Handbook of photomicrography

Front matter

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‘ A

HANDBOOK

Preface

This Handbook is the outcome of a series of articles originally published in the Photographic Monthly and intended as an introduction to photomicrography from a photographer’s point of view. In this volume the subject has been approached from its two aspects, with descriptions of both photographic and microscopic technique, and it is hoped that experts in either field will look with indulgence on an elementary treatment of the subjects with which they are familiar. At the same time, however, the processes are discussed in sufficient detail to be of use in research, for which the full possibilities of the combination of microscopy and photography are scarcely yet realised.

In a handbook devoted to general processes and methods it is impossible to give an adequate account of the many uses of photomicrography in technology and industry, but an attempt has been made to indicate briefly some of its main applications. Many, perhaps equally important, have necessarily been omitted. The methods described may, however, be applied by any worker with the microscope to his own particular investigations.

In the preparation of some of the more specialised portions, for reading of proofs, and for valuable suggestions, the authors have to acknowledge the kind help of several friends, in particular Mr. E. Collier Green, Drs. N. S. Finzi

VI

PREFACE and C. T. Holford, and Messrs. L. Archbutt, G. H. Broom, and R. Lyon. Their thanks are also due to manufacturers and their representatives for the loan of valuable apparatus and of blocks for many of the figures in the text.

Several of the illustrations are from photomicrographs of objects procured from slide-lending libraries. Access to slides which have been mounted by specialists is invaluable for practice in photomicrography and for the study of objects otherwise unobtainable, but the worker is strongly recommended to make his own preparations whenever possible. As a help in that direction the methods adopted in preparing some of the objects illustrated in the plates are described.

In conclusion we must recognise with thanks the great care and courtesy with which the Publishers have always met our wishes in regard to the illustrations and general arrangement of the book.

H. L. H.

W. B. R.

November, 1913.

Contents

•CHAPTER PAGES

I. Photomiceographic Apparatus .... 1-24 Horizontal cameras — Vertical cameras — The workroom — Prevention of vibration.

II. The Microscope ...... 25-46

Chapter I

A PHOTOMICROGRAPHIC Outfit Comprises a camera, microscope, and illuminating system, carried on a suitable baseboard or table. Each item is more or less elaborately fitted up with accessories, but the great majority of workers will find that quite a simple apparatus is adequate, and it may be said at the outset that the refinements in optical and mechanical details, which so rapidly run up the cost, are made primarily to facilitate difficult or high-power work, and to enable the most delicate adjustments to be carried out with rapidity and certainty. Difficult photomicrograph}^ can indeed be done with the simplest apparatus, provided the lenses are good, but the calls then made upon the skill and patience of the worker are much greater than when the adjustments are more or less automatic.

An ordinary stand camera, a student’s microscope, and an oil or incandescent gas lamp, with a bull’s-eye and Abbe condenser, low and medium-power objeetives, and an eyepiece, will do perfectly well for many purposes, and very few workers require anything more elaborate. But the photography of diatoms requiring very high resolution, or of baeteria neeessitating magnifications of

B p

PHOTOMICEOGRAPHY a thousand diameters or more, calls for lenses of high power and great perfection, and a microscope and camera with correspondingly delicate movements.

Photomicrography should not be regarded merely as a method of recording observations, but also as a valuable means of research. Its evidence must therefore he unimpeachable, and this can only be the case when the chances of false images, due to defects in the optical parts of the apparatus or to their improper use, have been ehminated as far as possible. The optical equipment is consequently the most important part of the outfit, and the aim of the designer and maker of the microscope stand, the camera, and optical bench, is to provide a framework which will carry the lenses, and to fit it with mechanical adjustments of sufficient refinement to bring them to bear on the object, and allow them to work to their best advantage. This does not involve the use of the most expensive objectives and condensers when slightly less accurately corrected, and consequently considerably cheaper ones, will serve, as they undoubtedly will in the majority of cases ; but it does mean that the accuracy of the fittings and the delicacy of the movements which carry and control the objectives, etc., must be commensurate with the corrections of the lenses, and that the latter be suitable for the work contemplated.

Although this view of the value of photomicrography is not lost sight of in what follows, the needs of the worker with restricted capital to outlay also have consideration. An endeavour is made to show how a considerable part of the apparatus can be made or adapted, and in the descriptions of camera, microscope, and illuminating system, the items which are luxuries, or refinements that may usually be dispensed with, are differentiated from those that are absolutely necessary.

Horizontal Cameras. — The Baseboard or Optical Bench. — There are two ways made use of for setting up the apparatus for photomicrography : the Horizontal, in which the optical axis is parallel with the table, and the Vertical, in which the camera is supported above the microscope. Preference is generally given to the former, particularly if a rather complicated illuminating system is employed. But many workers prefer the vertical arrangement, which has the great advantage that, with a simple lamp and condenser , an optical bench is unnecessary, as correct adjustment of the illumination can be carried out by means of the mirror. It is also more portable and compact, and with preparations in liquids it is generally the onty practicable method to use. Each method has its advantages, and if a camera has to be adapted the choice may depend on its greater suitability for one or the other system.

No doubt the most universally serviceable stands are those made for both horizontal and vertical work, but caution must be exercised to make sure that no loss of stability is involved in the arrangement, and every possibility of vibration of the camera should be avoided.

A photomicrograph may be taken after no further preparation than is involved in placing the camera, microscope, and lamp in -a straight line on a table, but the difficulties that have to be faced when using such an apparatus for anything but the lowest powers are at once apparent. Movement of the camera is almost inseparable from the action of drawing the slide, and renders accurate focussing of no avail. The readiest way to prevent this disturbance is to clamp the camera to the table ; but other difficulties connected with the proper centration of the various lenses are also encountered, and as a rule it takes much longer to get the apparatus properly together and the illumination adjusted than to take the photograph and develop it. When changes of magnification involving alteration of camera extension have to be made, there will be further loss of time, as either the camera or microscope, or possibly both of them, will have to be undamped and fixed in another position. These difficulties and others may be so easily overcome by the provision of a baseboard or optical bench of some kind that no attempt should be made to take photomicrographs without one, and a baseboard is so easily made by anyone having ordinary" proficiency in the use of tools that the first thing to consider, after the camera, microscope, and illuminating system are obtained, is how to design one suitable for the apparatus. It must be constructed so that the optical axes of the microscope, lamp, and condensers shall be truly in line and perpendicular to the focussing screen of the camera, and so that any of the pieces of apparatus can be placed in its exact position without loss of time. Above all, it must be so rigid as to prevent vibration as far as possible in any part of the apparatus. Illustrations are given of arrangements we have made ourselves to adapt apparatus already possessed. They have proved adequate, and are offered primarily as an indication of the lines to be pursued. A different camera, microscope, or lamp may involve a different arrangement, but very little ingenuity is necessary to devise one suitable to the materials at hand.

Illustrations are also given of several cameras on the market, each of which is fitted with an accurately made optical bench. They are not necessarily any more efficient than home-made apparatus, though they may be expected to be much better finished, to work with greater nicety and precision, and all these points become of great importance in difficult work, and add immensely to the ease with which photomicrography is carried out.

The Camera. — A camera specially made for photomicrography can hardly be described as a necessit}^ and many workers are content to adapt the one they use for other purposes to microscopical work, or else to fit

CAMERAS up a studio or field camera, and reserve it for photomicrography. Nor is there any need for discouragement when faced with these alternatives to a first-class professionally made outfit. If the camera fulfils the requirements set out below, it will answer the purpose admirably, and if set up in conjunction with the other apparatus with care and judgment, there is no reason at all why it should not serve for work of the very highest class.

In the first place, the base of the camera must lie perfectly flat on the table or board on which it is fixed, and the back must be capable of being adjusted so rigidly at right angles to this that the insertion of the dark slide, or the drawing of the shutter, causes no movement. The lens must be removable, as it is not used in photomicrography, and there must be a ground-glass focussing screen. In addition, a long extension will be found very advantageous, and if the camera can be extended from the back whilst the front remains in its original position, so much the better. A square bellows stand camera with a front of the same size as the back meets these requirements admirably, and it is easy to obtain an old-fashioned one without a lens for a very few shillings. A box camera fitted with focussing screen and dark slides can be used, and has the advantage of great rigidity, but the very great disadvantage that only one magnification can be obtained with one combination of objective and eyepiece, an objection that almost rules this kind of camera out for all-round work. A hand and stand camera of the type represented by the “ Sanderson,” “ Vaido,” or “ Una ” is perfectly suitable, but lighter types will be found rather tr3dng to use. Reflex cameras are in certain cases adaptable, and sometimes, as for instance in the photograph}' of living micro-organisms, are very useful. The apparatus illustrated in Fig. 4 includes a new camera, designed and patented by Mr. Lumley Gator, and marketed by Staley, called the “Convertible Britisher,”

which combines in itself the features of the last two groups of cameras. The reflex mechanism is contained in a separate segment that can be attached to the rest of the camera, or dispensed with at will, leaving an ordinary hand and stand camera.

The most generally useful size for the camera is half plate (6|"x4|"), or its American or Continental equivalents, but quarter plate (4j"x3J"), or better, 5" x 4", or 9x12 cm., is usually quite large enough. The 5" x 4" has an advantage over the others in that its shape is better proportioned to include a circle without waste. Larger cameras than half plate are seldom required, but 10" x 10" or 10" X 8" are often used on specially built outfits, and are very useful for enlarging purposes as well as photomicrography ; the dark slides of the larger sizes should be fitted with adapters to take the smaller plates down to lantern-slide size (3J"x3J'') and 3J"x2J".

Adaptation of an Ordinary Camera. — Fig. 1 shows a method of fitting up an ordinary half plate camera. The baseboard is of well-seasoned wood 4' x 9" x 2", with a groove t’V" broad doAvn its centre. Suitable blocks are cut to carry each component of the system, and strips of hard wood about |" deep are let into them along their centre line ; about of these projects and slides in the baseboard groove. The blocks may warp if not of well-seasoned wood ; their thickness is very carefully adjusted so that the optical axes of the camera, microscope, and lamp may be in line. The microscope carrier is first made, and depressions cut in it to receive the foot of the microscope, and to hold the latter so that the tube lies truly along the centre line and perfectly horizontal. If the microscope will not stand steadily when the tube is horizontal, some means must be taken to support it. A clamp or wooden turn-button pivoted between the horseshoe serves very well, and if nothing better is at hand a temporary support may be made by placing

HORIZONTAL CAMERAS a bottle and cork adjusted to the right height under the tube.

The optical axis of the microscope is then taken as the axis of the apparatus^ and the block to carry the camera is planed until the centres of the lens aperture and ground glass fall in this line. The means adopted for clamping the camera to its carrier will necessarily be dependent on the form of the camera base. Sometimes scrcAvs or turn-buttons can be used, in other cases a piece of wood may be placed over the baseboard and screv'ed to the block at either side to grip the camera tightly.

CAMERA WITH SLIDING CAIIUIEUS

It may be more convenient to provide two blocks for some cameras with long extension, the front and back being carried separately and sliding apart when the bellow'S are extended. If there is a centre panel three supports may be used.

Another block is adapted to the lamp. In the figure it will be noticed that this carries a rod on which the lamp slides up and down. The Liliput arc lamp shown is fitted with a condenser, but in other cases it may be necessary to cut a further block for the condenser.

Arrangement for an Enlarging Camera. — The arrangement shown in Tig. 2 differs from the first mainly in

PHOTOMICEOGRAPHY the method used for sliding' the carriers along the baseboard.

The camera is a large one (10" x 8"), with an extension of 26" and a breadth of 11", so that a baseboard 6' x 15" is used. A block to carry the camera is screwed on one end, and fitted with angle-irons at the front ; the camera has a rigid base, so that it can be readily placed in exact position against these, and held there by a screw clamp at the back.

Two strips of wood about 3' or 3' 6" long, and 1" square .section, are bevelled along one edge and screwed to the baseboard parallel to its length, and about 8" apart.

Fig. 2

ARHANGEME.S'T FOIl ENLARGING CAMEll.A to form runners between which the carriers for the microscope and illuminating apparatus slide. The carriers are made of inch-board bevelled to dovetail into the strips, and on them the microscope, lamp, and condenser are fitted as in the first example.

An ordinary optical lantern with 4" condenser is shown in the figure as the source of light. A cooling trough for water or fluid light-filters is carried in the holder provided for lantern slides. A bull’s-eye condenser is also shown, and can be used in conjunction with the large condenser, or in place of the latter ; it is screwed into a tube mount which can be pushed in or out of a wooden holder for focussing purposes. Colour screens and dia-

HORIZONTAL CAMERAS pliragms are carried in a recess on the other side of the holder, which is fitted with a dovetail slide for transverse motion on a cross-piece of wood carried on a vertically extensible brass tube — an ordinary telescopic bull’s-eye carrier. These two motions provide a rough means for centring the condenser.

An important point in both these arrangements is the means adopted for removing the camera when it is desired to examine the object direct^ through the microscope. In the first, its carrier slides off the baseboard ; in the second, the camera can be lifted off bodily, but in either case it can be replaced without any disturbance to other parts of the apparatus. In other cameras the microscope and illuminant are carried on a turn-table which can be readily swung in or out of the optical axis ; but this plan is rather difficult for an amateur to carry out satisfactorily unless well provided with tools. In professionally made outfits such as that of Mr. Barnard (Fig. 8), the turn-table works excellently.

Focussing Arrangements. — A long extension is of great advantage, in that it permits of a wider range of magnification from one combination of objective and eyepiece, and when a short camera has to be utilised it may even be advisable to add an extra bellows extension, such as is made by Messrs. Lancaster and other firms, to the back, or a conical tube blackened inside, to the front. A long bellows neeessitates the use of a mechanical focussing gear to reach the fine adjustment of the microscope while examining the image on the ground glass. In the camera above described it consists of a brass rod carried on two bearings screwed to the baseboai'd ; a milled head is soldered on one end ajid a pullej^ on the other. A piece of thread is used to form a band round the pulley and fine adjustment head, and motion is thereby conveyed with very fair delicacy to the fine adjustment, but no mechanical focussing device gives quite such delicate control over the fine adjustment as is obtained by direct manipulation of the milled head. Other focussing arrangements are shown at Figs. 41 and 46, and a well-known device for the purpose is a Hooke’s handle (Fig. 67). A little play must be allowed in the gear wheels connecting an unsupported handle to the fine adjustment, otherwise the focus will be upset when the rod is laid doAvn.

Screen for Light Adjustment. — An easel covered with white paper forms part of the apparatus shown in Fig. 2. The camera can be removed and replaced by this screen for the preliminary adjustment of the light, as it is easier to detect uneven illumination on the white surface than on the ground glass. The image is also more readily examined and focussed on the white paper, and it is possible to place the focal pla.ne of the camera exactty in the position originally occupied by the screen by means of a scale fitted along the baseboard, and pointers on the camera back and easel. The scale is also of use for determining by calculation from the camera extension the magnification produced by any objective and eyepiece whose powers are' Imown.

Light-tight Connection between Microscope and Camera. — The connection between the camera and microscope must be light-tight. A velvet sleeve with an india-rubber ring sewn into one end to fit over the eyepiece, and the other end joined to the camera, answers the purpose very well ; or a metal or cardboard ring about an inch deep can be fitted into the lens aperture of the camera, and a cell into which it slips placed over the eyepiece. One of these cells is shown in Fig. 1.

Focussing Screens. — The ground glass fitted to the camera back for focussing purposes should be as fine as possible, in order that delicate structures may be made out. Tyler’s micro-grain screens are very suitable, and they usually have a clear glass centre. If there is no clear space on the ground glass, a cover slip should be cemented with Canada balsam on to its centre, as it is so much easier to focus fine detail accurately on clear glass than on ground glass, however fine. A focussing lens is used to see the image under these conditions, and it is necessary to make sure that it is focussed on the glass and not on an aerial image in another plane. For this purpose a scratch is made on the clear glass, or lines drawn across the ground glass before the cover glass is cemented on, and these are kept in focus at the same time as the image. Some workers prefer a clear glass screen which can be substituted for the ground glass for final focussing, after the preparation has been searched and properly centred on the latter. Lines are ruled diagonally across the ground glass to aid in centring the light or the image, and it is also advisable to rule rectangles of lantern-slide size, and of the sizes of all the plates that may be used, to coincide with the positions of the adapters in the dark slides.

Centring the Apparatus. — The greatest care must be taken when making the apparatus to ensure that the optical axes of all the parts are truly in line ; if this is done at the outset all the components can be placed very rapidly in their proper positions, and only very small adjustments of the lamp or of the condensers, by means of their centring screws, are necessary before talcing a photomicrograph. The most careful measurements are hardly sufficiently accurate for this purpose, and the directions given on page 145 for centring the microscope, lamp, and condensing lenses should be followed out before their positions are definitely fixed.

Watson’’s Laboratory Camera. — The two cameras just described were made without any expert assistance,

VERTICAL CAMERAS and lack some of the delicate movements provided for centring eondensers, etc., in some of the better professionally made ontfits, and it is particularly in refinements of tliis sort and in general perfection of detail that the latter prove their superiority for difficult pliotcmicrographjL Fig. 3 shows a camera built by Messrs. Watson on the same lines as the other two, but with improvements in detail. The optical l)ench is of tiiangular section, and on it the condenser, diaphragms, and a Kingsford trough are carried by means of saddle stands with vertically extensible pilla.rs and centring screws for the condensers. The condenser is the Watson-Conrady system, as described later. The camera can be moved along its supporting rails — made of steel tubing — either from the back or front. Almost any sort of illuminant may be mounted on a suitable stand and used inside tlic case, and it may be well to point out that some such casing or lantern ought always to be used to enclose the illuminant, as it is much easier to focus when all extraneous light is shielded. The support next to the Mmp carries the condenser and iris, the next is the trough, and the third an iris diaphragm, so mounted that a supplementary condensing lens can be fitted into it when required.

Vertical Cameras. — Adaptation of Hand and Stand Cameras. — The stand, with blocks running in a groove, may very readily be used in a vertical position, as sho\vn in Fig. 4, provided that the back of the camera can be fixed rigidly at right angles to the baseboard. The camera shown is the “ Convertible Britisher,” arranged for reflex instantaneous photography. A slotted angle-iron is screwed on to either side of the carrying block, and two long screws are attaehed to the baseboard ; these pass through the slots in such a way as to allow the camera a considerable range of vertieal movement. Two thumbscrews suffice to clamp the block

PHOTOMTCROGRAPPIY rigidly at the desired height, their position limits the range of motion. The baseboard is screwed to a wall at such a height that the focussing screen is easily ac-

Fig. 4

KEFLEX VEKTICAL CAMERA cessible, the microscope and illuminating system being placed on a low table or box. The position of the focussing screen in reflex cameras is a great point in their favour, as it permits of the fine-adjustment head being reached even when long extensions are employed.

Van Heurck Camera. — Dr. Van Heurck was the greatest advocate of the vertical system and employed it very largely, and a camera named after him made by Messrs. Watson is a most rigid and efficient instrument. It consists of a box standing on four legs over the microscope, and connected thereto by a velvet sleeve ; it is inextensible, and hence no change in the magnification can be made otherwise than by a change of lenses. There is a door in the front of the camera large enough to allow of the hea,d being inserted to examine the object through the microscope, if desired, or to focus on a white card in place of the ground glass (Fig. 71).

Adjustable Vertical Cameras. — Fig. 5 shows a modification that can be made to fit any suitable camera that may be at hand. It consists of a four-legged support strongW and rigidly made. The top pieces are rebated, so that the camera fits exactly and firndy into them, but is removable at will. The baseboard of the camera also falls firmly into place on the stand, and supports the front rigidly and permits of any desired extension being made with ease. In this it offers an advantage over the Van Heurck model, and, to make use of different extensions easily, two bars aie screwed across the legs about 6" from the ground, and across these a removable board is clamped to carry the microscope, and, if desired, the illuminating apparatus. Another pair of bars can be fitted higher up, or blocks may be provided on which to raise the microscope still more when a shorter extension is used. This stand has proved very useful even for large pictures with an enlarging camera, but generally when long extensions are required the horizontal arrangement is better. In any case, when making use of a particular camera its adaptability to either method should be carefully studied, and the stand made in accordance with that to which it is best suited.

Many vertical cameras are on the niai’ket which permit

Fig.

VEUTIC.VL CAMERA

of a varying bellows extension ; some of them are sup-, ported by two arms running on a single upright metal pillar, and may be clamped at an}" desired position on the pillar. These cameras are very convenient to use, but have the drawback that unless very substantially

Photographs at Natural Size or small magnification with ordinary Photographic and Telephoto Lenses.

A Embryo CiircK in Shkll after 19 days incubalion x 1

Objeclive ... .\l)eiiurc ...

Plate

Devc]o})cr

... Imperial Special Rapid.

... Diffused daylight.

... I'yro-soda.

The chick was exposed hy carefully culling away Ihe upper part of the shell under water.

The ])hotograph was taken with the ohject completely immersed in water; the shell supported on a hase of plasticine.

The hlack background is a sheet of l)lack paper soaked in methylated spirit jirevious to immersion in wafer, to avoid air bubbles rising from the paper.

Fossils x oh

Objeclive ...

.{perl a re ...

Exiension

J>lale.

IlluininunI Exposure ...

Developer I’he fossils were usi'd.

... Dallmeyer's “_\don” Telephoto, with exiension tube.

... Iris in extension tube closed down to its limil. ... Image distance, 19^". Separation of lenses, 5^". ... Schleussner’s “Paragon. ” Fine grain ordinary. ... 4 amj). arc, diffused.

... B. 2 min. Dark-coloured fossil.

C. 1 min. White fossil.

... Metol-quinol.

laid on dead hlack paper and a vertical camera

The great restriction of aperture is necessary to get covering pow eland depth of focus, as the “ \don'’ was made for distant objects.

I To follow page 16.

B

C

UNIVERSAL CAMERAS made the}' arc verj’ liable to tremors transmitted from any vibration of the table. The arms that carry the camera must be very rigidly connected to the pillar,

Fig. (5

or after wear the back of the camera may fall away from its perfectly horizontal position. Cameras carried between two vertical columns are better in this respect.

Universal Cameras. — It is a natural transition from the last two arrangements to an apparatus that may be used in either a vertical or horizontal position. This is iindoubtedly the most universally applicable form of camera, and becomes an instrument of the highest precision when fitted with an optical bench on which the

GEOMETRIC SLIDE APPARATUS 19

microscope, condensers, and lamp are carried. Fig. 6 illustrates a camera made by Messrs. Bausch and Lomb, and Fig. 44 one by Zeiss. Suitable optical benches can be obtained separately, or are supplied with the camera as a complete photomicrographic outfit such as is illustrated in Figs. 7 and 8. Fig. 7 shows Leitz’ large photomicrographic apparatus with the camera raised into the vertical position. A wheel may be noticed beneath the steel tubes which form the optical bench ; it is used for vertical adjustment of the stand carrying the microscope when the camera is used in a horizontal position. A flexible rod is supplied for focussing purposes, carrying a little wheel with india-rubber round its circumference, and bearing on the side milled heads of the microscope. The bellows of the camera can be drawn away from the back, if it is preferred to focus on white card.