Elementary microscopy : a handbook for beginners

Preface

The groundwork of this little book originally appeared as a series of articles written by the author under the title of ‘ Microscopy for Beginners.’ They .were continued for eighteen months, and appeared to so far attain the purpose for which they were written as to justify their republication in book form. For this purpose they have been almost entirely rewritten and recast. The author desires to disclaim any idea of comparison with larger and more elaborate works, and has confined himself strictly to the microscope itself, leaving its applications to be sought for elsewhere. He has endeavoured throughout to keep in view the wants of the beginners for \yhom he originally wrote, and to give that practical information and advice as to the choice of a microscope and its accessories, their uses, and the more elementary methods of preparing objects for the microscope, which experience has taught him the beginner most needs. He has tried also to explain, without technicalities or too much detail, the principles underlying microscopical practice, methods, and technique.

Thanks are due to many firms for their ready courtesy in lending many of the illustrations, several of which have been especially made.

Jesus College, Cambridge,

February, 1905.

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Contents

CHAl'TER PAGE

I. INTRODUCTORY I

II. THE SIMPLE AND COMPOUND MICROSCOPE - - - 3

Chapter I Introductory

In few things 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 the result that they soon relegate them to comparative obscurity when the first novelty of possession has departed. There 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 are to be obtained on the subject, but they deal for the most part with more advanced microscopy and with its applications, whilst the keen competition amongst our leading opticians, and the constant change and

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INTRODUCTORY progress to which it gives rise, render a not inconsiderable portion of such books quickly out of date. The aim of the present work is simply first to indicate to the beginner, who, with much enthusiasm, little or no knowledge, and perhaps not too well lined a purse, proposes to take up study with the microscope, how he 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. Theory will not be more fully entered into than is necessary for explanatory purposes, and whilst an attempt is made to give instruction on elementary methods of preparing objects for the microscope, it has been decided that Nature, as revealed by the microscope, is quite outside the scope of this little 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 writer’s aim to so 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 enable him, if he so desires, to do real and serious work in medicine, in zoology, in botany, or in any other of the countless ways in which the modern microscope has become a necessity, and to use the microscope to the best advantage for that purpose.

Chapter II

The Simple Microscope.— Strictly speaking, a microscope may be either simple or compound. By a simple microscope is understood a lens, or combination ol lenses, which magnifies the image but once, and which is generally either held in the hand or provided with some simple form of 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. They may be obtained from

Fig. i. — Ebonite Pocket Magnifiers.

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.

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.

Aplanatic Magnifiers. — Of late years an entirely new form of simple hand-lens has come largely into use, and few working microscopists are without one or more

* 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.

of these, either for pocket use or for dissecting. These are achromatic triplets, which are also aplanatic. They are often spoken of as ‘ Steinheil loups.’ There are several formulae 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 flat field by means of which the whole of the lens can be

Fig. 3. — Platyscopic Lenses.

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

ELEMENTARY MLCROSCOPY makes these triplets under the name of ‘ platyscopic ’ lenses in magnifications of io, 15, 20, and 30 diameters (Fig. 3). 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. Leitz and Reichert supply them in dissecting mounts or in pocket mounts, whilst Bausch and Lomb’s form is known as the Hastings’ triplet. Any of the above lenses may safely be 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.

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 that they may be viewed by light transmitted through them so as 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 the light, and even accurately 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 has called forth. We propose to leave the explanation of the principles of microscopical vision and of microscopical optics until a later chapter, desiring first to assist our reader to choose his microscope, 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 being capable of coarse adjustment for focussing, either by sliding bodily in a sleeve or by rack and pinion, and 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 stability of the stand as a whole. There are practically two forms — the pillar and horseshoe form and the English 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 must be used in an upright position, as often happens 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 ad-

ELEMENTARY MICROSCOPE inner draw-tube outer draw-tube ruck -work to draw-tube

-coarse adjustment

-- body -tube eyepiece fine -- limb

-fiat

adjustment to sub stag ci am

-foot - -

Coarse - - adjustment to

Substage idmq bar mechanical Stage.

Substage -centrirx mirror tail - rod

Fig. 4. — Parts of a Microscope.

ditional 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. 12, p. 33), 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 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 not more than three, or a most irritating unsteadiness will result.

The stand must be capable of inclination. It is only 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 fingers will be unable to make the necessary delicate alterations in the position of the object. The surface 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 finger can be inserted beneath the slide so as to gently raise it when focussing with 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 glide or his objective, Mr. E. M. Nelson, who has done

IO so much for English microscopy, has suggested that the opening shall be cut away right to the front of the 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.

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,

Fig. 5. — Sliding Bar to Stage.

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. 6), though for ordinary work the fingers can be trained to a delicacy of adjustment that will cover most requirements. The mechanical stage has two milled

THE SIMPLE AND COMPOUND MICROSCOPE n 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

Fig. 6. — Detachable Mechanical Stage.

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. The graduations are generally ruled off in millimetres, and numbered from o 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 micrometric measurements (pp. 91 and 141).

Rotary Stage. — Microscopes for special purposes, such as petrology, have also a complete rotation to the stage, and the most complete 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; but, apart from the necessity of recentring the condenser, this only becomes important or noticeable in case of rotation of the stage. To correct this either the stage must be recentred 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.

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. 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 not infrequently provided instead, which is less expensive and often of service. A strong 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.

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 be 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, but this is generally 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 reflect and then focus the light of a lamp upon a piece of white paper placed upon the stage of the microscope, the mirror being moved up and down the rod until the requisite focus 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. This 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, be 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 (i'527 inches, or 38-786 millimetres). 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. 21, p. 42), 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. 12), and there may even be added an additional fine adjustment to enable the last delicate adjustments to be made without setting up any disturbing tremor. This is, however, a refinement that is only needed for the most critical work. Further, as the centring of objectives generally varies more or less, as already intimated, it is advisable to have some means of centring the condenser, a matter of no little importance in high-power and critical work.

Centring Sub-Stage. — This is provided by two centring screws working at right angles against a spring, or by ^proper rectangular movements. But whether a simple ring or a complete sub-stage be provided, it should be constructed in a workmanlike manner. There should be 110 tendency to shake, a weakness to which the swing-

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ELEMEN TA R V MICRO SCOP Y out and spiral-screw forms are somewhat liable, and the centring should remain the same whether the condenser be screwed or racked up or down. A means of rotating the condenser or other 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, and set just beneath the stage, so that by rotation each of the openings can be brought central with the opening in the stage. 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. They 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. Not the least of its disadvantages is, however, the increased danger of damage to either objective or slide, and we consider a thoroughly well-made rack and pinion coarse adjustment, without tine adjustment, often preferable to a sliding coarse adjustment and an additional fine adjustment. W. Watson and Sons have recognised this fact by putting such an instrument upon the market in their ‘ School ’ microscope. Whenever possible, however, 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 form. Mr. E. M. Nelson has quite recently 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.

Fine Adjustments. — The Fine Adjustment brings us to a subject upon which much controversy has taken place. It is scarcely necessary to explain that good work with high powers cannot be done without an efficient fine adjustment, and of all parts of the microscope this is the one that has most tendency to deteriorate and become inefficient. The modest compass and design of this book prevent a full discussion of the various types now in use, and of the improvements and refinements which the ingenuity of various workers has brought forth, but we can explain in detail the main i8

ELEMENTARY MTCROSCOPY points of the principal types. First of these in point of seniority comes Powell and Lealand’s form (Fig. 25). The coarse adjustment is a massive bar working vertically within the casting which supports the stage. To the top of this and at right angles is bolted a bar or arm which projects beyond the centre of the stage, and into which is screwed the body tube. Within this horizontal arm is a lever of nearly the same length, which is raised and depressed by means of a screw fitted with a milled head, and placed just behind the junction of upright bar and arm. The fulcrum of this lever is near the body-tube end of the arm, and within the body tube it moves a nosepiece which carries the objective. As made by Powell and Lealand in their famous but costly stands, this form of coarse and fine adjustment has, as a well-known writer says, ‘ held an unrivalled position for the past fifty years.’ But unless the reader proposes to buy a microscope made by this particular firm, we would strongly urge him to avoid absolutely any instrument in which this model is apparently followed, whether it be new or second-hand. As made and exposed for sale in the windows of the average optician, the coarse adjustment is a mean triangular bar with perhaps one edge converted into a rack- work, and showing distinct lateral play even before use, whilst the fine adjustment is a screw at the side of the body tube working on a bracket, and actuating an ill-fitting short tube within. There are hundreds of such stands for sale in London and the provinces, but they are only sold to the ignorant, and a maker of any present-day reputation would be chary of displaying even a second-hand microscope of this type in his window. The coarse adjustment is untrustworthy from the start, and rapidly becomes worse, whilst the fine adjustment deteriorates even more quickly. The next form of fine adjustment is that of which the J ackson model may be taken as a type (Fig. 33). Here there is a solid arm projecting over, and firmly bracketed to, the stage. The rack-work of the coarse adjustment is attached to the body tube, and works in V-shaped slides. We have already said that these bearings should be of ample length and properly

Fig. 7. — Watson’s Fine Adjustment.

‘ sprung.’ Between the slides of the coarse adjustment and the limb or arm is a second set of similar V-shaped slides forming the movement of the fine adjustment. The body is raised or lowered by means of a lever working against a spring, and actuated by a fine micrometer screw (Fig. 7), much as in the Powell and Lealand form, except that the whole body instead of an inner tube is moved. This is one of the best forms known ; it is rigid, extremely sensitive, and not liable to deteriorate rapidly even in unskilled or careless hands. In this form also it is apparent that the distance between objective and eyepiece remains unaltered. This fine adjustment is adopted by W. Watson and Sons in nearly all their stands, and by Charles Baker in some of his more recent models ; also by J ames Swift and Son in one or two models, but in the last maker’s stand the lever is bent downwards, and the milled head comes at the side, so that it cannot be actuated by either hand. Watson and Sons have also brought out a further refinement of this adjustment designed by Mr. Stringer.