Practical microscopy : an introduction to microscopical methods

Preface

In preparing a second, edition of ' Elementary Microscopy,’ published in 1905, much new matter has been added throughout, and the scope of the book considerably enlarged, the number of pages having been nearly doubled. The third chapter in particular, dealing with the choice of a microscope, has of necessity been largely rewritten, and to both this and the succeeding two chapters, dealing with accessories of the microscope, several descriptions of new instruments and apparatus have been added, with a view to bringing under the notice of workers apparatus which may be of service to them in their work, or to discussing the value of others which they may be thinking of purchasing. Chapter VI., dealing with the Practical Optics of the Microscope, has also been amplified in many directions — all, it is hoped, of practical service, but still contains no more theory than is necessary to understand the subject, and, like the succeeding chapter on the Manipulation of the Microscope, endeavours to provide that practical information and advice which experience has shown the author to be most needed.

Matters which have recently come prominently forward with regard to high magnifications and their value, the limits of vision and of resolution, photo-micro-

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PREFACE viii

graphy with ultra-violet light, and apparatus for examining ultra-microscopic particles, are fully dealt with in the present edition. An entirely new chapter on photo-micrography has been added, and the original short and elementary chapter on mounting has been replaced by a much longer one on Microscopical Technique, in which the author, without attempting the impossible task of describing special methods, has endeavoured to give a practical explanation of the broad principles of preparation, staining, etc., generally so little understood, and to leave their applications in zoology, botany, human histology, or pathology to be sought for elsewhere. Section-cutting has been dealt with at considerable length, in order to assist as much as possible the comparatively inexperienced worker, for whom the book is mainly written.

In view, therefore, of the great enlargement of the book and its more extended scope, it has been decided, after much consideration, that the retention of the original title would be misleading, and the new edition is accordingly published under the name of ' Practical Microscopy.’ It is hoped that this will not lead to confusion. In deciding on this title, which seemed to be the only one suitable, the author has not overlooked the fact that it has already been used by Mr. G. E. Davis ; but this latter book has been for many years out of print, and is of a different nature. In books on microscopy the choice of titles is, unfortunately, limited.

The author wishes to express his obligation to Mr. J. E. Barnard, F.E.M.S., Lecturer in Microscopy at King’s College, London, and to Dr. W. S. Lazarus- Barlow, M.D., F.R.C.P., Director of the Cancer Research Laboratories of the Middlesex Hospital, for kindly reading the proofs of Chapters VI. and IX. respectively,

PREFACE

IX and for making several valuable suggestions. He also wishes to acknowledge his indebtedness (like all other workers) to Mr. Bolles Lee’s ‘ Microtomist’s Vade- Mecum,’ to Professor Guyer’s ‘ Animal Micrology,’ and to Mr. Conrad Beck’s recent Cantor Lectures on the microscope, from which he obtained several useful suggestions. The major portions of Chapters \ III. and IX. appeared originally in Knowledge, and thanks are due to the proprietors for their kindness in permitting their reproduction here. Thanks are also again due to many firms of opticians for their ready courtesy in lending many of the illustrations, several of which have been specially made. The other illustrations arc drawn by the author.

Contents

I. Introductory - - - - - i

II. The Simple and Compound Microscope - - 3

III. The Choice oe a Microscope — Representative

Chapter 1 Introductory

In few tilings is a little initial advice and help more valuable than when one is first taking up the use of an instrument like the microscope. There are many who are desirous of purchasing a microscope, and then learning how to use it, who feel bewildered by the multiplicity of stands and accessories advertised by the various English and Continental makers. There are others who already possess microscopes, but have not yet mastered even the principles of an instrument which is essentially one of precision, and so fail to use their microscopes to advantage, with (he result that they soon relegate them to comparative obscurity when the first novelty of possession has departed. I here are yet others who use the microscope in scientific work, but even they have not mastered these principles, and scarcely realize how much their work might be facilitated by doing so. Several excellent manuals arc to be ol Rained on the subject, but they deal for the most part with more advanced microscopy and with its theory, whilst the keen competition amongst our leading opticians, and the constant change and progress to which it gives rise, render a not inconsiderable portion of such books quickly out of date. l he aim of the present work is first to indicate to the beginner, or to i

INTRODUCTORY the student, how lie may to most advantage spend his money on the necessary outfit — what, in fact, are the essential requirements he must look for in his instrument, together with its accessories — and then to explain in detail its manipulation and management, so that he may learn to use his instrument and its accessories to the very best advantage. Theory will not be more fully entered into than is necessary for explanatory purposes, the aim of the book being essentially practical, and whilst an attempt is made to give instruction on the elementary and broad principles underlying modern methods of preparing objects for the microscope, it has been decided that Nature, as revealed by the microscope, or technical applications, are quite outside the scope of this book. The suggestions as to the choice of a microscope are not directed to providing an outfit suitable only for whiling away a pleasant hour or two — for ‘ looking at pictures,’ in fact. Instead, it is the aim of this book so to advise the beginner that afterwards, when he has gained experience for himself, he may feel that what he has bought is not only capable of showing him the infinitely little in Nature as a pastime or pursuit, but will be equally serviceable for real and serious work in medicine, in zoology, in botany, or any other of the countless ways in which the modern microscope has become a necessity, and will enable him to use the microscope to the best advantage for that purpose. In spite of the universal use of the microscope in our science laboratories and medical schools, little practical instruction in the use of the instrument is generally given, and the author’s experience has been that there are large numbers of men who are only too glad to obtain the instruction which the following chapters endeavour to provide.

CHAPTER 11

The Simple Microscope. — Strictly speaking, a microscope may be cither simple or compound. By a simple microscope is understood a lens, or combination of lenses, which magnifies the image but once, and which is generally either held in the hand or provided with some simple form ol support. Of these, the best known are the ordinary reading-glass, made in all sizes, and the small pocket magnifiers, mounted in vulcanite or imitation tortoiseshell, containing one or more lenses (Fig. i). All these are of the simple double-convex type, and, though cheap, have the drawbacks and imperfections incidental to such a type. These drawbacks include loo. i.- Ebonite Pocket Mac.ni kicks much distortion of the image towards the edges of the field of view, and its further disturbance by fringes of coloui. 1 he details also are not as sharply shown as they should be. These imperfections may in the first place only be noticed when the lens is compared with one of a higher type, such as those about to be mentioned, but if the lens is used for prolonged work, supported by a stand, the resulting fatigue to the eyes becomes very marked. These magnifiers may be obtained from almost any optician at prices ranging from one shilling upwards. The watchmaker’s eyeglass is of this kind.*

Linen Tester. — The Linen Tester is of the same description, but is made with a folding support, at the bottom of which is a small square opening in which to count the number of threads to the inch. Another form of simple double-convex lens is mounted on three legs, and the focus is adjusted by means of a screw. This last is sometimes made of two lenses with a diaphragm between.

Coddington Lens. — The Coddington Lens is a single double-convex lens which has a groove cut all round the middle, this groove being blackened or filled with black cement so as to act as a limiting diaphragm. The lens itself is of considerable thickness, being really only the central portion of a glass sphere. The performance is very imperfect, and it is now less frequently met with than formerly.

* The author remembers that a good many years ago an ingenious street-vendor used to sell in the neighbourhood of St. Paul’s Churchyard a microscope of this sort made out of a pill -box, with a small hole in the lid, in which was a drop of clear balsam. A similar, but of course temporary, microscope can be made by boring a hole in a blackened card with a red-hot needle, and inserting therein a drop of water.

Stanhope Lens. — The Stanhope Lens is also a double-convex lens, but the curvatures are unequal. In use, the object to be examined is placed on the side with the least convexity, and in actual contact with it, and it is just in focus when the other side of the lens is turned towards the eye. It is obvious that its uses are very limited.

Fig. 2. — Steixiieil Lupe (Section).

Aplanatie Magnifiers. — Of late years an entirely new form of simple hand-lens has come largely into use, and few working microscopisls are without one or more of these, either for pocket use or for dissecting. These are achromatic triplets, which arc also aplanatie. They are often spoken of as ‘ Steinheil lupes.’ There are several formuke adopted by various makers, and there is not much to choose between them, but their construction is as illustrated (Fig. 2), and their distinctive features are exquisite definition, freedom from colour, a Hat field by means of which the whole of the lens can be utilized, the image remaining sharply in focus up to the very margin, and ample working distance. In the higher powers particularly they have therefore a marked advantage over the simpler form of lens. Browning makes these triplets under the name of ‘ platyscopic ’ lenses in magnifications of xo, 15, 20, and 30 diameters. R. and J. Beck supply them with magnifications from 6 upwards in either ebonite or German silver. Chas. Baker, James Swift and Son, and W. Watson and Sons, supply them either in a simple ring to fit into a dissecting holder, or in a mount for the pocket (Fig. 3). Lcitz and Reichert supply them in dissecting mounts or in pocket mounts, whilst Bausch and Bomb’s form is known as the Hastings’ triplet. Any of the above lenses may safely he selected, the most convenient power for everyday work being perhaps 6, and after that 10. For a pair 6 and 15 would be handy sizes, but the working distance of the latter will he found to be rather short. In use they are held close to the eye.

The Compound Microscope. — By a compound microscope is understood a combination so arranged that the image given by the first lens is itself magnified again before reaching the eye. These lenses are called respectively the objective and the eyepiece, or ocular. Opaque objects are examined by means of either natural or artificial light reflected from their surfaces, but the vast majority of microscopic objects are specially prepared so that they may be viewed by light transmitted through them in order to show their structure. The source of light can be looked at direct, but this would not be convenient in many cases, and therefore beneath the stage holding the object in position is placed a mirror capable of movement so as readily to reflect the light through the object into the lenses. Many microscopes, especially the cheaper Continental stands, make no further provision than this, but the improvement in microscopic objectives and the use of higher powers has necessitated the interposition between the mirror and the object of another lens or system of lenses with which to condense (he light, and even accurately to focus it upon the object. These are therefore the elementary essentials of a modern compound microscope, and the following explanations concern the elaborations of these essentials which precision of working lias called forth. The explanation of the principles of microscopical vision and of microscopical optics will be left until a later chapter, it being desirable to assist the reader to choose his microscope first, and to explain its principles and use to him afterwards.

Parts of a Microscope. —Leaving aside for the moment the optical parts of the microscope, it will be observed that the instrument consists of a foot or base (Fig. 4) ; a stage to hold the object; a mirror to reflect the light ; between this and the stage probably a sub-stage carrying a condenser to condense the light ; and a limb bearing a body tube. Into the lower end of this tube is screwed the objective, whilst into the upper end is inserted the ocular or eyepiece, the tube itself having a coarse adjustment for focussing, either by sliding bodily in a sleeve or by rack and pinion, and being further elaborated by a fine adjustment for use with high powers. We shall take all these in turn, explaining them as we proceed.

The Foot. -On the Foot depends the stabililv of the stand as a whole. There are practically two forms the pillar and horseshoe form and the Fnglish tripod. The advantage of the former is that it is generally a little cheaper, and the sub-stage apparatus is more conveniently manipulated when the microscope is used inner draw-tube outer draw-tube rack -work to draw-tube

-coarse adjustment

-- body -tube adjust me limb fine

adjustment - to substage

clam

coarse adjustment to Subs tail - rod mirror eyepiece

- stc ge

idmc bar mechanical Stage

SubsTage centring Screws

Fig. 4.— Parts of a Microscope.

in an upright position, as is often necessary with medical and zoological workers. In any other than the upright position it is much less steady than the tripod form, whilst for horizontal work, especially for photo-micrography, it is quite top-heavy. The additional weight put into this form of stand to remedy the instability is also a disadvantage. The importance of these points can only be fully appreciated and verified by the practical worker, but the tripod foot, either in its ordinary or modified form of bent claw (Fig. 14, p. qb), is daily coming more and more into favour — in England, at any rate— and we unhesitatingly recommend its choice. At the same time, the pillar and horseshoe foot is almost universal on the Continent, and American makers have borrowed the same model. As a result some of our own makers have been obliged to follow suit. Whatever form of stand be adopted, ascertain that the actual points of contact with the table are no! more than three, or a most: irritating unsteadiness will result.

The stand must be capable of inclination. It is onlv the cheapest form of Continental stand for rough laboratory use that is without means of inclination.

The Stage. The Stage should be large and rigid. In its simplest form it will be provided only with two springs to hold the slide in position. These should be adjusted so as not to press too strongly upon the slide, otherwise, the lingers will be unable to make the. necessary delicate alterations in the position of the object. I he surlace may be of brass, of vulcanite, or of glass ; for ordinary purposes vulcanite is preferable. The aperture should be large, not less than an inch in diameter, and preferably considerably more. By having a large aperture the end of the linger can be inserted beneath the slid.' so as gently to raise i! when focussing with

IO high powers, the slide being lowered again when the approximate focus is found, and the focus of the objective finally adjusted. By this means the practised microscopist guards against any risk of damaging either his slide or his objective. Mr. E. M. Nelson, who has done so much for English microscopy, has suggested that the opening shall be cut away right to the front of t he stage so as to make a horse-shoe opening, and the suggestion has been adopted in more than one stand, as will be seen when we come to deal with the instruments of individual makers.

Fig. 5. — Sliding-Bar to Stage, with Finder.

Sliding-Bar. — A great improvement upon the ordinary stage springs is a sliding-bar or carrier, which slips up and down either in grooves in the stage or outside its parallel edges (Fig. 5). It carries the usual springs, which can be turned aside or brought into use as required. By this means the adjustment of the slide can be carried out with greater precision, and in particular it can be systematically examined by moving alternately the slide horizontally and the bar vertically on the stage.

Mechanical Stage.— In a first-class instrument a further refinement called a mechanical stage is necessary (Fig. 4), though for ordinary work the fingers can be the simple AND COMPOUND MICROSCOPE n trained to a delicacy of adjustment that will cover most requirements. The mechanical stage has two milled heads which give rectilinear movements horizontally and vertically respectively. The milled heads should preferably be so arranged as not to travel with the stage, otherwise the screws are apt to foul the condenser. The largest and most elaborate stands have a fixed mechanical stage, which may or may not carry a sliding-bar in addition, but all the leading makers have now introduced detachable mechanical stages which can in many cases be added as accessories after the purchase of the instrument.

Graduations to Stage. -Yet another refinement is the addition of graduations to the stage, both horizontal and vertical, by means of which the position of a slide can be readily noted for future reference, as it is only necessary to replace the slide in the same position to insure a ready finding again of the same field of view. Such graduations arc shown in Fig. 5 as a simple and inexpensive but most useful addition to a stage fitted with a sliding-bar (sec p. 10). The graduations arc generally ruled off in millimetres, and numbered from 0 to 20 or 30. Where they are marked on a mechanical stage a stop is provided, removable or otherwise, against which to place the corner of the glass slide, and the almost universal adoption now of slides measuring 3 inches by 1 inch renders this easily practicable. In addition, verniers for more exact reading are provided, and by this means rough micrometric measurements can be made in conjunction with a line across the centre of the eyepiece. Other methods will be alluded to when we come to micromctric measurements (pp. 115 and njq).

Rotary Stage. Microscopes for special purposes, such as petrology, have also a complete rotation to the stage, and the most elaborate form of instrument is provided not only with this, but with a couple of milled heads just beneath the stage, by means of which the centre of rotation can be made accurately to coincide with the optic axis of the microscope in order to insure an absolutely true rotation. However carefully an objective may be constructed by its maker, it will be found that the optic axis of the microscope varies slightly with each change of objective, and this not only necessitates re-centring the condenser, and often re-finding the object, but becomes specially important and noticeable in case of rotation of the stage. To correct this, either the stage must be re-centred by means of centring screws provided for the purpose, or the objective itself must be adjusted by means of what is called a centring nosepiece. The latter way is cheaper, but naturally lengthens the tube; a point which will be dealt with later.

The last refinement of all is a rack and pinion by which the stage can be slowly rotated when necessary, and this should be capable of being readily thrown into and out of gear. It is seldom required, except in petrological work. There may also be graduations in degrees around the circumference of a circular stage to show the amount of rotation. In its most perfect form the whole of the stage, including the mechanical part, will rotate bodily, though a rotating top plate is sometimes provided instead, which is less expensive and often of service. A protest should be made against the absurdity of providing a microscope with a complete rotating stage, fitted with centring screws, and with elaborate graduations around its entire periphery, but which, owing to the projecting milled heads of the mechanical stage, is incapable of rotation for more than say, 300 degrees. The rotary stage is not often fitted to English students’ microscopes, and is sometimes carelessly made, so that there is a troublesome amount of ‘ play ’ in it when used with high powers.

Mirror. — The Mirror is carried upon a tail-rod, either square or circular, and sometimes with an extra joint in it. Under any circumstances it should be capable of being swung aside. In certain microscopes sufficient movement is allowed to enable the mirror to be brought above the stage so as to illuminate an opaque object, and we wonder this is not more frequently met with. For this reason we prefer a circular tail-rod when only one joint is provided, the support of the mirror itself having a corresponding circular clip. The mirror should have two faces, plane and concave ; they should lie of good size, and be hung in a gimbal so as to give universal movements. The concave mirror having a definite focus, it is necessary that there should be a means of focussing it vertically, and generally this is provided. We have, however, come across microscopes in which the movement was too short to allow of the concave mirror being properly adjusted. The best way to test this is to rellect and then locus the light of a lamp upon a piece ol white paper placed upon the stage of the microscope, the mirror being moved up and down the rod until the requisite locus is obtained. This focus will, however, strictly speaking, vary somewhat according to the inclination of the mirror, due to an alteration in the inclination of the microscope or height of the lamp.

The plane mirror should preferably be ‘ parallel worked,’ otherwise an annoying double or treble reflection will be observed when the condenser is in focus. 1 his may be obviated in most mirrors by rotating them in their cells, as suggested by Dr. Dallinger, until a certain point is reached, when all the images are superimposed. All mirrors might with advantage be mounted so as to admit of this rotation. The optical reason given for this duplication of the image is that an ordinary mirror is somewhat wedge-shaped, and there are, accordingly, distinct reflections from the glass surface, from the silver surface, and from the silver and air surfaces. To obviate this, certain workers use a rectangular prism instead of a mirror, but this has the disadvantage of requiring that the prism should be large enough for use with a modern condenser with its wide back lens. It is also, as might be expected, much less convenient to use, and is therefore comparatively seldom met with.

The Sub-Stage. — This is now a most important adjunct to the modern microscope. Its office is to carry the condenser and other apparatus. Without such a fitting no microscope-stand can be considered complete or capable of doing good work with other than low powers. It is the condenser that makes the modern microscope so enormously superior to its predecessors, and it is second only in importance to the invention of the achromatic objective. Its office, as already intimated, is to gather together, concentrate, and focus the light upon the object from beneath. No microscope should be purchased that has not fittings for such a condenser, or to which the condenser cannot, at any rate, lie subsequently readily added. In its simplest form it is merely a ring fitted beneath the stage, and centred with the optic axis of the microscope. The inside diameter of this ring should be of the ‘ universal ’ size adopted by the Royal Microscopical Society— i.e., i£ inches full (1-527 inches, or 38-786 millimetres). 1 his gauge is not uniformly observed on the Continent, Reichert, with his usual enterprise, adopting it, but

Lcitz retaining one of 30-5 millimetres (1-2 inches), and Zeiss one of 36'S millimetres (145 inches). The condenser, in a suitable mount, slides up and down in this ring, which is often made to swing aside when not required.

Focussing Sub-Stage. — But as the condenser requires focussing like the objective, a focussing adjustment becomes more than a convenience. This in its cheapest form is provided by means of a left-hand screw and milled head working vertically (Fig. 17, p. 39), which is so arranged as to swing the condenser aside when it is quite out of focus and clear of the stage. But in the best instruments a complete sub-stage with rack and pinion is provided (Fig. q, p. S), and there may even Ire added an additional line adjustment to enable the last delicate adjustments to be made without setting up any disturbing tremor. 1 his is. however, a refinement that is only needed for the most critical work. Further, as the centring of objectives generally varies more or less, 1 as already intimated, it is advisable to have some means ol centring the condenser, a matter of no little importance in high-power and critical work.

Centring Sub-Stage. 1 his is provided by two centring screws working at right angles against a spring, or by piopcr lect angular movements. It is generally found only in compound sub-stages (Fig. 4), though Swift fits centring screws to a simple ring beneath the stage. But whether a simple ring or a complete sub-stage be provided, it should be constructed in a workman-like manner. There should be no tendency to shake, a weakness to which the swing-out and spiral-screw forms aie somewhat liable, and the centring should remain the same whether the condenser be screwed or racked up 01 down. A means ol rotating the condenser or othei i6 fitting by rack and pinion is sometimes provided, but in few cases is this worth the additional cost.

Diaphragm. — The Diaphragm is a means of adjusting the light so as to bear some ratio to the apertures of the various objectives. In its simplest form it is a circular plate containing apertures of various sizes, set just beneath the stage, so that by rotation each of the openings can be brought central with the opening in the stage. An iris diaphragm is also not infrequently fitted in this position. The most unsatisfactory form of all is where one or more rings are provided to fit inside the stage opening. But the widespread use of the condenser has necessitated the placing of the diaphragm immediately beneath it, and in this position the plate of diaphragms has given place to a set of loose stops which can be slipped into a suitable carrier, or to the now familiar iris diaphragm.

Condensers themselves are dealt with in Chapter V.

The Limb. — The Limb is the arm carrying the body tube, and on its construction much depends. There are practically three forms now in use — that originally designed by Ross, and known as the bar movement, the solid limb, and the Continental limb. 1 hey are so closely bound up with the coarse and fine adjustments that we must consider them under these headings.

Coarse Adjustments. — The Coarse Adjustment in its simplest form is simply a broad ring in which the body tube slides. It is manifestly only fitted to the least expensive instruments, though when an efficient fine adjustment is provided in addition it is capable of good work up to a certain point, but as it cannot rotate truly in its ring, it necessarily increases troubles due to de-centring. Not the least of its disadvantages is, however, the increased danger of damage to either objective

TIIE SIMPLE AND COMPOUND MICROSCOPE 17

or slide, and we consider a thoroughly well-made rack and pinion coarse adjustment, without fine adjustment, often preferable to a sliding coarse adjustment and an additional fine adjustment. W. Watson and Sons have recognized this fact by putting such an instrument upon the market in their ‘ School ’ microscope. Whenever possible, and certainly in all stands intended for good work, the coarse adjustment should be by rack and pinion. Many years ago Swift and Sons introduced a ‘ diagonal ’ rack and pinion, which has now been adopted by all the leading makers, which works with exceeding smoothness, and is less sensitive to wear and consequent ‘ back-lash ’ than the older rectangular form. In fact, the fitting of any other than a diagonal rack and pinion at once stamps a microscope as inferior. Mr. If. M. Nelson has introduced a ' stepped rack and pinion, formed by two racks, one of which is capable of adjustment, but this is, of course, more costly than the ordinary type. The bearings of the coarse adjustment, or the slides in which it works, should be ‘ sprung,’ and provided with screws for adjustment for wear and tear, and there should be ample bearing surface. The milled heads should be large.