point-counting system is based on statistical distribution of random probes and that statistical tests are to be made whether a given component of a sectional area can be measured with a sufficient degree of reproducibility. General reviews of different aspects of the point-counting method are given by Elias et al. (1971) and Weibel (1972).

Even when hits are registered with mechanical or electronic counting devices, point-counting remains a rather labourious task. In many situations it has become possible to perform the measuring of areas which can be sufficiently discriminated against their surroundings with different kinds of electronic image-analysing equipment; the instrumentation for this will be dealt with briefly on page 270. From the point of view of the biological


microscopist, it may be stated that these fully automatic techniques cannot replace the point-counting system, as - in contrast to the circumstances in much geological and metallurgical work - the various components often differ insufficiently in contrast to enable a sharp discrimination on the basis of contrast level. The point-counting itself can often be reduced to a minimum by careful planning and the performance of statistically controlled trial measurements. Moreover, it is possible to reduce the strain of the counting procedure without the need for large series of photomicrographs by using a microscope with a projection head on the screen of which a transparent print of the test lattice is mounted. Especially when this is combined with an automatic or semi-automatic stepping stage such as described by Weibel (1970), a highly efficient semi-automatic system for the gathering of point-counting data is created (fig. 11.11), in which the image interpretation, however, remains completely the task of the investigator.

Fig. 11.11. Projection microscope, in combination with an automatic steppmg stage according to Weibel, ·provided with an electronic counting device for semi·automatic pointcounting.

Measurement afvalumes; stereolagy As has been explained in the previous sections, many problems exist with regard to the measurement of one- or two-dimensional parameters; when an attempt is made to measure directly three-dimensional microscopic objects, the difficulties quickly become insurmountable. Only the volume of objects which have a simple geometrical form can be measured with


some degree of accuracy. In the case of a spherical cell nucleus - a favoured subject in the pioneer period of biological morphometryl - the situation seems rather uncomplicated as by measuring the diameter the volume follows by simple calculation. For different reasons the value thus obtained for the volume of the nucleus has comparatively little meaning, however. Even when such a nucleus has a perfectly spherical shape (which will only seldom be the case), this measurement is subject to large errors as can be shown with the following example. Suppose that in measuring the nuclear diameter a reading of 11 divisions of a micrometer scale has been taken instead of the correct value, 10. When the real volume and that obtained on the basis of the false reading are compared, it appears that they differ by over 30%, starting with an error in the basic measurement which falls within reasonable limits (cf. page 255). It is self-evident that when such a nucleus deviates somewhat from a perfect spherical shape, as usually will be the case, the situation would become much worse. With a more elongated form of such a nucleus it is possible, of course, to apply the geometric formula for a rotation-ellipsoid. In most cases, however, this hardly yields more reliable results than simply averaging the long and short axis and treating the nucleus as a sphere, as of course no perfect ellipsoid is involved and anyhow only rather coarse differences can be demonstrated (e.g. a doubling of the nuclear volume). Moreover, in this field of measuring techniques for cell nuclei sometimes called kG/Tomet}}', differences in populations of nuclei are often investigated by just measuring diameters instead of converting these into volumes, now that the great inaccuracies in determination of volumes have become generally known. With objects diverging still further from perfect geometrical shapes, the possibilities for direct measurement of the volume begin to fail almost completely; eventual results, calculated on the basis of all kinds of extrapolations and with mathematical formulae founded on different suppositions have often hardly any value at a1l 2•

1. (Micro) morphometry, literally measuring of (or on the basis of) shape, is a term which can be considered as an analogue to (micro)photometry, the measuring of light absorbed by a microscopic specimen. (micro)refractometry, etc. The term morphometry as used in the field of histology. also called histometry can be considered as embracing all the kinds of measuring techniques dealt with so far. Whether the more specialized techniques of random sampling used in stereological analysis of three-dimensional configurations, as dealt with later on, are kept out of the notion morphometry (as some authors do, reserving the term morphometry for direct measurements in situ or with models), is a matter of terminology. 2. Quite another approach to the measurement of isolated microscopic objects, such as free cells in suspensions, is the use of a Coulter-counter. With this instrument the changes in electrical resistance are measured when the individual cells pass a thin capillary; to a


Only in some situations is another approach possible, i.e. in the case of small objects having a flattened form, so that the surface can be taken as a (rather crude) measure of their size. Under certain circumstances, such as with smeared flat cells or isolated nuclei, this can be of value for demonstrating some differences, but the limits of direct measurement of volume has here been reached. As for larger objects, indirect measurement of volumes of more complicated structures is possible sometimes via reconstructions from serial sections; this very tedious approach cannot be considered to belong to the field of microscopic measurements proper.

From the foregoing, it would seem that the prospects for the measurements of volumes in microscopic objects are rather dim and without much future for obtaining reliable data with regard to the complex interrelations of different components as observed in most biological specimens. It appears, however, than the possibilities are quite good, due to a development of indirect measuring techniques which has made use of principles which have been known for rather a long time in branches of science other than microscopy. Essentially, the basic idea goes back to the so-called principle of Delesse, formulated as early as 1848 by a French geologist of that name and demonstrated in later years to be mathematically correct. This principle states that in a mixed rock formation the relative volumes of the different components can be determined by measuring the relative surface areas of the different components in a number of random cut surfaces, instead of a direct measurement of the volumes which cannot be performed.

This very same principle can be applied to biological material consisting of different components. In measuring a number of random cut slices from a complicated three-dimensional structure, the volume fraction of the different components can therefore be found by measuring their relative surface area fraction in the slices, i.e. the sections. This dimensional reduction is a very important step, which has made possible the development of stereology in the study of biological material, as surface areas can - in contrast to volumes - be measured with great accuracy, as has been explained a few pages before. A section can of course be considered as a random cut slice; its thickness is not infinitely small, however (here the situation differs from the swface in the original Delesse model), so that the Holmes effect (see fig. 11.7) should be taken into account. This effect is due to the fact that sections have a finite thickness, so that certain structures which are

large degree this change in resistance parallels the volume of such free cells. Strictly, this is not a direct measurement of volume nor is it a microscopic technique. To a certain extent, the same holds true for volume analysis of microscopical particles via light scattering techniques (cf. Brunsting, 1974).


small in comparison with the section thickness are overestimated due to projection of the slice content. The Holmes effect becomes negligible when the section thickness is very small as compared with the structures to be measured (e.g. in most cases with electronmicrographs); Weibel and Elias (1967) mention an overestimation of the area of opaque structures of 15~~ when the diameter of the section is one tenth of that of the structure to be measured, whereas this fraction falls to 5% when this relation is 1/30. In many cases, therefore, a correction is necessary in light microscopic work.

As has been explained, the application of the Delesse principle makes it possible to reduce the measurement of volumes to the measurement of surface areas; to this end all techniques treated in the previous section can be applied, such as drawing with planimetry or cutting out and weighing, linear integration, point-counting methods and, where possible, determination of surface areas with automatic image analysis (see next section) in any form.

As explained before, in most cases where the volume fraction of different components of a complex tissue or organ have to be measured, the pointcounting system cannot be avoided. Whatever method is used for determination of the surface area, however, it should be kept in mind that the components of a structural pattern to be measured should have more or less a random distribution and even then any single plane of transection cannot be considered to be representative of the whole structure. Sample representation has to be achieved by adopting rigorous sampling procedures; underestimation of the difficulties enclosed in this entire measuring principle has often led to erroneous results (cf. Mayhew and Cruz, 1974).

Apart from these methods enabling an estimation of the volumetric composition of tissues, the so-called volume density of tissue components in a given volume, another stereological technique is to estimate the 3-dimensional surface areas or the sUlface density of a component in a given volume. This value can be measured with a lattice of linear probes. The number of intersections with these probes can be shown to give an estimate for the length of the border-line in a section of a three-dimensional structure; the length of these border-lines in a series of random sections can again be considered to give a measure of the surface area. Unlike the situation with determination of areas by point-counting, the orientation of the linear probes with regard to the three-dimensional object does matter for the result obtained. Fig. 11.12 shows clearly the large difference in intersection points obtained with the same line lattice with different orientation. For the situation where the structure to be measured does not have a random orien-


--~

_r\ n

1 '.J

/' "\ J '- ~ "" / /)..

\ ./ ~ r

,~ -~ }

V '-.J ~

A B

Fig. 11.12. Differences between numbers of intersection points with various positions of an object with regard to a lattice of parallel lines; with the same circumference, 24 and 8 intersection points are found with A and B, respectively.

tation, a special test system of non-systematically orientated lines or a test system with parallel curved lines such as that designed by Merz (1967) is used for measurement of intersection points (fig. 11.13). On the other hand,

Fig. 11.13. Test grid with parallel curved lines according to Men, as used for determining intersection points of non-randomly orientated surfaces.

systematic differences in intersection points with a test system of parallel lines in different rotatory positions can detect a certain orientation in the structural pattern of a three-dimensional objectl.

1. The same holds true for the measuring of intersection points by a television scanning system which measures with parallel image scan-lines.


In many instances a sUi/ace-lo-volume ralio, an estimate of the contact area of the structure per mass unit, is a very useful parameter which often gives an impression of the physiological circumstances prevailing in a biological structure. It can be determined by combining surface estimation by intersection with point-counting volumetry. The test system in common use for this type of measurement consists of a number of short lines with interruptions as long as the lines (fig. 11.14). The number of intersections falling

Fig. 11.14. Test system for determining surface-to-volume ratios.

over the short lines is counted, and with the same position of the test system the number of end points falling on sections of the structure is to be determined. A measure for the surface-to-volume ratio then follows by simple

division; strictly its value equals Ni , in which Ni and Np are the Np x Z

number of intersections and the number of 'hits' over the interior of the structure, respectively, and Z the length of the short lines.

As in most circumstances changes are more important than absolute values, often only the quotient NijNp is used in determining surface-tovolume relations; likewise, often only a relative value is determined in estimating volume- or surface-densities without calculating the relations per [Lm3. This is simply the same situation as with conventional 'absolute' and 'relative' length measurements, as dealt with at the beginning of this chapter.


AUTOMATIC AND SEMI-AUTOMATIC IMAGE ANALYSIS

As has become clear from the previous section, it cannot be denied that the point-counting method is time-consuming and laborious, although, by careful statistical planning, the counting can often be drastically limited. In some cases it is possible to perform the measurements of surfaces or interfaces by fully automatic techniques, instead of by point-counting or line intersection counting. In many cases, however, this is not possible, as it appears that a particular component of the specimen cannot be unequivocally discriminated on the basis of specific absorption characteristics. What the eye of an experienced observer recognizes at a glance by a complex of factors can often not be coded into simple criteria of contrast to which electronic detecting systems primarily respond. With the more sophisticated types of image analyzing computers, the possibility has arisen to program for discrimination of object parts on the basis of criteria of differences in shape or pattern (so-called texture analysis, cf. Klein and Serra, 1972). Moreover, some of the newest systems developed can perform also other types of measurement than the gathering of strictly morphological data, such as microdensitometric analysis, as performed on a television image.

Treatment of details of the various image analyzing systems which have been developed and become commercially available would fall outside the scope of this book (see Schaefer, 1972 and Fisher, 1972 for general information). The different systems vary widely, moreover, from more simple TV-type scanning integrators to highly complicated and versatile image-analyzing computers. A general outline of the basic ideas of such apparatus is indispensable, however, in view of the fact that they are used more and more in solving different types of problems in quantitative microscopy. All automatic image analyzing systems have in common that the speed is exceedingly high in comparison with point-counting by an observer (about 107 points/sec against maximally 5 points/sec with the human eye). Although this is undoubtedly of great advantage, the value of speed in itself if sometimes overemphasized. Apart from special circumstances, it is utterly unimportant whether a given reading can be produced in two seconds or thirty milliseconds when considerable time has to be spent in finding a correct field and adjusting the equipment at each measurement.

Generally speaking, instruments for image analysis can be subdivided into the following types, depending on the way the object is scanned: A. Flying-spot scanning; in this system the specimen is scanned by a small

light spot. This system has been used for earlier developments in image

AUTOMATIC AND SEMI-AUTOMATIC IMAGE ANALYSIS 271

analysis. For different reasons it has been abandoned with most recent systems.

B. Image-plane scanning; in contrast to the preceding system, in which the scanning movement is made by the light source, the scanning here is performed by a sensor which moves over the microscopic image. The

image is generally photometrically evaluated with a TV-system; imageplane scanning instruments are not necessarily TV-systems, however.

C. Specimen-plane scanning; here sensor and light source are static, whereas the specimen is moved on the stage in such a way that it is scanned systematically by the sensor. This type of instrument is usually somewhat slower than the fully electronic image-plane scanner, as the movement of the stage is fully mechanical. As stated before, however, speed is not always of primary importance, whereas it is often possible with specimenplane scanning to deal with a larger field than the object field of an objective.

Apart from individually adapted programmes for specific problems for which complicated additional equipment is necessary, all image analyzing devices from a simple TV-scanner to the most complicated machines can perform the following basic functions: 1. Counting of objects, as displayed on a television screen. These objects

have to show a sufficient degree of discrimination against the background. In a more or less complicated way, it is usually also possible to produce data with regard to the frequency-distribution of particles on the basis of diameter or area of the particles. This has been used e.g. for counting and analysis of dust particles and animal cells in suspension.

2. Measurement of sUifaces of discriminated areas. In this measurement of areas the contrast level often forms the central problem; as mentioned before, the variations in absorption in the image over a given area often do not have the sharp differences in contrast with regard to the surroundings required for sufficient discrimination. Even when this problem has been solved more or less satisfactorily, it appears that other elements of the specimen are counted together with the area to be measured. This can be illustrated with the following example. Fig. I 1.15 gives an over-all view of one of the more simple types of electronic image analyzers of the image-plane scanning type. On the screen of the television monitor an image of a section of a human lung is shown as seen with a magnification of 250 x. In fig. 11.15, lower left, an enlarged view of the microscopic image is shown as it is seen on the television screen and at right a discriminated area of the same image switched to the measuring position


Fig. 11.15. Upper image: General view of an image-scanning instrument for image analysis of simpler type with the television camera mounted on the microscope and the monitor on top of the control panel; a box with electronic circuits is not visible. Lower left: Screen of the monitor, displaying an image of a section from human lung tissue. Lower right: the same image with the instrument SWItched into measuring position: the discriminated (white) area can be read as a percentage of the total surface of the screen.

showing (in white) the surface area which can be read as a percentage of the total screen surface. When the relation lung tissue/air space has to be determined in sections, this could be done with point-counting e.g. with an equipment as shown in fig. 11.11. It would seem, however, that an electronic measuring system can do the job much better, as the


contrast lung tissue/air space is high, even with the routine-stain used here: no necessity exists for making any differentiation within the lung tissue. In practice, however, even in this very simple situation a few typical sources of error appear, which are not very disturbing in this particular case, but which could easily become so in other circumstances. On the one hand, some alveolar phagocytes lying free in the air space (which are in some instances much more numerous than in this particular field) are measured together with the lung tissue. On the other hand, open spaces in the lower of the two blood vessels visible (minimal here, but this can be rather important in some cases) are measured together with the air space. It would be possible with one of the more sophisticated image analyzers to correct this with a light pen used on the screen, 'wiping out' the alveolar phagocytes on the screen and filling up the blood vessels. Apart from a loss in the objectivity (often said to be an advantage of automatic analysis) of the measurements thus corrected, it can amount to quite a deal of work in some circumstances.

3. Determination of intersection points; circumferences. In the more simple TV-scanning systems, this consists in an electronic counting of intersection points of the border of a discriminated object with the scan lines. For different reasons this is not necessarily a measure for (half) its circumference. As shown already in fig. 1l.l2, the form of the object can influence the number of such intersection points, be it with a test system for visual counting or with electronic scan lines. A reading with the scan movement in only one direction is of very limited value, therefore. By rotating the object and taking readings of the intersection points recorded, it is possible to trace certain orientations in an object, such as the course of bone trabeculae in spongious bone. With the more highly developed systems for image analysis, length measurements of a circumference and numbers of intersections can be measured independently from one another.

4. Other parameters. The image analyzing computers as they have been developed in the last five years are capable of a wider range of parameter analysis, such as pattern recognition, automatic selection of object parts with certain spectral properties, and in addition possibilities have been created to apply image analyzing systems for microdensitometry and microfluorimetry (see next section). In most cases, the facilities required for a given case where image analysis on a large scale has to be performed should be carefully weighed before buying such a large image analyzer. The price of such equipment can easily exceed that of a modern electron microscope, so that it is worth careful consideration before


Fig. 11.16. Photographs, as taken from the television screen of the image analyzing apparatus shown in fig. 11.15, displaying isolated rat liver cells stained with Feulgen Naphthol Yellow-S. Upper left: aspect of cells on the screen as seen with green lIght (560 nm maximum transmittance) with very low image contrast in the cytoplasm and dominating cell nuclei. Lower left: the same image, clear discrimination of the nuclei in the measuring position. Upper right: the same cells, as seen on the screen with light of a wavelength of 430 nm maximum transmittance showing a dark cytoplasm due to bound Naphthol Yellow-S, with below the same image with the apparatus in measuring position, showing clear discrimination of the entire cellular area (From Tas, Oud and James, 1974).

buying a 'complete' equipment, having probably quite a few possibilities which will never be used, but have to be paid for.

Apart from the instrumentation, it should never be forgotten that the specimen itself should reveal its information in its most clear way to the image analyzing instrument. This does not mean that a staining technique


which does quite well for visual observation would also be suited for image analysis. It is sometimes necessary to develop special techniques which are not very impressing under the microscope or have too high a contrast, but can produce - in combination with light of an appropiate wavelength range - a high contrast enabling good discrimination with an image analyzing system. An example of this is the use of Naphthol Yellow-S as investigated by Tas, Oud and James (1974) which, in combination with the Feulgen technique makes it possible to discriminate selectively the surface area of an entire cell as well as the nuclear area with image analysis from the same specimen (fig. 11.16). These types of stains are more suited for use with image analyzing systems than classic stains like haematoxylin-phloxin, which has been used in fig. 11.15 only for demonstration purposes. The applications of image analysis can be widened and the results made more reliable with specific types of specimen preparation. It is simply not fair to reproach an image-analysis apparatus that it cannot cope with a specimen in which an infinite array of gray tones exists which all have another meaning in relation to structural characteristics in the specimen. Even with the newest developments in image analyzers, it remains highly improbable that the experienced human eye and brain will be superceded by an imageanalyzing computer, so that in many cases point-counting will remain indispensable.

MICROSPECTROPHOTOMETRY AND MICROPHOTOMETRY

Microspectrophotometry A microspectrophotometer is an instrument for measuring the light absorption ofa part ofa microscopic object at different wavelengths. As a matter of fact, this is nothing but an analogon in the microscopic field of the cuvettemicrospectrophotometer long used in analytical chemistry. Essentially, such an instrument consists of a powerful light source in combination with a socalled monochromator which produces light of narrow spectral range, a microscope provided with special stops so that a small region can be focussed in the specimen, a sensitive photocell (presently a photomultiplier tube) combined with an amplifier and finally an output device, usually a recorder.

A monochromator would ideally produce light of a single wavelength, e.g. by the isolation of a spectral emission line; for different reasons, however, this is not feasable in practice. Usually light of a certain bandwidth (as narrow as possible, a few nm, or even less) is selected by dispersion on a prism from the light emitted by a powerful source with continuous spectral


emittance, e.g. a xenon burner. The specimen is focussed (often with ordinary illumination) and located with regard to the monochromatic spot of light and the spectrum is recorded. With the larger types of recording microspectrophotometers, the movement of the prism in the monochromator is fully automatic, so that the wavelength of the light sent through the specimen changes at a determined speed. When the instrument has been built according to the dual-beam optical system, a control specimen (with the same slide, cover-glass and mounting medium etc.) should be brought in the beam of a control microscope; a chopper then divides the illumination beam into two components which pulse alternatively in time, before reaching the detecting device.

In principle, the whole measuring procedure is analogous to that with a cuvette system; only the pathway through the specimen is much shorter and the band of monochromatic light passing the specimen has to be as narrow as possible, in order to select a given spot in the usually very heterogeneous object. The light bundle which passes the specimen is in most cases of the order of 1 [Lm diameter; a very powerful amplification is necessary, therefore, to enable a recording of the very weak signal coming from such a minute spot.

Fig. 11.17 gives an illustration of the possibilities which exist with microspectrophotometry. The two spectral absorption curves shown were recorded with a round measuring spot of 1 [Lm diameter through the cytoplasm and the nucleus of a red blood cell of a salamander. The haemoglobin present in the cytoplasm of this cell causes first an absorption maximum near 545 nm, whereas a second, much higher, absorption maximum occurs at 416 nm, the so-called Soret-band of the haem. In the ultraviolet, the absorption at first recedes, increasing at 300-320 nm to a third plateau, mainly due to a more aspecific protein-absorption. In the cell nucleus also an absorption maximum is observed at 416 nm; this is due for a large part to haemoglobin-containing cytoplasm, lying over and under the nucleus which is met by the passing band of light. In the lower wavelength region, a very conspicuous absorption can be observed in the ultraviolet which increases clearly to a maximum in the 260 nm region. Apart from the protein absorption, this second plateau is caused especially by the high concentration of nucleic acids in the cell nucleus in the 260 nm region. The two photomicrographs in fig. 11.17 have been made in the micro spectrophotometer at a wavelength of 416 and 260 nm, respectively, with a bandwidth of a few nanometers; they illustrate great differences in image contrast. The very high image contrast in the cell nucleus at 260 nm in the right hand photograph has even caused a total loss of all details in the nucleus, still visible

MICROSPECTROPHOTOMETRY AND MICROPHOTOMETRY 277

Fig. 11.17. Transmission curves as registered with a microspectrophotometer with a measuring spot of I lim 0 through the cytoplasm (I) and the cell nucleus (II) of a red blood cell of a salamander (Triturus sp.). Both photomicrographs made with a panchromatic emulsion at 416 and 260 nm have been localized in their corresponding positions at right and left, respectively. The photomicrograph at 416 nm indicates the aspect of the cell as could have been observed with the eye with a sufficient brightness of the image; at 260 nm an image converter would have been necessary to observe this image.

in the other photograph. These are of course the same rules of contrast formation which have been dealt with in chapter 10.

Due to the necessity for a huge amplification of the very weak signal from the measuring spot which has a surface of the order of I !J.m2, the precision is less than can be reached with conventional analytical spectrophotometry, so that very small shifts (of one or a few nanometers) would escape detection. On the other hand, a great advantage lies in the fact that micro spectrophotometry can be used as an analytical tool with a variety of biological materials in which spectral absorption can only be investigated in situ.

Absorption curves as shown in fig. 11.17, which are usually made over only a part of the spectrum, can be used to identify unknown substances on the basis of their absorption characteristics. This has been applied by Morselt, Cambier and James (1973) for the identification of breakdown products of haemoglobin during intracellular digestion of engulfed erythro-


Fig. 11.18. Electron micrograph of an ultrathin section from a macrophage with erythrocytes in different stages of digestion, 8000 x ; inset: the same cell in the immediately following 1.5 !J.m thick section, as photographed at a wavelength of 416 nm. The absorption spectra below are recorded from a measuring spot of 1 !J.m 0 centrally in cell parts with the corresponding figures (From Morselt, Cambier and James, 1973).

MICROSPECTROPHOTOMETR Y AND MICROPHOTOMETRY 279

cytes in macrophages, on the basis of combined light- and electronmicroscopic images of these cells (fig. 11.18).

Microphotometry When the absorption characteristics of a certain light absorbing substance (a chromophore) in a solution has become known by spectrophotometric analysis, it is in most cases comparatively easy to extend this to obtain quantitative data about the amount of such a chromophore in a solution by so-called absorption analysis or photometry. This is a widely used technique in analytical chemistry which was applied to microscopy by Caspersson in the late thirties, originally mainly for use in the ultraviolet region of the spectrum, but applied now more often for absorption analysis in the visible region. The circumstances in which chromophores are bound in a microscopic specimen, be it a naturally absorbing substance such as haemoglobin, or a dyestuff bound in the specimen, are totally different from those with a dissolved substance in a cuvette. The instrumentation for microphotometry or cytophotometry, as the photometric analysis of microscopical objects is called, asks for entirely specialized equipment, therefore. The reasons for this will be explained below.

When light falls through a cuvette with a homogeneous solution of a sample of a light absorbing substance, a portion of the incident light is reflected, a portion is scattered, a portion is absorbed, whereas the remainder is transmitted. Under normal conditions in analytical photometry with a parallel light beam, reflection and scatter form only a minor portion of the difference between incident and transmitted light with a substance showing any degree of absorption near the wavelength of the incident light. As for absorption, the Lambert-Beer set of rules! applies. Essentially, the basic principle of this law states that the proportion of the incident light absorbed by a medium is independent of its intensity and that each successive unit layer of the medium absorbs an equal fraction of the light passing through; it is an exponential phenomenon, therefore. The ratio of the intensity of the

transmitted light to that of the incident light ~ is called the transmission, T 10

and when expressed in percent the percentage transmittance, % T. The

reciprocal value of the transmission _1_, sometimes called the opacity, is T

1. In his 'Essai d'optique sur la gradation de la lumiere' (Paris, 1729) the French physicist Bouguer, professor of hydrography in Le Havre, was the first to describe these basic rules. Johann Heinrich Lambert, in his 'Photometria' (Augsburg, 1760) credits Bouguer with the discovery of the law which is, however, mostly called after the former and Beer, whose contribution dates from the nineteenth century.


related to the light absorbed, but cannot be used as a direct measure for the amount of chromophore (absorbing matter), due to the exponential relation just described. When certain conditions are met (such as homogeneity of the solution and absence of certain concentration effects) the logarithm to base 10 of this value appears to parallel the concentration of the chromophore under standard conditions (wavelength of the light, path length). This factor is called extinction (E) or optical density (OD) and it can thus be defined as the logarithm to base 10 of the ratio between the intensities of the light beam before and after leaving a sample:

To I E = loglo- = log- = -log T.

It T This means that the extinction can always easily be found on the basis of the transmission recorded. Table Xl lists a few representative conversions of

I T,-andE.

T Provided that the chromophore in solution obeys the Lambert-Beer law (which is not always the case and should be checked by measuring a series of concentrations), the following general formula will apply in absorption photometry:

E = K.C.L.

in which K is a constant factor which is characteristic for the chromophore under the conditions of solution and the wavelength of the light (extinction coefficient), C the concentration of the chromophore and L the path length.

K and L (the cuvette inner diameter) remaining constant, E and C will vary proportionally; in other words, at half the pathway and twice the concentration, the reading should remain constant. Readings are mostly taken at the top of the absorption curve for those substances showing a specific absorption spectrum, to obtain a high signal/noise ratio.

The measurement of a certain amount of chromophore in a microscopic specimen is far from simple, even when the bound dyestuff (or a natural pigment such as haemoglobin) has an exactly known absorption spectrum and has been shown to obey the Lambert-Beer law. Not only is the optical density in such an object very inhomogeneous, also its form is irregular and the path length unknown. The circumstances which approach most closely that of a cuvette is that in which the form of the object part to be measured is a regular geometrical one. This occurs e.g. with a cell nucleus which has a globular shape in most cases; it is with cell nuclei that the first cytophoto-


TABLE XI. ABSORBANCE, TRANSMITTANCE AND EXTINCTION.

% Absorbance % Transmittance Extinction

0 100 0.000 5 95 0.022

10 90 0.046 15 85 0.070 20 80 0.097 25 75 0.125 30 70 0.155 35 65 0.187 40 60 0.222 45 55 0.260 50 50 0.301 55 45 0.347 60 40 0.398 65 35 0.456 70 30 0.523 75 25 0.602 80 20 0.699 85 15 0.824 90 10 1.000 95 5 1.301 99 1 2.000 99.9 0.1 3.000

100 0 00 (opaque)

metric measurements have been made. In theory, a Feulgen-stained nucleus could be considered as a solution of the magenta dye in a globular cuvette amidst an unstained cytoplasm. In determing an average extinction near the absorption maximum of the dye and measuring the volume of the nucleus by one of the methods described in the preceding section, it would seem possible to find a measure for the total amount of dye present in the sphere.

This technique, in which the average extinction of a nucleus is determined with a plug-like sample has been much used in the early development of microphotometry (plug-method, also called method of Lison). Apart from the fact that this method cannot be used for objects with an irregular shape, the technique suffers severely from an important source of error which is typical for microphotometry of biological objects, the so-called distribution error. This phenomenon is a consequence of the fact that the chromophore is not homogeneously distributed; the average transmission recorded (out ofa number of unequal local transmissions), gives rise to a calculated extinc-


tion which is less than the mean of the logarithms of the different transmissions occurring in the object. This error may be quite considerable; it varies with the product of the mean optical density and the variance of optical density.

Several approaches have been developed to overcome this effect of heterogeneity of chromophore distribution of which the most important are the photographic method, the two-wavelength method and the use of scanning and integrating cytophotometers (for a review see Mayall and Mendelsohn, 1970 and Wied and Bahr, 1970). With scanning instruments, which are presently mainly used, the object is divided into subunits so small that they can be regarded as homogeneous. By summating a whole series of extinction readings, an integrated extinction is obtained of the whole object giving a value (in arbitrary 'machine' -units as the extinction coefficient of the dye as bound in the nucleus is unknown) for the total amount of chromophore. This is reached by shifting the stage in a scanning movement with the specimen on a fixed sensor (object-scanning instruments) or by scanning the image with a moving hole in front of the photomultiplier (image-scanning instruments). In both cases the instrument converts the transmissions recorded into extinctions and integrates the large number of readings taken over the object. Different scanning instruments are on the market now and it may be stated that they have a high degree of precision and accuracy; a high degree of precision (reproducibility of results) does not necessary mean that the values obtained are accurate, i.e. close to the 'true' value (cf. Goldstein, 1970, 1971). When a high degree of accuracy and precision has to be reached, a number of circumstances have to be rigorously under control, such as stray light, monochromaticity of the illumination, absence of refraction phenomena at the borders of the object to be measured. The stray light can be reduced greatly in reducing the illumination to the object proper or object part to be measured (in a certain sense an exaggeration of the principle of the Kohler-illumination). Narrow band filters or a monochromator will prevent errors due to too great a diversity of wavelength in the illumination beam, whereas it is necessary to mount the specimen in a liquid or hardening medium which matches the object to be measured rather closely (see appendix I). Anyhow, it should not be forgotten that in scanning photometry (and many other types of measurement) the image to be analyzed does not necessarily show sharp contrasts or other signs of good image quality. Due to the fact that a low condenser aperture is often applied to avoid oblique rays in the illumination beam and - especially in image-scanning instruments - the image is often enlarged beyond the limit of useful magnification, such an image may look quite unpromising to the

MICROSPECTROPHOTOMETR Y AND MICROPHOTOMETRY 283

human eye. Electronic scanning of an image is not in the least disturbed by e.g. unsharp borders, however; on the contrary, it may even be that an optimal deduction of absorption data from the image with a moving sensor can only be derived from an image in empty magnification.

Using one of the modern image- or object-scanning and integrating microphotometers and paying attention to the different sources of error mentioned, it is possible e.g. to measure directly amounts of haemoglobin in the individual erythrocytes of 30 pg (30 X 10-12 grams) with a reproducibility of a few percent (cf. Morselt and James, 1971). Nuclear DNA (about 6 pg per nucleus in mammals) can be measured with a similar accuracy and precision via the specific Feulgen staining ('Feulgen-DNA content'), or alternatively on the basis of the natural absorption of the nucleic acids corrected for interference of proteins (cf. Sand ritter, 1958; Gledhill et ai., 1966). In particular it has been proven possible with the cytophotometric FeulgenDNA measurement to detect minor changes in certain cell nuclei of a cellular population showing early signs of DNA-breakdown which would effect insufficiently the mean values of data gathered with a large population of cells (cf. James, 1968). This is a clear example of the advantage of me asurement of individual cells with cytophotometry; other examples can be given from the analysis of red cell populations (cf. Morselt and James, 1971; James and Goldstein, 1974).

MICROSPECTROFLUOROMETRY AND MICROFLUOROMETRY

Just as a distinction should be made between the qualitative spectral absorption pattern at different wavelengths obtained by microspectrophotometry and the determination of the amount of chromophore at a fixed wavelength with microphotometry, a differentiation has to be made between the two analogous measurement techniques in fluorescence microscopy. Microspectrojluorometry essentially deals with instrumentation and results of the determination of excitation and emission spectra in fluorescent microscopic objects. In analytical microspectrofluorometry unknown substances may be identified on the basis of their excitation- and emissionspectra (cf. Rost & Pearse, 1974). In quantitative fluorescence microscopy (also called micro fluorometry or fluorescence cytophotometry), the quantity of the fluorescent substance is determined on the basis of the amount of fluorescent light given off by the specimen. Although problems related to adequate filters, lack of specificity of fluorescent dyes and different instrumentation problems have limited its application, it may be stated that at


the present time, microfluorometric histochemical tests have found an expanding field of application in protein histochemistry, fluorescent antibody techniques and even with enzyme reactions (longkind et aI., 1974).

Generally speaking, fluorescence cytophotometry has a few very obvious advantages over cytophotometry on the basis of light absorption. In the first place, the method is highly sensitive to extremely small quantities of fluorescent substances, when the background is sufficiently dark. A second advantage, briefly touched upon in chapter 8, is of quite another nature; as fluorescent molecules function as self-Iuminating objects, the number of light quanta radiating from the object will yield the same reading on the photomultiplier, independent of the regular or irregular distribution of the fluorescent molecules over the specimen. This means that the techniques for avoiding the distributional error (e.g. scanning and integrating devices) are not necessary for fluorescence cytophotometry, so that the instrumentation can be comparatively simple. Reviews of this field are given by B6hm (1972) and Sengbusch and Thaer (1973). Scanning techniques have been introduced recently in cytofluorometry for quite another reason, i.e. the possibility of measuring extremely small quantities of fluorescent substance, which would be lost when measuring an entire field.

Quite another application of fluorescence microscopy in the field of cytophotometry is its application in flow system analysis. This measurement via fluorescent light pulses can be considered as a further development of existing electronic cell-sizing apparatus (Coulter) and optical sensing flow systems dealt with on page 266. With this new system, cells are stained with a fluorochrome, binding to specific cell constituents (DNA, proteins); when the cells flow in a capillary across a beam of excitation light, the emitted pulses of fluorescent light are sensed by a photomultiplier. These systems, called impulse cytophotometry (Dittrich and G6hde, 1969) or jlow microjluorometry (Steinkamp et aI., 1974) have a great advantage in their speed, so that huge series of cells can be measured in a few minutes. They can be used only with homogeneous cell populations, however, as the sensor can make no distinction between e.g. a clump of small cells and a single large one. So far, the most promising application has been the clinical investigation of blood cells in leukaemia and related disorders.



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E. R. Weibel and H. Elias (eds.): Quantitative methods in morphology. Springer Verlag, Berlin-Heidelberg-New York 1967.

G. L. Wied and G. F. Bahr (eds.): Introduction to quantitative cytochemistry, II. Academic Press, New York-London 1970.

CHAPTER 12

MICROSCOPY WITH INVISIBLE ELECTROMAGNETIC RADIATION

MICROSCOPY AND THE ELECTROMAGNETIC SPECTRUM

The image forming agent with all microscopic techniques described in the preceding chapters was light, i.e. electromagnetic radiation with a wavelength within certain limits. The range of wavelength which can be perceived with the human eye, visible light, forms only a small fraction of the wavelength range of what is called light and this again is only a very small segment of the electromagnetic spectrum, as can be seen from the review scheme of fig. 12.1. The differences between the various types of electro-

\ vIsible / radio waves micro waves '~Igh~... X-rays

-------"--- ~---.. ',I' ,.--A---------.. Infrared : iultralilolet 1) - rays cosmIc rays

_---"-------, :: -A. __ , .. ~ _______ ~ , It'· I I I • I I I I =:::

10-2 10-4 10-6 10-8 10-10 A In meters

=lmm .lfJm =lnm

Fig. 12.1. Schematic view of the electromagnetic spectrum; AB is the region (from infrared light to hard X-rays) which is applied in microscopy.

magnetic radiation are not as sharp as suggested in this scheme, however; in fact this largely historical nomenclature is more related to the different sources of radiation than to its actual physical nature. The overlap between the different types of radiation is physically unimportant, therefore; properties like propagation speed are the same throughout the whole range, the only difference being found in vibration frequency and consequently wavelength: the higher the frequency, the shorter the wavelength and the greater the energy. These different wavelength classes of electromagnetic

MICROSCOPY AND THE ELECTROMAGNETIC SPECTRUM 289

radiation are generated by widely different sources: radiowaves and microwaves from electrons moving in conductors, infrared from heated objects, visible light from very hot objects and heated gasses, ultraviolet from arcs and gas discharges, X-rays from electrons striking a target and gamma rays from nuclei of radio-active atoms.

With these rather unsharp limits of what should be called light, between microwaves and X-rays, the limits of visible light also are not entirely fixed. The sensitivity of the human eye does not fall to zero at the limits set in fig. 12.1; from the area of highest sensitivity near a wavelength of 550 nm it tails off gradually to nihil in the range 360-380 nm, passing into the invisible ultraviolet and in the 730-760 nm range at the other end of the visible spectrum (at first still seen as deep red) into infrared. Radiation from the infrared, as generated by moderately heated bodies cannot be seen beyond a wavelength of 760-780 nm (this is individually different) but is felt as heat radiation, up to a wavelength of the order of 1 mm, where it is overlapped by microwaves and radiowaves which have found no application in microscopy.

Powerful ultraviolet radiation seems to be observed as colourless grey. This is a situation which should be avoided by all means, by the way, even for a short moment, as it can - even when the eye has been exposed from the side - damage the cornea. When it has passed a few layers of glass, ultraviolet light is generally sufficiently attenuated to be safe for not too long an exposure. Ultraviolet radiation which enters the eye causes visible light to be formed by fluorescence, especially in the eye lens. It is somewhat dubious, therefore, whether the grey 'seen' by some people in the 360-370 nm region, really is due to direct perception of that radiation. At the shortwaved limit of the ultraviolet, there is an overlap with X-rays which reach down toan order of wavelength of 10-3 nm. X-rays as used in microscopy usually are of a wavelength range of a few tenths of a nm, a factor thousand under the deepest violet which can be perceived by the human eye. Seen in the broad scala of the electromagnetic radiation as a whole, however, only a minor fraction of it is applied in the field of microscopy (A-B in fig. 12.1).

Another type of radiation which also has a wave-character is produced by accelerated electrons; these electron rays have become of great importance as they have made possible the development of electron microscopy. It should be emphasized that this type of radiation is not an electromagnetic radiation, although it behaves as such in many respects. Several physical properties of electron rays have enabled the development, in less than 30 years, of the complex technical universum of electron microscopy, which

290 MICROSCOPY WITH INVISIBLE RADIATION

will not be dealt with in this book!. A few remarks about the fundamental differences between light- and electron-microscopy which are often overlooked will be made, however. The first and all-important basic fact is that the wavelength of accelerated electrons as they are used in electron microscopy is of the order of 1 :100.000 of that of visible light. When placed in the spectrum of fig. 12.1 (where they do not belong) they would fall, at a very commonly used beam voltage of 60 KV, in the range of the extremely shortwaved y-rays and cosmic rays with a wavelength of the order of 0.005 nm. As the formulae for minimum resolvable distance in light microscopy appear also to hold true for electron microscopy, one could expect a corresponding increase in resolving power, with a minimum resolvable distance of e.g. 0.002 nm or so. This conclusion is far from the realisable possibilities, however, because of the fact that the electron lenses used to deflect electron beams in forming the image, can easily be made of extremely short focal length, but suffer severely from lens aberrations which are entirely comparable with those occurring with light in ordinary lenses. As the various types of aberration which occur with electron rays (of which spherical aberration is the most important) have different relations to the aperture of the electromagnetic lenses used, a compromise has to be sought. It has been found that the aperture has to be reduced drastically for optimal functioning of the electron-optical system. Curiously enough, this is exactly the means which microscopists in the distant past had to apply when uncorrected lenses were used in the compound microscope! The reason for van Leeuwenhoek's superiority with his simple microscope suffering only once from lens aberrations stems from the same phenomenon. Apart from astigmatism, aberrations like spherical aberration cannot be compensated for in electron lenses except by aperture reduction: the aperture angle of an objective lens in the 50-lOOK V beam voltage range is of the order of t o ; with a modern 40 X light microscopic objective an average value is 800 • As for the resulting situation, however, the circumstances are totally different, in that the extremely short wavelength of the electron rays amply compensates for the small aperture dictated by the electron optical system. In conclusion, it may be stated that the limits for the maximum resolving power of a modern

1. A few recent introductory monographs cover this subject adequately (Meek, 1970; Wischnitzer, 1970; Hayat, 1972; Huxley and Klug, 1971; Agar et ai., 1974). This is one of the reasons why, in contrast to an earlier version of this book published in Dutch in 1969, electron microscopy has been treated in this edition only in a bird-eye's view. Faced with the choice between a very considerable extension and revision of the section on electron microscopy in its original form on the one hand and the limitation of this revised version to light microscopy with a chapter on the application of invisible electromagnetic radiation on the other hand, the second approach has been chosen.

MICROSCOPY AND THE ELECTROMAGNETIC SPECTRUM 291

electron microscope have been set indirectly by spherical aberration and not directly by diffraction, as is the case for the light microscope. With a theoretical minimum resolvable distance of 0.3-0.5 nm (under optimal conditions and in special types of crystal lattices which are far removed from most practical situations), the gain is stilI of the order of 2000.

The comparatively strict analogy between deflected electron beams and

light rays bent by refraction (even the classical lens formula 2- +~ = ~ o 1 f

as dealt with in chapter 1 holds true 1) is the more remarkable, as the way in which contrasts are formed when both are used as image-forming agents is totally different. With the light microscope, absorption is as a rule the main factor in contrast formation, apart from contributions of refraction and diffraction (cf. chapter 7). In the electron microscope, the image formed is of course invisible; it can be revealed with a fluorescent screen or photographic material. The contrasts in the image are determined, under the conditions of conventional transmission electron microscopy, mainly by local differences in density in the object, i.e. differences in the power to scatter electrons. A prerequisite for this is the use of sufficiently thin sections or other specimens (of the order of 50 nm), reducing absorption phenomena below a certain level, so that they no longer playa role of any importance in image formation. Heavy metals used to enhance contrasts do this on the basis of their high electron scattering power.

Electron rays are used not only in imaging objects by transmission, but also in studying the response from the surface of a specimen struck with incident electrons. In contrast to the circumstances with incident illumination with light microscopy, this is not made by radiating an object with a broad bundle of accelerated electrons. Although in the past many experiments have been made with such mirror-electron microscopes, the large difference in the energy (and consequently, wavelengths) of the reflected electrons created a confusing picture with a low quality image of the surface to be studied. Between 1945 and 1965, important technical developments have been made which have led to the commercial construction (since about 1965) of another type of electron microscope, the scanning electron microscope (or for short, S.E.M.) which, in contrast to the conventional transmission electron microscope (T.E.M.) is used to study surfaces. With this very important new instrument, the specimen surface is scanned systematically with a saw-tooth movement by a very narrow ('" 0.01 !lm) bundle of accelerated electrons; this saw-tooth movement is brought over simultaneously to the deflecting coils of a cathode-ray tube. The electrons striking the surface generate a secondary emission of electrons (a phenomenon which can


be compared with fluorescence of light) which are attracted towards a collector by its positive charge. As there is a point-by-point correspondence of the electron probe striking the object surface and the pattern of the spot on the cathode-ray tube, the brightness of which can be made to relate to the electrons striking the collector, the variation in brightness of the displayed image corresponds to differences in emission of secondary electrons of the region of the specimen scann~d, so that an impression may be gained about its surface pattern. The resolving power of such a system is lower than that with conventional transmission electron microscopy; due to fundamental laws of electron optics and the circumstance that some penetration of electrons into the specimen cannot be avoided (even with a thin metal coating) the limit of the minimal resolvable distance is in the 0.01 [lom (10 nm) range. The real advantage of the S.E.M. lies not primarily in its resolving power (in which it does not equlli the T.E.M., although surpassing the light microscope with a factor 25), but in its ability to study surfaces in a magnification range from 20 x to 10 5 x with a very considerable depth of field. This latter can be considered as the outstanding advantage of the scanning electron microscope, even in comparison to a light microscope with incident illumination. Details about the functioning ofa scanning electron microscope, specimen preparation and fields of application are treated in a number of recent reviews!.

Another field of application of accelerated particles as an imaging agent is that based on the point-projection principle. Essentially, this comes down to making an enlarged image of a specimen by placing it in front of a point source of illumination (or irradiation). This principle can be used with light or electrons (where it has no advantage over existing imaging systems) or X-rays, as will be dealt with further on in this chapter. Another development which will be mentioned very briefly in this connection is that in which the structure of a metal tip is imaged at high magnification by means of electrons (field-emission) or ionized gas atoms (field-ion) emitted from the object under the influence of a powerful electric field. This interesting technique has been used to image the atomic arrangement of tips of many different metals with an exceedingly high resolving power (separating distances as small as 0.2 nm) in the case of field-ion microscopy (F.I.M.) with suitable gasses such as helium (cf. MUller, 1970). It has not been possible to use this principle with biological material, although attempts have been made to apply the high resolution of the F.I.M. in the study of certain macromolecules. I. Hearle, Sparrow and Cross (1972), Oatley, 1972; Hollenberg and Erickson (1973).

UL TRAVIOLET MICROSCOPY 293

ULTRA VIOLET MICROSCOPY

In using ultraviolet light of below 380-360 nm for the forming of an image of a specimen, problems start right away with the material for object slides, coverslips and lenses: most kinds of ordinary glass start to absorb so heavily from the 340 nm range downwards, that it quickly becomes impossible to form an image with glass lenses. Virtually all transparent plastics have a similar transmission limit and are likewise of no use in this connection. One is obliged, therefore, to use costly materials like quartz (transparent for UV down to 200 nm), fluorite (transparent down to 185 nm) or lithium fluoride. It has been shown possible to construct ultraviolet microscopes with objectives of short focal length and high N.A. Apart from such special optics use has to be made of a light source emitting a sufficient amount of radiant energy in the ultraviolet (any conventional incandescent lamp has an output of zero here, cf. chapter 6). The specimens have to be mounted between a quartz object slide (usually made smaller and less thick than ordinary glass slides, 25 x 37.5 X 0.5 mm) and a quartz cover glass which have to be made thicker than glass ones ('" 0.025 mm) and for which ultraviolet objectives have to be corrected. Finally, the mounting medium and contingent immersion fluid should be transparent for ultraviolet light; for both use is often made of anhydrous glycerin.

It is clear from the foregoing that the use of ultraviolet light as an imaging agent entails a series of complications in practical microscopy, as compared with conventional circumstances. Although this technique has been introduced originally to enlarge the resolving power, it is applied at present mainly for micro spectrophotometric and microphotometric investigation of substances showing selective absorption in the ultraviolet.

In the years around 1900, when Abbe's theories about the influence of the wavelength of the light on the resolving power were well-known and nobody had even thought of electron microscopy, the shortening of the wavelength of the light seemed the only possible means to reduce the minimum resolvable distance of the microscope. As early as 1904, the pioneer A. Kohler managed to construct a quartz objective for use in the ultraviolet. With just quartz at his disposal as material for lenses, he could only reach a certain correction for spherical aberration in his objectives; with a combination of lenses from the same material no correction for chromatic aberration is possible, however. Kohler has drawn the ultimate consequence of this: his lenses were constructed for use at one wavelength only, the 275 nm line isolated from a light arc. For many years, these so-called monochromatic objectives were the only ultraviolet objectives known. The results obtained


with this equipment, rather advanced for the period 1900-1930, showed indeed a measurable gain in resolving power; yet this development has not fulfilled the expectations of that pioneer period. This is due to two main factors; in the first place, the increase in resolving power which could thus be reached has become overshadowed by the rise of electron microscopy.1 In the second place, it appeared that even with optimal instrumentation as has become possible in later years, the results in this direction tend to be somewhat disappointing. Every book on microscopy mentions with more or less emphasis the gain in resolving power to be reached with ultraviolet light. It makes undoubtedly a clear difference (fig. 12.2), but only seldom

Fig. 12.2. Photomicrograph, made at a wavelength of 265 nm of an unstained smear of human bone marrow, using a 100 x N.A. 1.25 glycerin-immersion objective, 2000 x ; note the strong absorption by nucleic acids in the cell nuclei. In spite of the high final magnification in the print, there is no empty magnification. Out-of-focus areas are due to the extreme shallow depth offield and curvature of field. Note the heavy absorption of a contamination at lower left.

the theoretically expected gain (i.e. a factor of exactly two with light of 275 nm instead of 550 nm) has actually been measured. This is due to different causes. In the first place the wavelength has a direct influence on the depth of field (see the formula on page 88), so that enhancement of the resolving power becomes evident only with very thin objects. When a similar factor is kept as in chapter 5 for visible light microscopy (max-

1. Ruska demonstrated his first 'Uebermikroskop' with a minimum resolvable distance of 0.1 f.Lm in 1933, and four years later this value had already been brought back to 0.001 f.Lm.

UL TRA VIOLET MICROSCOPY 295

imal thickness of about ten times the minimum resolvable distance), a theoretical limit in resolvable distance of 0.1 fJom could be reached only with a maximal object thickness of I fJom. Under the most favourable conditions and with high contrasts such values have been measured incidentally, but apart from the depth of focus, factors like stray light, small deficiencies in the correction of the objectives, and problems with contrast and illumination reduce all these theoretical values considerably.

Although it may not have fulfilled its expectations for the resolving power, ultraviolet microscopy is indeed possible with suitable means. As the human eye cannot perceive light rays with a wavelength below 360-380 nm, the image should be registered by photomicrography or other means. Most standard emulsions have a sufficient degree of sensitivity down to the 230-250 nm range, so that the photographic material does not form a problem, at least in this proximal ultraviolet region. The focussing of the image is the real difficulty here; apart from pure guesswork (as Kohler had to perform with his monochromates, corrected only for 275 nm), a pre-focussing with visible light is of limited value only, even with the newest types of ultraviolet objectives which are corrected from about 250 nm far into the visible range (i.e. a kind of super-apochromate). The slightest change in focussing plane (unavoidable with any refraction objective at a wavelength shift of e.g. 300 nm) will lead to an unsharp image, however, and as a consequence of the extremely thin depth of field discussed before, a through-focus series is often necessary. Moreover, the object of fig. 12.2 (photographed with a high-power glycerin-immersion ultraviolet lens 100 x N.A. 1.25) is unstained and shows no contrast in the visible range which does not facilitate matters. Specially designed UV-focussing eyepieces with a fluorescent screen at the intermediary image plane have such a low brightness below 350 nm that they can give no more than a somewhat better approximation than a pre-focussing in the visible range. The ultraviolet image can be made visible (both for focussing and observation) by a so-called image converter, shown schematically in fig. 12.3. Essentially this is a vacuum tube, closed towards the image plane by a cathode plate, made from a material that emits electrons where it is struck by ultraviolet light rays (photo-emission). These electrons are attracted by the potential difference to the positively charged anode which has a fluorescent screen. The small size of the fluorescent screen requires magnification, however, and the image therefore shows light graining which may interfere with the observation of some of the finest details, but the image obtained is usually sufficient for correct focussing. In enlarging the potential difference between

296 MICROSCOPY WITH INVISIBLE RADIA TION

Fig. 12.3. Image converter for ultraviolet or infrared; further explanation in text.

anode and cathode, images with a relatively low brightness can be made visible. A comparable system, although based technically on another principle, is the television-camera with an UV-sensitive phototube. When such a camera is provided with a quartz window, it can be made sensitive down to 230 nm. The advantage is of course that the image can be followed on one or more receivers and an eventual video-recording can be made.

As for the optical aspect of ultraviolet microscopy and photomicrography, the developments made in the last twenty years have made it possible to solve the problem of chromatic aberration, either by making use of reflection objectives, which do not suffer from chromatic aberration but are difficult to construct for higher apertures (cf. chapter 3), or refraction objectives constructed from different UV-transparent refracting materials mentioned in the foregoing. The main problem has been the finding of nonabsorbing and non-fluorescent lens cements for these compound refracting systems. A solution has been found for this, but these cements are sometimes sensitive to greater changes in temperature. Condensers and eyepieces (projectives) have to meet less high optical demands, and are usually made from quartz. As for the light source, this can only be a gas-discharge burner. Special spark discharges are known which emit in a determined part of the UV spectrum but have often a rather low yield. The more powerful highpressure mercury and xenon burners emit light of such a broad spectrum, on the other hand, that filters or a monochromator are necessary to isolate light from the desired part of the ultraviolet. This, of course, brings about a considerable loss oflight energy.

Attempts to use a wavelength considerably below the usual 250-350 nm range run into many practical difficulties. Not only are suitable sources of radiation difficult to make, all kinds of material from which lenses can be made tend to become intransparent below the 200 nm range. Even the use of a microscope based totally on reflecting systems does not solve the

UL TRAVIOLET MICROSCOPY 297

problems, as the specimen itself shows progressive absorption and even quartz object slides can no longer be used. Moreover, air gradually absorbs so much of the ultraviolet that the whole microscope has to be placed in a vacuum system or in an atmosphere of nitrogen, while also the gelatine of photographic emulsions becomes intransparent for this shorter waved radiation. All this may be interesting from a physical point of view but the value of this far ultraviolet for biological microscopy is virtually nil. The region where biological subjects show an absorption is found mostly in the spectral range which can be studied more easily, while the value of shorter waved UV-microscopy for obtaining a higher resolving power has been long rendered out of data by use of other imaging agents than light.

It has long been known that ultraviolet light, especially in the 260-320 nm region, has a great influence on many biological materials. It is not surprising, therefore, that certain substances occurring in living nature show a strong selective absorption. Curiously enough, no one seems to have wondered why Kohler obtained any contrast in unstained biological specimens with his monochromatic objectives at 275 nm, long before Caspersson showed between 1936 and 1950 that precisely the biologically important proteins and nucleic acids absorbed ultraviolet in that region. The contrasts in fig. 12.2 made at a wavelength of 265 nm, are due to a selective absorption of especially nucleic acids (compare also fig. 11.17 on page 277). This kind of natural absorption can be used as the major asset of ultraviolet microscopy: the revealing of the distribution and quantitative analysis of materials which show a selective absorption in the 250-380 nm region, i.e. UVmicro spectrophotometry and microphotometry. Apart from the analysis of nucleic acids (main absorption at 265 nm) and proteins (280-320 nm region), which can be considered as the main field of application of this technique, it has been applied also for the metabolism of uric acid in certain yeasts (Janicek & Svihla, 1968) which shows a selective absorption at 290 nm. The limitations of micro spectrophotometry mentioned in chapter 11 hold true as well for the application of this technique in the ultraviolet region. Biochemically known, typical absorptions in the UV which can easily be detected in the cuvette spectrophotometer, can often not be shown or measured with ultraviolet photomicrography or microspectrophotometry, due to the large difference in path length through a cell organelle and that in a cuvette. The extinction of a certain particle is often overestimated, moreover, by defocussing effects which occur early as a consequence of the shallow depth of field. As contrasts such as shown in fig. 12.2 and fig. 11.17 are related entirely to natural absorption of certain substances without

298 MICROSCOPY WITH INVISIBLE RADIA nON

any treatment of the cells, it is possible to perform ultraviolet microscopy on unfixed or even living cells when they are brought into an UV-transparent medium between quartz. In view of the high sensitivity of cells for ultraviolet light of certain wavelengths, caution has to be taken to limit the exposure to a minimum when studying living cells, i.e. only during a photographic exposure. In using television or a flying spot system, the lighting intensity in the object plane can be held at a low level, but often it is advisable to do the screening work, e.g. with phase contrast using visible light.

Quite the opposite effect is aimed at in making experimental lesions in cells using an UV-microbeam of only a few [Lm diameter. This technique, which has been used for over 60 years, has always remained rather limited in its application because of a large spot size (minimum 2-3 [Lm) and a comparatively low power density which can be obtained in using conventional UV-sources. Recently, laser sources have come into use in this field (cf. Berns, 1971, 1974), whereas Cremer et al. (1974) have described an ultraviolet laser microbeam with exceedingly high irradiance power density for 257 nm (i.e. near the maximal absorption range of nucleic acids), allowing radiation with an effective spot size as small as 0.5 [Lm.

INFRARED MICROSCOPY

Much that has been stated about ultraviolet microscopy holds true as well for microscopy with infrared as an imaging agent; on the other hand very evident differences exist and the fields of application are widely separated. In comparison with the important place infrared spectroscopy has acquired in chemistry and technology and the wide use of infrared in technical photography, the applications of infrared microscopy have so far remained modest. Technically the differences in using infrared light as compared with visible light are not much greater than with ultraviolet: up to a wavelength of 1500 nm ordinary standard objectives can be used, although the image quality begins to suffer at a wavelength range of over 1000 nm. This is mainly due to spherical aberration; chromatic aberration becomes evident over 1200 nm, even in using apochromatic objectives which are calculated for red light. As glass becomes intransparent for infrared in the 3000 nm range, use has to be made of special materials for designing lenses such as thallium-bromide-iodide. Although in this way refracting objectives can be computed which can be used far into the infrared, it is difficult to design lenses which are corrected for a sufficient wide range of wavelengths with these unorthodox lens materials. For the range of over 1500 nm, use is

INFRARED MICROSCOPY 299

often made of reflecting systems or reflection-refraction (catadioptric) objectives. Theoretically, with a complete reflecting microscope, an image can be formed with infrared up to 20 [Lm wavelength. It is difficult, however, to construct reflection objectives for higher apertures, as already stated in chapter 3. A low aperture is the more disadvantageous as the resolving power, dependent on the aperture, diminishes proportionally with the wavelength; even in the proximal infrared, the loss in resolving power quickly becomes sensible.

As for the instrumentation for infrared, most incandescent lamps send out more infrared than visible light (cf. chapter 6 and 10). In this connection, the colour temperature is again an important parameter: with a filament temperature of e.g. 2400oK, the maximum light emission lies around 1200 nm and with 33000 K around 800 nm. Using special filters it is possible to isolate infrared of a desired wavelength. Observation of the image can be made with image converters or specially designed television-scanning tubes. The image converters can be of the type shown in fig. 12.3 or of a 'solid' type, consisting of a photoconductor and an electro-luminescent layer sandwiched between two thin transparent conducting layers through which an alternating current is fed. The electro-luminescent layer, comparable with the anode of the vacuum tube in fig. 12.3, will radiate visible light opposite the area where the photoconductor layer has been struck by infrared radiation. As an alternative, television tubes have been designed which are sensitive up to a wavelength range of 3500 nm. Infrared photomicrography using emulsions which have been treated with special sensitizers is possible in the lower ultraviolet, up to about 1300 nm.

Certain substances, intransparent when studied with visible light, become transparent in the lower infrared. This effect has been used in studying the chitin layer impregnated with melanin found with certain insects. The characteristic absorption of certain organic substances in the wavelength range of 2-30 [Lm has virtually not been applied in qualitative or quantitative microscopic studies of biological material. Apart from problems of instrumentation and recording of the image, this is also related to the loss in resolving power, which becomes quite noticeable in this wavelength range. The minimal resolvable distance (under optimal conditions of illumination, cf. chapter 6) of an objective with a N.A. of 0.6 being about the same as the wavelength of the light used, implies that it would hardly be possible with a reflecting objective of such an aperture to observe at all a cell with a diameter of about 10 [Lm with infrared light of 10 [Lm wavelength!


USE OF X-RAYS

Some fundamental properties of X-rays and their applications The wavelength range of roentgen rays or X-rays! is roughly about 0.01-100 A and as such these rays are to be found in the electromagnetic spectrum between y-rays and ultraviolet rays (fig. 12.1), without distinct boundaries with these other types of radiation, as previously discussed. X-rays are generated when accelerated electrons strike a target. In a so-called Rontgen tube, an evacuated tube made from glass or other material, the target consists of a piece of metal which is struck by accelerated electrons coming from a heated filament (cathode); the electrons coming from the cathode are attracted towards the target (anode) by an acceleration voltage, the potential difference between the filament and the target, i.e. cathode and anode (fig. 12.4). The electrons impinging on the anode generate X-rays

E

Fig. 12.4. Schematic view of a conventional X-ray tube with at left the filament opposing the anode with a potential difference of E. The beam of X-rays leaves the evacuated tube through a window.

by two mechanisms, the stopping of accelerated electrons and the actual removal of electrons bound to the inner orbits or ionization, when the striking electrons have sufficient energy. With the first process, the radiation energy is generated by the reduction in energy-level of the electrons coming in collision with atoms within the anode; the originally German term

1. The latter name being given by W. C. Rontgen, who discovered this mysterious (hence "X") radiation in 1895-1896. He was awarded with the first Nobel prize for physics in 1901.

USE OF X-RAYS 301

Bremsstrahlung which is also used in the English language (literally: braking radiation) refers to the process of deceleration of the electrons when striking the target. The radiation produced by the latter mechanism forms a continuous spectrum, whereas when the electrons expel orbital electrons in the target material, X-ray-emission lines are excited.

The wavelength range of the continuous spectrum (also called white radiation in analogy with 'white' light from a source emitting a continuous spectrum) bears an inversely proportional relation to the energy of that radiation. Obviously, this energy is related to that of the electrons impinging on the anode and this is again dependent on the potential difference E applied between filament and anode. The wavelength of the continuous radiation can be calculated as follows:

A~E.~ - E

when E is expressed in kilovolts, A follows in Angstrom-units. This means, therefore, that with a potential difference of e.g. 12.4 KY, the continuous radiation may include rays of any wavelength longer than 1 A. When the incident electrons are sufficiently energy-rich to ionize atoms by expelling orbital electrons in the target material, characteristic X-ray emission lines are excited, which are imposed as small peaks on the continuous spectrum. The wavelength of such typical emission lines can be applied to identify atomic configurations in the target material (X-ray microanalysis).

X-rays can be subdivided into different categories on the basis of their wavelength and thus their energy. Usually, division is made between hard and soft rays, in which X-rays with a wavelength of below one Angstromunit are called hard (those below 0.1 A ultra-hard) and soft rays those with a wavelength of over one A (over 10 A ultra-soft, merging ultimately with ultraviolet rays). In X-ray microscopy and other applications of X-rays of importance in fundamental biological work usually soft or not too hard Xrays are used; ultra-hard and ultra-soft rays find little application because of their too great or too small energy and penetrative power, respectively.

When animal or plant tissues are traversed by X-rays from the wavelength region of one to several Angstrom-units, a comparatively strong photoelectric absorption occurs; scattering, i.e. change of direction of incident rays, also takes place but this phenomenon can be considered much less important under the conditions in which X-rays are applied in X-ray microscopy and related fields. It should be noted in passing that the situation with electron rays under the usual conditions of transmission electron microscopy is just the opposite with regard to absorption and scattering.


Apart from X-ray microscopy proper, in which the X-rays form the imaging agent and which will be dealt with separately in the next section, a few other applications of X-rays at the micro-level will be dealt with briefly at the end of this section. Detailed reviews of these fields are given by Engstrom (1956) and Hall et al. (1972).

X-ray absorption analysis makes use of the fact that the absorption curves for different elements at various wavelengths show sudden changes due to electron transitions between distinct orbits; these jumps always occur at the same wavelength for a given element. This phenomenon is applied in analyzing qualitatively certain elements with soft X-rays, as well as in the measurement of masses of specimens of mixed composition (e.g. cells). Its applications - apart from determination of inorganic salts in mineraiized tissues - have been overshadowed to a certain extent by other methods of quantitative analysis which make use of a simpler type of instrumentation.

X-ray fluorescence analysis is based on the phenomenon whereby X-rays incident on a specimen excite a weak X-ray spectrum characteristic of the constituent elements, which can be analyzed spectroscopically. In analogy with light fluorescence (chapter 8), the detector of the spectroscope must be placed in such a position that the directly transmitted radiation does not fall on it. The fluorescent radiation, which can be sampled from areas as small as 10 [.Lm in diameter, is always very weak and the detector signal has to be amplified considerably. This technique has theoretically many advantages, as it is possible to measure the total mass of a given element in a biological structure of non-uniform thickness and with irregular distribution. The freedom from distributional error, as known from fluorescence microscopy (cf. chapter 8) also appears to hold true for X-ray fluorescence. Usually there is no danger of specimen damage, as may occur with use of the electron-probe as described in the next paragraph. Electron-probe X-ray microanalysis (X-ray spectrography). This very important new technique, which is increasingly used as an analytical tool in electron microscopy (both with transmission and scanning) has undergone a stormy development in the last decade. Its physical basis, shortly touched upon at the beginning of this section, is the phenomenon whereby a beam of electrons with a certain energy strikes a material, X-rays both from the continuous spectrum and the line spectrum are generated. Only the latter X-rays are of importance in this connection, as the wavelength of each specific line (strictly a narrow spectrum) correlates with a particular atom bombarded, the wavelength being directly related to the atomic number of

USE OF X-RAYS 303

the element concerned. Thus, analysis of the X-ray emiSSIOn can yield detailed information about the constituent elements of the specimen. To enable a more precise localization of elements to be investigated in an object, a thin probe of electron rays is focussed onto the specimen and the X-rays generated are analyzed with a spectrograph and represented graphically by a complicated electronic analyzing device. This can be applied in a scanning electron microscope with incident electrons (SEM + X-ray analysis), while on the other hand a logical extension of transmission electron microscopy is to combine high resolution imaging of ultrathin sections with X-ray microanalysis (TEM + X-ray analysis). These arrangements combine methods for the study of ultrastructure using incident or transmitted electrons with microanalysis on the subcellular level for detailed cytochemical analysis. The information which can thus be obtained is unique for many types of biological work; although these analytical electron microscopes with all their accessory equipments demand heavy investment, their field of application is still widening. In principle any element such as phosphorus, calcium or iodine can be detected with high specificity (the characteristic emission spectrum usually consists of one or a few closely connected lines) and can also be measured quantitatively within reasonable limits. With a diameter of the probe of 1 !Lm, minimal masses to detect a given element are of the order of 10-16 g. Heavy metals used as contrastenhancing 'stains' in electron microscopy can easily be discerned thus on the basis of their X-ray emission, which opens many possibilities in the further development of electron microscopic cytochemistry (cf. Chandler, 1974)

X-ray diffraction. As early as 1912, some fifteen years after Rontgen's discoveries, the German physicist Max von Laue suggested that, the wavelength of X-rays and the spacing of atoms in a crystal being of the same order, a crystal might act to X-rays in a way similar to the effect of a grating on light rays. It was shown shortly afterwards that when a beam of X-rays passed a crystal of copper sulphate (CuS04), a photographic plate behind the crystal shows direct rays (zeroth order maximum) and a typical pattern of X-rays around it as a result of diffraction by the crystal lattice.

X-ray diffraction is a technique in which the short wavelength of roentgen radiation can be exploited in full for detecting distances and spatial relationships at a truly molecular level. It is by no means a direct imaging technique and as such bears no relation to microscopy. The technique is not only important for crystallographists (for whom this technique has been a major tool for many years), but has proved its value also in biology


and medicine: the fundamental work for the double helixstructure of DNA (the Noble-prize winning achievement of Watson and Crick in 1953) was based for a large part on X-ray diffraction pattern analyses with comparatively simple equipment (cf. Watson, 1968).

X-ray microscopy By their great penetrative power compared with light rays, the fact that they are absorbed selectively and their extreme short wavelength, X-rays seem to be very suitable for microscopy. They are invisible to the eye, but can blacken photographic emulsions and can be made visible, moreover, by fluorescent screens or image converters. The cardinal problem in the application of X-rays as an imaging agent in microscopy lies in the fact that these rays cannot be refracted, or deflected in any manner (as can electron rays). The refractive index of virtually all materials for X-rays lies around 0.999998; if a lens was to be constructed on the basis of this minimal refraction, its focal length would be something like 10.000 times the radius of the lens curvature (Nixon, 1956). The fact that the index of refraction is slightly below unity means, however, that X-rays can be totally reflected if the angle of incidence is low enough. If the reflecting surface is part of a sphere, a focussing action on the X-rays can be obtained. It has been possible to build an X-ray reflection microscope on this principle; due to a number of technical difficulties and imaging errors, however, its resolution does not surpass that of the light microscope, notwithstanding a wavelength of some 1000 times shorter than that of visible light rays. For general use in biology and medicine the X-ray reflection microscope is far too complicated an instrument. The two other means for applying X-rays as an imaging agent, contact-microradiography and projection-X-ray microscopy, which will be dealt with in the next pages, make use of un deflected X-rays and have found a fairly wide application.

Contact-microradiography This is the most simple and oldest form of microradiography; it has become possible only with increasing refinements in the manufacture of photographic emulsions, as the microscope is not used for studying the image itself but an X-ray photograph of the image. The object, e.g. a tissue section, is brought into contact with a special photographic emulsion with extremely high resolution. X-rays from a small source are allowed to pass through the specimen to form an image on a photographic emulsion which is in direct contact or at least extremely close to that emulsion. The developed negative consequently forms an unmagnified image of the object, the

USE OF X-RAYS 305

contrasts in which are determined by local differences in absorption of the X-rays. Due to the high resolution of the emulsion, the image on the developed film can be studied with an optical microscope. The results of this technique can be excellent, and the high penetrative power and specific absorption of the X-rays exploited to the full (fig. 12.5A, B). Yet it is clear

Fig. 12.5. Contact-microradiograms (negative images) of a transverse section of bone tissue of the femur of a monkey, made on a spectroscopic film wIth extremely fine grain (theoretical limit of resolution corresponding with 1000 lines/mm). A Magnification 15 x ; review with 'reserve' in resolved details. B Magnification 48 x ; higher magnification of the first image, dark holes of bone cell lacunae clearly visible. C Magnification 150 x; the limit of further magmfication has been reached, the grain of the film having become visible (Photographs made by Dr. R. Steendijk).

that with this set-up, the short wavelength (and therefore the high resolution) of the X-rays cannot be brought to expression, as the limiting factor is formed by the film grain of the recorded image and not by the image itself (fig. 12.5C).

Under favourable circumstances, a minimum resolvable distance of 0.5-1 [Lm can be attained with a magnification range of 100-200 x. This corresponds with a lower resolving power than can be reached with conventional light microscopy with a theoretical minimum resolvable distance of 0.25 [Lm. In comparing fig. 12.5 with fig. 5.6 on page 84, the difference between a limitation of the resolving power by technical circumstances in the registration of the image and that by the properties in the image-forming agent can be observed. The limiting factor, as said before, in the resolv-


ing power with contact-microradiography is the film grain. As discussed in chapter 10, it is not so much the actual size of the grains themselves (which can be brought down to a diameter of the order of 0.1 (lom) but the distribution of the grains in the emulsion and other technical circumstances, in combination with the penumbra-effect (see next section), which sets the limits of resolution here. If the film resolution could be extended further, the potentialities of contact microradiography could be enhanced considerably. Even if the grain of the emulsion could be made infinitely small, however, the short wavelength of the X-rays could not be exploited in full, as the resolving power of the microscope used in studying the contact microradiography would then become the limiting factor.

From the instrumental point of view, it should be noted that an essential condition for microradiography is bringing the specimen (in a special holder, the so-called camera) in such close contact with the film, that the specimen - film interspace is negligible, otherwise geometrical blurring of the image occurs. The specimen is mounted on an extremely thin, X-ray transparent collodion membrane or formvar film (with a thickness of the order of a few (lom maximally), when it is not applied directly against the photographic emulsion. Depending on the acceleration voltage of the source, exposure times vary from a few minutes to about an hour. The actual radiation source should be small to avoid the penumbra-effect (see page 308-309).

As explained in the previous section, when X-rays of medium wavelength traverse animal or plant tissues, energy loss mainly occurs by absorption, scattering being relatively unimportant. Contrasts in X-ray microscopy are brought about mainly by local differences in absorption. The laws of the absorption of X-rays are rather complicated (they are dealt with from the viewpoint of biological application in the reviews by Engstrom, 1966, and Hall et aI., 1972), but it may be stated as a general rule of thumb that the absorption coefficient (J of an absorber can be described by the following formula

(J = C X Z3 X )...3

in which C is a constant, Z the atomic number of the absorber and )... the wavelength of the incident radiation. Consequently, in microradiograms of thin biological specimens, the highest contrast can be expected when soft X-rays are used in areas where heavy atoms like calcium are found. As soft and ultrasoft X-rays (wavelength region 5-25A) are readily absorbed by air, the camera of the contact-microradiography equipment should be evacuated. With thicker specimens, such as the 100 {lom thick ground section

USE OF X-RAYS 307

from calcium-rich bone tissue shown in fig. 12.5, much shorter wavelengths are used, which would produce a very low contrast with a thin section of soft tissue. Finally, it should be noted that inorganic substances have about the same absorption coefficient as water. This means that, in view of their high water content (65%-80%) water should be removed from tissues, in order to show contrasts on the basis of inorganic substances. This excludes the possibility of studying living cells (which would have been difficult anyhow because of the necessity of evacuating the camera when using soft and ultrasoft radiation).

In studying microradiograms from biological structures, a technique sometimes called historadiography, information can be gained about the fine structure and distribution of dry weight (mass) within soft tissues. With a somewhat higher energy level of the incident radiation, distribmion and density of calcium, as it occurs in bone, otoliths etc. and other metals in tissues and cells, can be detected. In a situation as that of fig. 12.5, the micro radiogram enables estimation of the degree of calcification, the size of the 'holes' in the calcified matrix such as those from the bone-cell lacunae, etc. With a properly chosen acceleration voltage the calcium containing parts are seen preferentially in microradiograms which, however, do not reveal much detail in the soft parts. It is also possible to enhance the contrast by introducing in the tissues or organs substances having a high atomic weight, so that they stand out clearly in the microradiograms. An often used technique where this enhancing of contrasts is applied, is the technique of microangiography which consists in bringing a contrast medium into the blood vascular system before fixing the organ concerned and making a microradiograph to study the pattern of the vascular bed (microlymphangiography is the same technique applied to the lymph-vessel system). The contrast medium should be of small particulate or colloidal nature, so that it can flow through the smallest capillaries and other narrow canals in the vascular bed, and are readily miscible with blood. A contrast medium frequently used for this purpose is Micropaque, a colloidal suspension of barium sulphate with a particle size of 0.1-0.5 (.lm, which is sufficiently radiopaque to produce a good contrast between the vascular bed and the surrounding tissues so that even the vessels of smallest caliber are clearly demonstrated (fig. 12.6). Although contact microradiography has mostly been used for microangiography, projection systems are finding increasing application.


Fig. 12.6. Microangiogram, made by contact-microradiography, showing small vessels in the parathyroid gland of a dog; injection with micropaque, 45 x (Photograph made by Prof. P. J. Klopper).

Projection-microradiography In contact microradiography, quite another factor limiting the resolution is the blurring of the image when the object is removed somewhat from the emulsion. This factor, which has been briefly mentioned in the preceding section, is due to the fact that the radiation source has certain dimensions. As a consequence of this, rays coming from one part of the source come to fall in the 'shadow' formed by the object with regard to the rays originating from another part of the source (fig. 12.7). This blurring has an extent which depends on the size of the source and the relative distance between source and object (a) and object and emulsion (b) respectively, as can easily be seen from the scheme of fig. 12.7. This blurring half-shadow or penumbra can be reduced considerably by bringing the object in close apposition with the photographic emulsion, so that the distance b becomes negligible. This is exactly what is done in contact microradiography, where the penumbraeffect can be considered as very small, so long as the radiation source is not too large and the thickness of the object small in comparison with the distance from the source. In moving the object towards the radiation source, however, the effect becomes progressively more significant and rapid deterio-

USE OF X-RAYS 309

Fig. 12.7. The penumbra-effect ~u with a radiation source with diameter S, an object 0, a distance between source and object a and an object-emulsion distance b; e photographic emulsion.

ration in the quality of the image occurs. At the same time a very desirable effect happens, however: the image is enlarged when distance b increases. As a whole, this effect is the same as that which occurs when shadows are formed on a wall with a hand in front of a torch or candle; as almost everyone will have discovered in his childhood, any object of the same size as the source will form a poorly defined shadow; smaller objects often produce no shadow at all, unless the light source is very small. This general phenomenon is called the point-projection principle and it can be applied with light, X-rays, or even electron rays. In all these situations the magnification changes with the quotient: image distance over object distance

( a: b in fig. 12.7) , whereas the illumination intensity varies with the

square of the distance from source to emulsion. It is of little use building a light microscope based on this principle, which would ultimately be limited in its resolving power by the same diffraction effects as the ordinary, more conveniently built, light microscope. In X-ray microscopy, however, the situation is quite different, as the possibility exists here to obtain an enlarged image without deflection or reflection of the X-rays, which is impossible


or only possible on a very small scale, respectively, as discussed in the previous section.

The problems in constructing such a projection microscope for X-rays are concentrated on the production of a sufficiently small and intense source, for reasons clear from the foregoing discussion. The application of the 'camera obscura' principle in screening off a small bundle from a larger source is of limited value, as this will always lead to a source with a very low radiant intensity. The most direct way of producing a divergent powerful bundle of X-rays emanating from a very small area is to focus a bundle of fast electrons so that they strike a point-like part of a target metal. Focussing electron beams is a well-known technique worked out in electron microscopy; the technological knowledge gathered here can therefore be applied to focus an accelerated beam of electrons with (usually two) magnetic electron lenses on an area as small as 0.5 fLm. When a thin metallic target foil is used, the X-rays generated emerge from its other side (fig. 12.8).

EG

ELI

IP Fig. 12.8. Schematic view of an X-ray projection microscope; EG electron gun, ELl and EL. first and second electron lens, 0 object plane, IP image plane; a and b: source-to-object distance and object-to-emulsion distance, respectively.

USE OF X-RAYS 311

The specimen can now readily be placed rather close to the source; as the diameter of the source in fig. 12.7 can be considered to be approaching zero, virtually no penumbra effect will occur. In theory, a rather high magnification can be reached with such an X-ray projection microscope, the image of which can be viewed on a fluorescent screen, or photographed; in practice some technical difficulties appear which again limit a fu\I exploitation of the short wavelength of the X-rays. The minimal resolvable distance which can be attained is mostly in the order of the size of the focal spot, i.e. between 0.3 and 0.4 (.Lm, whereas the performance is limited also by the intensity of the X-ray source. In most cases the object is studied in air, but when the rather soft rays (in the order of 10 A wavelength and over) are used in visualizing the fine details of rather low absorption, e.g. in thin objects, the image space may have to be evacuated (vacuum camera) in view of absorption and scattering of the radiation. In studying the mineral distribution in undecaIcified sections of bone with a thickness of 100 [Lm, X-rays with a wavelength of 0.5-1 A may have to be used, which have a sufficient penetrating power to pass the air in the object space.

The actual instrumentation of an X-ray projection microscope is more complicated than would appear from fig. 12.8, as the electric leads, vacuum pumps etc. are not shown. The whole system is , however, not as complicated as an electron microscope. In practice, the object is brought to within a short distance of the source (i.e. the target foil), usua\Iy not less than a few millimeters, owing to heat production of the source, which produces more heat than X-rays. The focussing is made by changing the current of the magnetic lenses until the image is at its sharpest, which means that the source-spot is at its smallest. Strangely enough, there is no focussing of the object necessary or even possible when the spot on the target has been focussed correctly, e.g. with a grid; the object remains sharp when it is moved up and down the beam, only the magnification changes. The primary magnification, as well as the size of the field is determined by the factor object distance a . . . . . . (fig. 12.8). Under normal condItIOns WIth an Image Image dIstance b distance (target to emulsion, also called camera length) of 10 cm, an object distance of 1 cm would produce a geometric magnification of 10 diameters, whereas a magnification of 1000 x would theoretically be reached with an object distance of 0.1 mm. Object distances of under a few mm lead to heat damage to the specimen and very long exposure times. Magnifications over a few hundred times would moreover come into the empty magnification range with a minimal resolvable distance, under the most favourable


conditions, of about a few tenths of a [Lm. In moving the object towards the emulsion, the magnification approaches unity when the image distance becomes zero; this is of course nothing but the situation of contact-microradiography.

Fig. 12.9 shows a projection-microradiograph of the leg of a frog. Under the conditions of wavelength, thickness and distribution of densities and elementary compositions of the different parts of the specimen, the bones of the skeleton are shown with fine details in their internal architecture, but structural details can also be observed in e.g. the muscular tissue. The wavelength is too short and the specimen too thick, however, to bring out more structure in the soft parts. This figure gives an example of the study of an 'unstained' specimen (it only has been dried) in contrast to fig. 12.6. In most circumstances, the basic problem in projection-historadiography is to obtain sufficient contrast, apart from situations where keratinization or mineralization enhances a pronounced absorption of X-rays. Histological sections, even when using soft X-rays, exhibit rather little contrast which can sometimes be improved by certain treatments using metal salts with high atomic weights. When such treatments are specifically bound in the tissues so that a kind of X-ray histochemistry is developed, a wide range of biological applications may be found. An example of this is the study of nerve cells impregnated with metal salts (cf. Hall et aI., 1972). Although the X-ray projection method is superior in many respects to contact-microradiography in resolving power and the facility of changing the magnification, the differences remain rather small and neither technique can surpass light microscopy in this respect. As the projection technique demands a much more sophisticated and costly instrumentation, the application of contact-microradiography, e.g. in the study of mineralized tissues and also in technology out with biology, is much more widespread than that of projection-microradiography. In one particular respect, however, the projection method has very obvious advantages. As explained previously, once the spot has been focussed so that the X-ray radiates from a minute point, an object will give a sharp image at any place in the beam. This tremendous depth of field, due to the peculiar nature of the shadow-image of the point-projection principle, is also demonstrated in fig. 12.9, where different parts of the skeleton superimposed over each other stand out simultaneously in sharp focus. This imaging of all layers of a thick specimen (which has nothing to do with stereoscopic observation, as it is a two-dimensional projection) can produce very confusing images in unfavourable specimens. On the other hand, e.g. in studying vascular relations in microangiography and in studying thick specimens, this can be very advantageous as spatial

USE OF X-RAYS 313

relations are not disturbed. It is also possible to make pairs of stereo X-ray micrographs which can be studied as light microscopic stereo-photomicrographs (cf. chapter 10) to bring back the third dimension.

Fig. 12.9. Articulation of the wrist of a frog, photographed with an X-ray projection microscope with an acceleration voltage of 30 KV; magnification 12 x (Photograph made by Dr. W. Boersma).


A. W. Agar, R. H. Alderson and D. Chescoe: Principles and practice of electron microscope operation, in: Practical methods in electron mircoscopy, vol. 2, ed. Audry M. Glauert. North-Holland Publ. Cy. Amsterdam 1974.

M. Arif Hayat: Principles and techniques of electron microscopy. Biological applications, vol. 1. Van Nostrand Reinhold, New York-Cincinnati-Toronto-London-Melbourne 1972.

M. E. Barnett: Image formation in optical and electron transmission microscopy. Review article J. of Microsc. 102 (1974) 1-28.

M. W. Berns: A simple and versatile argon laser microbeam. Exp. Cell Res. 65 (1971) 470-473.

M. W. Berns: Recent progress with laser microbeams. Int. Rev. Cytol. 39 (1971) 383-411. T. Caspersson: Cell growth and cell function. Norton, New York 1950.

J. A. Chandler: The use of wavelength dispersive X-ray microanalysis in cytochemistry, in: Electron microscopy and cytochemistry, eds. E. Wisse, W. Th. Daems, I. Molenaar and P. van Duijn. North-Holland Pub!. Cy. Amsterdam 1974.

C. Cremer, C. Zorn and T. Cremer: An ultraviolet laser microbeam for 257 nm. Microsc. Acta 75 (1974) 331-337.

A. Engstrom: X-ray microscopy and X-ray absorption analysis, in: Physical techniques in biological research, vo!. lIlA, ed. A. W. Pollister. Academic Press, New York-London 1966.

T. A. Hall, H. O. E. Rockert, R. L. de C. H. Saunders: X-ray microscopy in clinical and experimental medicine. Charles C. Thomas, Springfield 1972.

J. W. S. Hearle et a!.: The use of the scanning electron microscope. Pergamon Press. OxfordNew York-Toronto-Sydney-Braunschweig 1972.

M. J. Hollenberg and A. M. Erickson: The scanning electron microscope: potential usefulness to biologists. J. Histoch. and Cytoch. 21 (1973) 109-130.

H. E. Huxley and A. Klug: New developments in electron microscopy. The Royal Society, London, 1971.

L. E. Janicek and G. Svihla: Ultraviolet micrography in biological research. J. Bioi. Photogr. Ass. 36 (1968),59-66.

A. Kohler: Mikrophotographische Untersuchungen mit Ultraviolettem Licht. Z. wiss. Mikr.21 (1904) 129-165.

G. A. Meek: Practical electron microscopy for biologists. Wiley Interscience, London-New Y ork-Toronto-Sydney 1970.

E. W. MUlIer: The atom-probe field ion microscope. Naturwiss. 57 (1970) 222-230. W. C. Nixon: X-ray microscopy, in: Modern methods of microscopy, ed. A. E. J. Vickers.

Butterworth, London (1956) 92-102. C. W. Oatley: The scanning electron microscope. part I, The instrument. Cambridge Uni

versity Press, 1972. F. Pliquett: Anwendung des UV-Mikroskops MUF-6 in der Histochemie. Acta Histochem.

34 (1969) 27-37. J. D. Watson: The double helix, a personal account of the discovery of the structure of

DNA. Weidenfeld and Nicolson, London 1968. S. Wischnitzer: Introduction to electron microscopy, 2nd ed. Pergamon Press, New York

Toronto-Oxford-Sydney-Braunschweig 1970.

APPENDICES

APPENDIX I

Refractive indices (n D '·, i.e. measured at 20°C at the yellow natrium D-line) of some media used in microscopy for mounting, immersion, etc.

air 1.0003 Malinol* 1.520 distilled water 1.333 methyl benzoate 1.520 seawater 1.343 Apathy's gum syrup (W) 1.524 ethyl alcohol (W) 1.362 gum damar 1.520 - 1.530 Aquamount (W) 1.435 clove oil 1.531 water-free glycerol (W) 1.473 methyl salicylate 1.535 liquid paraffin 1.482 Permount* 1.530 - 1.540 Gelatinol* (W) 1 .480 - 1.490 Canada balsam* 1.530 - 1.540 toluene 1.495 Euparal* 1.535 xylene 1.497 nitrobenzene 1.535 benzene 1.502 (very toxic) cedar oil* 1.510 Cedax* 1.550 - 1.560 non-resinifying mono bromo benzene 1.560

immersion oil 1.515 anilin oil 1.586 ani sol 1.517 Clearax* 1.660 Eukitt* 1.510 - 1.520 mono bromo naphthalene 1.660 Clearmount* 1.510 - 1.520 Hyrax* 1.710 cristallite 1.5 10 - 1.520 methylene jodide 1.738

Media indicated with * become hard after a shorter or longer period, cementing object glass and cover glass together. This hardening process is caused by the evaporation of a solvent (xylene, toluene) and/or as a consequence of a process of polymerization (with synthetic media) or resinification (with natural resins). During this process, which may take from one or two days to over a week, the refractive index gradually increases until a final value is reached. In the table only an average final value is listed; this is often not rigidly constant everywhere in the specimen, as in the centre the evaporation/polymerization process is sometimes stopped at an earlier stage than at the border, due to sealing off of the central part.

Non-hardening media in the list usually are sharply defined chemical substances with constant physical characteristics: their refractive indices immediately have the desired value (which can easily be controlled with a refractometer). Especially in

* hardening mounting medium W miscible with water

APPENDIX I 317

photometry, where the adaptation of medium and object may be very critical, but also with more unusual specimens such a pure liquid mounting medium may be indicated. As these substances have a low viscosity and are often volatile, the cover glass has to be sealed and fixed with a ringing cement. Different types of quickly hardening sealing media are on the market, but they are often soluble in benzene derivates and non water-miscible substances like methyl salicylate with its favourable refractive index, so that they cannot be used to seal off specimens mounted in these media. In these circumstances, an excellent sealing can be obtained by using a 10% (wjv) solution of gelatin in water to which 10% (wjv) sucrose is added with gentle heating. When this mixture is kept in an incubator at 37°C, it remains liquid enough to be applied with a brush, but has a sufficient viscosity to seal off immediately a volatile medium. It dries in a few hours at room temperature in forming a stone-hard cement which can be removed easily with lukewarm water. It is not dissolved by xylol, petrol etc., so that immersion oil on the cover glass can be removed freely with these liquids.

Apart from the pure substances mentioned in the list, use can be made of mixtures of different compounds to achieve a certain refractive index; these can also be obtained commercially with virtually any desired refractive index (Cargilleoil). It should be kept in mind in using any type of mixture that the refractive index of a stock solution may change considerably with time, due to differential evaporation of components.

318

APPENDIX II FOUR-LINGUAL VOCABULARY OF SOME COMMONLY

ENGLISH

adjustment air bubble angle of aperture angle of view aperture stop attachable mechanical stage attachment camera

beam of (light) rays bellows camera birefringent bright field brightness of illumination built-in illumination

calibration case cement centering device chromatic difference of magnification

coarse adjustment

coating, blooming collector-lens colour temperature comparison eyepiece comparison microscope compensating eyepiece compound lens

concave mirror condenser condenser with swing-out frontlens

FRENCH

ajustement bulle d'air angle d'ouverture angle de vue diaphragme d'ouverture surplatine amovible chambre microphotographique oculaire

faisceau lumineux chambre photographique a soufflet birefringent fond clair force d'eclairement eclairage incorpore

etalonnage coffret lut dispositif de centrage difference chromatique de magnification

mouvement rapide

traitement anti-reflet collecteur temperature de couleur oculaire comparateur microscope de comparaison oculaire compensateur lentille compo see

miroir convergent condensateur, condenseur condensateur a lentille frontale

escamotable

319

USED TECHNICAL TERMS IN MICROSCOPY

GERMAN

Einstellung Luftblase Offnungswinkel Sehwinkel Aperturblende ObjektfUhrer Aufsatzkamera

(Licht) BUschel Balgenkamera doppelbrechend Hellfeld Beleuchtungsstarke Einbaubeleuchtung

Eichung Schrank Kitt Zentriervorrichtung chromatische Differenz der

Vergrosserung Grobtrieb (Zahntrieb)

ReflexschUtz Kollektor, Sammellinse Farbtemperatur Vergleichsokular Vergleichsmikroskop Kompensationsokular verkittete Linse, zusammengesetzte

Linse Hohlspiegel Kondensor Klappkondensor

SPANISH

ajuste burbuja de aire angulo de apertura angulo visual diafragma de apertura platina m6vil camara fotografica acoplada al ocular

haz luminoso camara con fuelle birrefringente campo claro intensidad de i1uminaci6n i1uminaci6n incorporada

calibraci6n caja 6 Estuche masilla dispositivo para el centraje diferencia cromatica de aumento

tornillo macrometrico (enfoque rapido)

tratamiento antireflectante colector temperatura de color ocular comparador microscopio de comparaci6n ocular compensador lente compuesta

espejo convergente condensador condensador con lente frontal abatible

320 APPENDIX II

ENGLISH

continuous running interference filter

control knob correction collar cover-glass critical illumination cross-wires curvature of field

dark field illumination defects of image deflecting prism

depth of field diffraction image dissecting microscope distortion division drawing apparatus (camera lucida) dry objective

electron dense electron microscope empty magnification entrance pupil exit pupil exposure time extinction (optical density) eyepiece eyepiece micrometer

field lens field number field stop (- diaphragm) field of view film holder final magnification fine adjustment

flash flat-field objective

fluorescent screen focal length focal plane to focus

FRENCH

monochromateur interferentiel

bouton de commande monture de correction lamelle couvre-objet eclairage critique reticule en croix courbure de champ

eclairage a fond noir defaut de I'image prisme de renvoi

profondeur de champ image de diffraction microscope a dissection distorsion eche\le graduee appareil a dessiner (chambre claire) objectif a sec

dense pour electrons microscope electronique grossissement vide pupille d'entree pupiIIe de sortie temps de pose extinction oculaire micro metre oculaire

verre (lentille) de champ index de champ diaphragme de champ champ visuel chassis grossissement total mise au point (mouvement lent)

eclair electronique objectif a champ plan

ecran fluorescent distance focale plan focal mettre au point

GERMAN

Verlauffilter-Monochromator

Bedienungsknopf Korrektionsfassung Deckglas kritische Beleuchtung Fadenkreuz Bildfeldwolbung

Dunkelfeldbeleuchtung Abbildungsfehler Umlenkprisma

Schiirfentiefe Beugingsbild Prapariermikroskop Verzeichnung Gradeinteilung Zeichenapparat (camera lucida) Trockenobjektiv

Elektronenmikroskop elektronendicht leere Vergrosserung Eintrittspupille Austrittspupille Belichtungsdauer Extinktion Okular Okularmikrometer

Feldlinse, Kollektivlinse Sehfeldzahl Leuchtfeldblende Gesichtsfeld Kassette Endvergrosserung Feintrieb (Mikrometerschraube)

Elektronenblitz Plan-objektiv

Fluoreszenzschirm Brennweite Brennebene Scharfeinstellen

APPENDIX II

SPANISH

filtro continuo desplazable de interferencia

tornillo de control collar 0 montura de correcci6n cubre objetos iluminacion critica reticulo en cruz curvatura del campo optico

iluminacion de campo oscuro defecto de imagen prisma de desviacion

(para proyectar y dibujar) profundidad de campo imagen de difraccion

321

microscopio de diseeci6n 0 estereoscopico distorsion escala graduada camara clara para dibujar objetivo 0 sistema en seeo

microscopio electronico electrodenso aumento en vacio pupila de entrada pupil a de salida tiempo de exposicion extinction ocular ocular micrometrico

lente de campo numero indicador de campo diafragma de campo campo visual chasis (portapeIiculas) aumento total tornillo micrometrico

(enfoque de precision) rayo electronico objetivo de campo plano 0 con optica

plana pantalla fiuorescente distancia focal plano focal enfocar

322 APPENDIX II

ENGLISH

focussing eyepiece focussing microscope, auxiliary

microscope focussing plane foot of the stand front lens frozen section

glare, stray light graticule, grating ground glass plate

high power magnification high pressure mercury vapour lamp

illumination illumination apparatus illumination field image image-formation immersion-objective incandescent lamp incident illumination intermediate piece iris diaphragm

lamp lens light diaphragm, stop light-refracting light source limb long-focus condenser low power magnification low voltage lamp

magnification main connection lamp measuring eyepiece mechanical stage microscope microscope stage (object stage) mirror mirror image mount

FRENCH

oculaire de mise au point microscope auxiliaire

plan de mise au point base du statif lentille frontale coupe Ii congelation

lumiere parasite reseau plaque en verre depoli

grossissement fort lampe a vapeur de mercure haute

pression

illumination appareil d'eclairage champ d'eclairage image formation de l'image objectif a immersion lampe Ii incandescence eclairage incident bague intermediaire diaphragme iris

lampe lentille diaphragme refringent source lumiere potence condenseur a longue focale grossissement faible lampe a bas voltage

agrandissement lampe a branchement direct oculaire de mesure platine Ii chariot microscope platine du microscope miroir image refiechie monture

APPENDIX II 323

GERMAN

Einstellokular Hilfsmikroskop, Einstellmikroskop

Einstellebene Stativfuss Frontlinse Gefrierschnitt

Streulicht Strichplatte, Gitter Mattglasscheibe

starke Vergrosserung Quecksilberhochdrucklampe

Beleuchtung Beleuchtungsapparat Leuchtfeld Abbildung, Bild Bilderzeugung Immersions-Objektiv Gltihbirne, Gltihlampe Auflichtbeleuchtung Zwischensttick Irisblende

Lampe Linse Blende Iichtbrechend Lichtquelle Tubustrager, Arm des Stativs Kondensor langer Schnittweite schwache Vergrosserung Niedervoltlampe

Vergrosserung Netzanschlusslampe Messokular Kreuztisch Mikroskop Mikroskoptisch, Objekttisch Spiegel Spiegelbild Fassung

SPANISH

ocular para ajustar microscopio auxiliar

plano de enfoque pie 0 base del estativo lente frontal corte por congelacion

luz parasita red, reticula placa de vidrio esmerilado

gran aumento lampara de vapor de mercurio a alta

presi6n

iluminaci6n aparato de i1uminaci6n campo de luz 0 de iluminaci6n imagen formaci6n de la imagen objetivo de inmersi6n lampara de incandescencia iluminaci6n incidente pieza intermedia diafragma de iris

himpara lente diafragma refringente fuente luminosa brazo del estativo condensador de gran distancia focal pequeno aumento himpara de bajo voltaje

aurnento lampara de conexi6n directa a la red ocular para medir platina de carro microscopio platina 0 mesa del microscopio espejo imagen reflejada montura

324

ENGLISH

mounting medium

neutral density filter numerical aperture

object field objective lens object marker oblique illumination optical axis optical path difference (o.p.d.) over-exposure

parafocal objectives particle path of rays phase contrast phase plate phase retardation phase ring phase shift photomacrography photomicrography plane mirror pointer eyepiece polarizing microscope

(polarisation m.) power supply

rack and pinion movement range of adjustment refractive index resolving power revolving nosepiece

scanning electron microscope scratch screw micrometer eyepiece section semi transparent mirror sharpness single lens slide, object slide sliding sleeve sliding stage smear speed (photographic emulsion)

APPENDIX II

FRENCH

milieu d'inclusion

filtre absorbant neutre ouverture numerique

champ d'objet object if marqueur d'objet eclairage oblique axe optique difference de chemin optique sur-exposition

objectifs parafocaux corpuscule marche des rayons contraste de phase plaque de phase retard de phase anneau de phase changement de phase macrophotographie microphotographie miroir plan oculaire indicateur microscope polarisant

connexion electrique

mouvement a cremaillere domaine de reglage indice de refraction pouvoir separateur revolver

microscope electronique a balayage eraflure oculaire micrometrique it tambour coupe miroir semi-transparent nettete lentille simple lame porte-objet douille platine it frottement gras frottis sensibilite (emulsion photographique)

GERMAN

Einschlussmedium

Neutralfilter numerische Apertur

Dingfeld Objektiv Objektmarkierer schrage Beleuchtung optische Achse Gangunterschied Uberbelichtung

Abgeglichene Objektiven Korperchen Strahlenverlauf, Strahlengang Phasenkontrast Phasenplatte Phasenverzogerung Phasenring Phasenverschiebung Makrophotographie Mikrophotographie Planspiegel Zeigerokular Polarisationsmikroskop Netzanschluss

Zahntrieb Einstellbereich Brechungszahl Auflosungsvermogen Objektivwechsler (Revolver)

Raster-Elektronenmikroskop Kratzer Schraubenmikrometerokular Schnitt halbdurchlassige Spiegel Scharfe Einzellinse Objekttrager Schiebehlilse Gleittisch Ausstrichpraparat Empfindlichkeit (Photoschicht)

APPENDIX II

SPANISH

medio de inclusi6n

filtro neutro apertura numerica

campo del objeto objetivo marcador 0 sefialador iluminaci6n oblicua eje 6ptico diferencia de camino optico exposici6n larga

objetivos parafocales particula, corpusculo trayectoria de los rayos luminosos contraste de fases placa de rase retraso de fases anillo de fase differencia de fase macrofotografia microfotografia espejo plano ocular indicador microscopic de polarizaci6n conexi6n al corriente electrico

desplazamiento por cremallera zona de ajuste indice de refracci6n poder separado 0 resoluci6n revolver

microscopio electr6nico de barrido rascador, raspador ocular micrometrico de tambor corte espejo semitransparente enfoque, estar a foco lente simple porta objetos vaina corrediza 0 tubo desplazable platina 0 mesa por deslizamiento frotis sensibilidad

325

326

ENGLISH

stage micrometer stain, dye staining stand stop (diaphragm)

telescopic mount time lapse

transmission range

transmission electron microscope transmi ttance transmitted illumination transparency turning table

ultrathin section under-exposure useful magnification

vernier, nonius

wavelength wide field condenser

wide field eyepiece (wide angle eyepiece)

working distance

APPENDIX II

FRENCH

micrometre object if colorant coloration statif diaphragme fixe

monture telescopique intervalle entre les prises de vue

domaine de transparence

microscope electronique a transmission facteur de transmission eclairage a lumiere transmise transparence platine tournante

coupe ultra-mince sous-exposition grossissement utile

vernier

longueur d'onde condenseur a grand champ

oculaire a grand angle (a grand champ)

distance de travail

APPENDIX II 327

GERMAN

Objektmikrometer Farbstoff Farbung Stativ Lochblende

Federfassung Zeitraffer

Durchlassigkeitsbereich

Transmissions-Elektronenmikroskop Transparenz, Durchlassigkeit durchfallende Beleuchtung Durchsichtigkeit Drehtisch

Ultradiinnschnitt Unterbelichtung fOrderliche Vergrosserung

Nonius

Wellenlange Ubersichtskondensor

(Brillenglaskondensor) Weitwinkelokular (Groszfeld-Okular) Arbeitsabstand

SPANISH

objetivo micrometrico colorant coloracion, tinte estativo diafragma fijo

monture telescopica observacion 0 microfotografia a intervalos

(microcinematografia) intervalo de transparencia

(grado de transparencia) microscopio electronico de transmision trans parencia iluminacion por transmision transparencia platina 0 mesa giratoria

corte ultra fino exposicion corta aumento uti!

nonius

longitud de onda condensador de gran campo 0 gran

angular ocular de gran campo

(ocular gran angular) distancia 0 separacion de trabajo

INDEX

Abbe, Ernst 25, 41 -, condenser according to - 102 -, dispersion index of - 11 -, drawing apparatus according to - 242 -, illumination apparatus according to -

32, 145 - test plate 54 -, theory of image forming of - 166-168 aberration -, chromatic - 8-11 -, monochromatic - 8 -, spherical - 9 absorption - filters 114-117 absorption-objects 116 absorption of X-rays 301 accommodation 4-6, 8 - range of the eye 87-88 achromatic-aplanatic condenser 102 achromatic doublet 11 achromatic objective 49-50 adjustment length 49 Ahrens prism 194 Airy disc 76 Amici, Giovanni Battista 25, 42 - -Bertrand lens 173 amplitude - contrast 178, 191 - grating 166 - objects 116, 165 analyzer 197 angle of vision 6-7, 68-69 Angstrom-unit 3 angular aperture 9, 40 angular magnification 7 ani sol 124 anisotropy 193-196 anoptral contrast 172 apertometer 55 aperture diaphragm of condenser 94-97,

125 aperture error 9 aplanatic 102 apochromatic objective 52

arm of stand 29 ASA-system 221 astigmatism 9, 133, 290 attachment camera 211-213 auxiliary lens of condenser 99 autofluorescence 153 automatic exposure control 214-216 axial resolving power 87 axial setting 256 azimuth 199

band-spectrum III bandwidth 224, 276 barrier filter 154, 158-159 bellows extension camera 212 binocular eyepiece 34-35 birefringence 193-196 black body radiation 231-232 Bremsstrahlung 301 bright field 146-147, 178 - fluorescence 156 brightness 17 - of image 126

calcite 194,201 camera -, attachment - 211-213 -, bellows extension - 212 - length 217 - - in projection X-ray microscopy 311 - lucida 241 candela 17 capillary microscope 37 carbon arc 111 cardioid condeser 149 cargille-oil 317 catadioptric systems 57 catoptric systems 57 cedar wood oil 124 centering device of condenser 101 - - stage 31 - of objective 123 chromatic aberrations 8-11

INDEX 329

- difference of magnification 10 - polarization 199-201 chromophore 280 cinemicrography 236-239 cleaning of optical surfaces 132-137 coating of lenses 18 co-axial controls 31 coherence 78-79, 126-127 collector 100, 109, 292 colour balance filter 232 - circle 225 - distortion 232-233 - in relation to wavelength 225-226 - photomicrography 229-233 - sensitivity of photographic emulsions

218-219 - temperature, Kelvin scale for - 231 coloured phase contrast 179 colposcope 37 coma 9 combination of lenses 11 comparison eyepiece 74 compensating eyepiece 63 compensator 201 compound microscope 7 condenser 92-99, 101-103 -, achromatic-aplantic - 102 -, aperture diaphragm of - 94-97 -, auxiliary lens of - 99 -, cardioid - 149 -, centering device of - 101 -, Heine - 171, 173 -, mirror - 149 -, N-A. of - 94-97 -, pancratic - 98 -, paraboloid - 148 -, working distance of - 94 -, Richter - 98 - according to Abbe 102 - aperture in fluorescence microscopy 156 - immersion 96, 125 - iris diaphragm 94-97 - with long working distance 105 - with swing-out frontlens 98 conoscopic microscope 202 contact-microradiography 304 continuous running interference filter 225 continuous spectrum 111, 301 contrast - filter 224 - formation 115-117 - micrometer 252 - in image 222

- in X-ray microscopy 306 - of photographic emulsions 220 conversion filter 232 correction collar 47 correction grade of objective 49 Coulter-counter 265 counting grids 262-264 cover glass effect 45-49 coverglass, thickness of - 46-47 critical illumination 99-102, 120 crossed prisms 195, 198 crown glass 11 Cuff stand 22 curvature of field 9, 55 cut-off filter 154, 158-159 cytofluorometry 163, 283-284 cytophotometry 279-282

dark field - fluorescence 160 - illumination 145-151 Davies shutter 90, 149 daylight - film 231 - filter 128 - illumination 108 Delesse, principle of - 266-267 density 220, 233 depth - of accommodation 88, 256 - of field 86 - -, geometrical 87 - - in projection X-ray microscopy 312 - of focus 86 - measurements 256-259 detachable mechanical stage 31 dichroic mirror 161 dichroism 196 - induced - 196 - intrinsic - 196 differential interference contrast 185-191 diffracted light 168-169 diffraction 75-78 - disc 76 -, X-ray - 303 DIN number 221 dioptres 7 discussion head 72 dispersion 9, 11 - index of Abbe 11 distortion 9 distribution(al) error 281-282, 284 Dollond eyepiece 61

330 INDEX

drawing device 239-243 - of Abbe 242 drawing prism 242 draw-tube 27, 48 Drliner-camera 234 Dyson, interference microscope according

to - 184-185 -, objective 58

electromagnetic spectrum 288-289 electron lens 290 electron microscope 290-292 -, mirror- - 291 -, scanning - 291-292 -, transmission - 291 electron probe X-ray microanalysis 302-

303 electron rays 289-292 elliptical polarization of light 192 emission spectrum 153-154 empty magnification 83 emulsion -, orthochromatic - 218 -, panchromatic - 218 entrance pupil 15 excitation filter 153-154, 158-159 excitation spectrum 153-154 exit pupil 15, 64-66, 83-84, 210 exposure 213-216 - latitude 220 - meters 214-216 extinction 280 - factor 204 extraordinary ray 193, 198 eye clearance 65 eye lens 4 eyepiece(s) 60-74 -, comparison- - 74 -, compensating - 63 -, Dollond - 61 -, Huygens - 61 -, image-shearing measuring - 255-256 -, integration - 261-264 -, Kellner - 61 - magnification 85 - micrometer 250-252 -, negative - 61 -, orthoscopic - 62 -, photographic - 208 -, pointer - 71 -, positive - 61 -, projection - 208 -, Ramsden - 61

-, screw-micrometer - 253-254 -, spectacle - 133 -, wide field - 68 - -grid 260, 264 - number 63 eyepoint 64-66

Farmer's liquid 242-243 Feulgen-DNA measurement 283 field diaphragm 101 field-emission 292 field-ion 292 field lens 60 field marker 130 field number 66, 69 field of view 66 filar eyepiece micrometer 253 film-cassette 216 film -, daylight - 231 -, reversal - 229 -, roll- 213 -, sheet - 213 film polarizers 196 filters, -, absorption - 224 -, colour balance - 232 -, contrast - 224 -, conversion - 232 -, daylight - 128 -, heat-absorbing - 244 -, interference - 224 -, liquid - 224 -, neutral-density - 232 flash tube 233 flow birefringence 195 flow system analysis 284 fluorescence - cytophotometry 283-284 - -free objectives 157 - microscopy 152-163 - -, polarized - 162 fluorite 50, 293 - objective 50-52 fluorochromes 154-155 flying-spot scanning 270 focal point 9 focussing frame 222 focussing of image 222-223 - -, in ultraviolet microscopy 295 focussing telescope 173, 1237 foot of stand 32 form-birefringence 195, 203

INDEX 331

formvar film 306 frame-lines of TV-system 245 free working distance 37, 40

gamma of film 220 gelatin absorption filter 224 geometrical depth of field 87 ghost-image 180-181 glare 96-97 gradation 220 grain size of photographic emulsions 219 Greenough-type stereomicroscope 35 grey-values 225 ground glass screen 212

halo (in phase contrast microscopy) 176-177

heat-absorbing filter 244 heat radiation 110 height of seat 118 Heine-condenser 171, 173 high pressure gas-discharge burners 111-

112 high-speed camera 238 high voltage lamp 108-109 hinged stand 26 histometry 265 historadiography 307 Holmes effect 260, 266-267 holographic microscope 235 homogeneous immersion 43 Hooke, Robert 19-22 Horn, interference microscope according

to - 183-184 Huygens, Christiaan 26 Huygens eyepiece 61

illuminance 17, 212 illumination -, bright-field - 146-147 -, critical - 120 -, dark-field - 145-151 -, daylight - 108 -, incident - 105-106 -, incident dark-field - 150-152 -, Kohler - 120-121 -, oblique - 143-145 -, Rheinberg - 151 -, vertical - 106 - apparatus 31 - apparatus of Abbe 32, 145 - cone 94-97, 143 illustrator 239

image -, final - 15 -, intermediary - 15 -, inverted - 15 -, latent - 227 -, isometric - 235 -, real - 15 - analysis, automatic - 270-275 - convener 295-296, 299 - errors 8 - forming agents 4 - of light source 97-99 - scanning photometer 282-283 - -shearing measuring eyepiece 255-256 immersion 121 - fluid 42, 124 - objectives 42-45 - oil 124 - -, non-fluorescent - 156 immunofluorescence 162 impulse cytophotometry 284 incandescent lamp 127, 227 incident - dark-field illumination 150-152 - illumination 105 inclination of image plane 144 induced dichroism 196 infinite tube length 45 infrared microscopy 298-299 infrared radiation 110, 289 instrumental depth of field 88 integrating microinterferometer 185 integration eyepiece 261-264 interference 75-78 - colours 200 - fil ters 224 - -, continuous running - 225 - -, precision line - 224 - -, monochromator 226 interference microscope 180,258-259 - according to Dyson 184-185 - according to Horn 184-185 - according to Smith 184-185 intermediary image 15, 60 intersection points 268, 273 intrinsic dichroism 196 inverted image 15 inverted microscope 104 iris diaphragm in objectives 90 isometric image 235

Jamin-interferometer 183 Jentzsch prism 34

332 INDEX

karyometry 265 Kellner eyepiece 61 Kelvin scale for colour temperature 231 Kohler illumination 99-102, 120-121

Lambert-Beer, law of - 279-280 lamp -, high pressure mercury-vapour - 111 -, incandescent - 127,227 -, high voltage - 108-111 -, low-voltage - 109-111 -, tungsten filament - 108-111 -, tungsten halogen -110-111 laser source 298 latent image 227 lateral chromatic aberration 10 Leeuwenhoek, A. van 20-22 lens - Amici-Bertrand - 173 -, electron - 290 -, macro- - 207 - aberrations 8 - formula 13, 291 - paper 134 Lieberkiihn mirror 92 light filter 127 light scattering 146-148 light source, image of - 97-99 lighting intensity 17,216,244 limb of stand 29 linear analysis 262 linear magnification 7 linearly polarized light 192 liquid filter 224 Lison, system for photometry of - 281 lithium fiuoride 293 longitudinal chromatic aberration 10,63 loupe 7,8 low voltage lamp 109-111 luminance 17, 108 luminars 59, 207 luminous flux 17, 109 lux 17

Mach-Zehnder principle 183 macro-lens 59, 207 macrometer (coarse adjustment) 29 magnification -, angular·- 7 -, chromatic difference of - 10 -, eyepiece - 85 -, linear - 7 -, nominal - 16

-, total - 16 -, transverse - 7 -, useful - 85 - changer 70 magnifying glass Maltese crosses 199 marking and re-locating 128-130 meander-like search 129 measuring beam 183 measurement - of areas 259-264 - of distances along optical axis 256-259 - of lengths 250-256 - of volumes 264-269 mechanical stage 30 -, detachable - 31 Merz-grid 268 microfilm 236 microflash 233-234 microfluorometry 163,283-284 micromanipulator 30 micrometer -, contrast - 252 -, object - 251-252 -, stage - 251-252 - value 251 - (fine adjustment) 29 - (unit of length) 3 microphotograph 207 microphotometry 279-283 microprojection 243, 247 microscope 7 -, capillary - 37 -, compound - 7 -, conoscopic - 202 -, electron - 290-292 -, Greenough-type stereo - 35 -, holographic - 235 -, inverted - 104 -, interference - 180-192 -, infrared - 298-299 -, orthoscopic - 202 -, operation - 37 -, photo - 212-213 -, polarization - 196-205 -, plankton - 104-105 -, precision micrometer - 258 -, setting-up of - 119-121 -, simple - 7 -, slit lamp - 37 -, solar - 108 -, stereoscopic - 33-37 -, travelling - 32

INDEX 333

-, ultraviolet - 293 -, X-ray reflection - 304 microrefractometry 175 microspectrofluorometry 283 microspectrophotometry 275-279 microtechnique 115-118 millimicron 3 minimum resolvable distance 5, 81-83,291 Mired 231 mirror 31 -, dichroic - 161 -, electron microscope - 291 -, Liberkiihn - 92 -, substage - 29,93 - condenser 149 - fork 93 - objective 57 modulation contrast 192 monochromatic aberrations 8 monochromatic objective 293 monochromator 226, 275 morphometry 265 mounting media 116-117 -, refractive index of - 316

nanometer 3 near point 4-6 necessary magnifying power 83 negative eyepiece 61 negative phase contrast 171 neutral density filter 108, 232 Newton objective 57 Nicol prism 194-195 Nomarski, interference system according

to - 186-187 nominal magnification 16 non resinifying oil 124 normal incident illumination 106 normal film format 238 nosepiece, revolving (rotating) - 27 numerical aperture 40-45

object - beam 183 - field 66 - -, illumination of - 97, 99 - finder 131-132 - marker 130 - micrometer 218, 251-252 - scanning photometer 282-283 - slide 117-118 - stage 29 - thickness 115

objective 39-59 -, achromatic - 49-50 -, apochromatic - 52 -, centering of - 123 -, correction grade of - 49 -, Dyson - 58 -, fluorescence-free - 157 -, fluorite - 50-52 -, immersion - 42-45 -, level of focus with high power - 123 -, mirror - 57 -, monochromatic - 293 -, Newton - 57 -, oil-immersion - 121-125 -, parafocal - 49, 122-123 -, photomicrographic - 58 -, plan-achromatic - 52 -, plan-apochromatic - 53 -, reflecting - 57 -, Schwarzschild - 57 -, semi-apochromatic - 50-52 -, strain-free- 201 -, testing of - 54-55 -, ultraviolet - 58, 293 oblique illumination 143-145 ocular: see eyepiece oil-immersion objectives 121-125 ordinary ray 193, 198 orthochromatic emulsion 218 orthoscopic eyepiece 62 orthoscopic microscope 202 opacity 279 operation microscope 37 optical activity 193 optical density 280 optical object distance 40 optical path difference 174, 182 optical rotation 193 optical section 89 optical thickness 174, 182

pancratic condenser 98 panchromatic emulsion 218-219 paraboloid condenser 148 parafocally adjusted objectives 49, 122-123 parallel prism 195, 198 partially polarized light 192 particle size analysis 260 pattern recognition 273 penumbra 308-309 percentage transmittance 279 phase annulus 169 phase-changes 165-168

334

phase contrast microscopy 165-180 phase difference 166 phase grating 167 phase objects 166 phase plate 169-173 phase-refractometry 175 phase reversal 176 phase retardation of anisotropic objects

199 phosphorescence 152 photographic emulsion -, colour sensitivity of - 218-219 -, contrast of - 220 -, grain size of - 219 -, resolution of - 219 -, speed of - 221 photographic eyepiece 208 photomacrography 207-235 photometer -, accuracy of - 282 -, image scanning - 282-283 -, object-scanning - 282-283 -, precision of - 282 photometry 17, 279 photomicrographic objective 58 photomicrography 207-239 -, colour - 229-233 -, infrared - 299 -, stereo - 234 -, ultraviolet - 294-296 photomicroscope 212-213 photomicrosysthesis 235 photomultiplier 214, 275, 296 plan-achromatic objective 52 plan-apochromatic objective 53 plane of polarization 192 planimeter 261 plankton microscope 104-105 pleochromism 196 plug-method 281 point-counting method 261 point-projection principle 292, 309 pointer eyepiece 71 polarization -, chromatic - 199-201 - microscopy 196-205 - -, quantitative - 200-202 - of light 192 - -, circular - 192 - -, elliptical - 192 polarized fluorescence microscopy 162 polarizer 197 polaroid filters 197-198

INDEX

polychlorinated biphenols 125 polystyrol-foam 134 Porro prism set 36 positive eyepiece 61 positive phase contrast 171 precision line-interference filters 224 primary fluorescence 153 principal plane 13-14 principal section 193 prism -, Ahrens - 194 -, drawing - 242 -, Jentzsch - 34 -, Nicol - 194-195 -, parallel - 195, 198 -, reinverting - 36 -, Thompson - 36, 194 -, Wollaston - 186-187 projection - area 260 - distance 244 - eyepiece 208 - head 73 - microradiography 308 - television 245 punctum proximum 4 pupillar border 8

quanta 192 quarter wave plate 201 quartz 293 quartz-rod illuminator 107

rack-and-pinion movement 28, 31 radiation -, black body - 231-232 -, electromagnetic - 288-290 -, heat - 110 -, infrared - 110, 289 -, ultraviolet - 159, 289 -, white - 301 Raleigh's criterion 77 Ramsden circle 64-66 Ramsden eyepiece 6 I ray -, extraordinary - 193, 198 -, ordinary - 193, 198 - tracing 12 reading glasses 132 real image 15 reciprocity failure 215, 230 reflecting objective 57 reflection 10

refraction 114-117 refractive index 116-117, 180-192 -, of X-rays 304 reinverting prism 36 research stand 32 residual aberration 49 resolution 81, 126 -- of photographic emulsions 219 resolving power 5, 55. 75-82 reversal film 229 revolving nosepiece 27 Rheinberg illumination 151 Richter condenser 98 ringing cement 317 Rontgen rays 300-307 roll film 213 rotating nosepiece 27

scanning -, flying-spot - 270 -, image-plane - 271 -, specimen-plane - 271 -, television - 271 - and integrating cytophotometer 282 - electron microscope 291-292 - microfluorometer 284 scattering 196 - of X-rays 301 SchwarzschiJd effect 215, 230 Schwarzschild objective 57 screw-micrometer eyepiece 253-254 secondary - electrons 291 - fluorescence 153 - spectrum 49, 52 section thickness 115 semi-apochromatic objective 50-52 semi-transparent beam splitter 237 semi-transparent plate 106-107 sensitation process 221 sensitivity curve of eye 128 sensitization of colour films 231 sensitometry 221 setting accuracy 254-255 setting-up of microscope 119-121 shading-off 177 sharpness of image 222-223 sheet film 213 sheet polarizer 197-198 SJ-system 3 simple microscope 7 sitting position 118 sliding stage 31

INDEX 335

slit lamp microscope 37 slow-motion cinematography 236-237 Smith, interference microscope according

to - 184-185 soft X-rays 301 solar microscope 108 Soret-band 276 specific refractive increment 181-182 specimen-plane scanning 271 spectacle 132 - eyepieces 133 speed of photographic emulsions 221 spherical aberration 9 - of electron lens 290 spot measurement of exposure 215 spring-mount 56, 121-122 stage -, detachable mechanical - 31 -, sliding - 31 - micrometer 251-252 staining 117 stand 26 -, hinged - 26 - with upright (straight) tube 26 - with oblique tube 26 star test 54 statoconia 201, 204 stereo-ocular 234 stereo logy 264-269 stereo photomicrography 234 stereoscopic microscope 33-37 Stilb 17 stops 14 strain birefringence 195 strain-free objectives 201 stray-light 43, 96-97 substage condenser 31 substage mirror 92-93 super-8 film format 238 surface area fraction 266 surface density 267 surface-to-volume ratio 269

television microscopy 245-247 television scanning 271 Thompson prism 194 thyristor resistance 109 time-lapse recording 236-237 tissue section 115 total magnification 16 transmittance 233 transmission 224, 279 - electron microscope 291

336 INDEX

transverse magnification 7 travelling microscope 32 trinocular tube 212 tube 27 - diameter 68 --length 16 - -, infinite - 45 tungsten-halogen lamp 110-111 tungsten incandescent lamp 108-111 Tyndall-effect 147

ultramicroscopy 147 ultrathin sections 291 ultraviolet - microbeam 298 - microspectrophotometer 297 - microscopy 293-298 - objectives 58, 293 - sensitive phototube 296 - radiation 159, 289 upright image 7 useful magnification 83, 85

valuation of eyepiece micrometer 251-252 varia ble phase contrast 172 vertical fluorescence illumination 161 vertical illumination 106 video-recording 246 virtual image 7 visible light 4 visual angle 60 visuflash 234 volume density 267

volume fraction 266

wavefront 76 - reconstruction 235 wavelength - of X-rays 301 - in relation to colour 225-226 white radiation 301 wide-field eyepiece 68 Wollaston-prism 186-187 working distance of condenser 94 working table 119

X-ray 300-307 -, absorption coefficients in - 307 -, hard - 301 -, soft - 301 -, wavelength of - 301 - absorption analysis 302 - emission lines 301 - fluorescence analysis 302 - microanalysis 302-303 - reflection microscope 304 - microscopy - 308-313 - projection microscope 308-313 xenon high pressure burner 112 xylol 134

Z-prism 36 Zeiss, Carl 25, 42 zone of action 177 zoom-system 70 Zernike, F _ 167

light microscopic techniques in biology and medicine

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