Chapter III (3)

The silver side-reflector is usually made with a ball-and-socket joint, so that it can be turned in any direction. It is secured to the stage of the microscope by the pin, which drops into a hole purposely drilled to receive it, and facility given for turning up and down, or in any position. If daylight is used the microscope should be placed in such a position that the light from a white cloud falls upon the speculum, but the light of the lamp is far more manageable for use with the reflector.

The Lieberkühn is only intended to be used with low powers--a 2-inch, 1/2-inch and a 2/3-inch. Such objects as the elytra of the diamond and other beetles are well suited for examination.

While experimenting with a parabolic reflector (Fig. 158), Mr. Sorby saw the value of examining objects under every kind of illumination. As on viewing specimens of iron and steel with this reflector he found that, from the great obliquity of the illumination obtained, the more brilliantly polished parts of the specimen reflected the light beyond the aperture of the objective, and these could not be distinguished from those parts which absorbed light, he thereupon proceeded to place a small flat mirror in front of the objective, and cover half its aperture, and at the same time stop off by means of a semi-cylindrical tube the light from the parabolic reflector. This arrangement produced the reverse appearance of that first employed, and it proved to be a useful aid in determining structure.

The Bull’s-eye Condensing Lens.

This accessory is brought into constant use for the purpose of converging rays from a lamp or mirror; or, for reducing the diverging rays of the lamp to parallelism with the parabolic illuminator, or silver side-reflector. The form in use is a plano-convex lens of about three or four inches in focal length (Fig. 159). It is usually mounted on a brass stand, so that it may be placed and turned in any direction, and at any height. When used by daylight, its plane side should be turned towards the object, and the same position maintained when used for converging the rays of light from the lamp; but when used with the side-reflector the plane side must be towards the lamp. Much attention has been paid to this very necessary accessory, the bull’s-eye lens. A doublet has been brought into use which has increased the value of the bull’s-eye condenser in bacteriological research, and in micro-photography generally.

“During a recent investigation of the spherical aberration in doublets, it was believed to be impossible to construct a doublet of the form known as ‘Herschel’s doublet’ free from aberration, although these doublets figure in many books on optics. In a condenser made by Baker the aberration is reduced to a minimum, 27 per cent. less than Sir John Herschel’s. This doublet, it appears, differs from Herschel’s both in the ratio of the radii of the meniscus, and also in the ratio of the foci of the two lenses; indeed, the only point of similarity is in the first lens, which is crossed. To test this, project the image of the flat lamp-flame on a piece of white card with a plano-convex lens (the field-lens of the Huyghenian eye-piece), use first the convex side and then the plane side towards the card, the lamp being placed about 6 feet from the lens. Focus the lamp-flame as sharply as possible, and a circular halo of misty light will be seen to surround the lamp-flame; but when the plane side of the lens is made to face the card this halo of misty light will be seen to be greatly reduced, and the brightness of the image of the flame proportionately increased. If the lens, then, were strictly aplanatic there should be no misty halo, all the light being concentrated in the image of the lamp-flame, and the image of maximum brightness. In short, the diameter of the halo or misty light is the measure of the spherical aberration. If the condenser referred to above, having the form of minimum aberration for two planes, be compared in the same manner with an ordinary single bull’s-eye of the same focus, the diameter of the misty halo will be found reduced to a radius of about 1/5-inch, but, with this new condenser there is a further reduction, so that the radius of the misty halo measures only 1/20-inch. These experiments are instructive, because the brightness, or the mistiness of the microscopical image is an associated phenomenon.”[29]

A sectional view of the optical arrangement of Baker’s aplanatic bull’s-eye doublet is shown, together with lamp, in Fig. 148.

_The Microscope Lamp._--The introduction of paraffin into household use has somewhat modified our views with regard to the most suitable artificial source of illumination. Good paraffin burns with a whiter and purer flame than colza oil, and consequently is less liable to fatigue the eyes. The first cost of the lamp is trifling; for a moderate sum a handy form of lamp can be had, mounted on an adjustable sliding ring stand, and with a porcelain, metal or paper shade, to protect the eyes from scattered rays of light. All opticians supply accepted forms of lamps.

To give the increased effect of whiteness to the light (“white cloud illumination” as it is termed), take a piece of tissue paper, dip it into a hot bath of spermaceti, and, when nearly cold, cut out a circular piece and secure it over the largest opening in the diaphragm plate. This will be found to materially moderate and soften the light.

_Beck’s Complete Lamp_ is constructed especially for delicate microscopical work. It has a burner giving a flat flame; this can be rotated to enable the edge or the flat of the flame to be used; likewise a metal chimney with two apertures, in which 3 × 1 glass slips slide; either white or coloured glasses may be used. A Herschel aplanatic condenser is carried on a swinging arm, which rotates around the lamp flame as a centre, and can be clamped in any position. The whole lamp has a raising and lowering motion, with a spring clamp to hold it in any position. The lamp is so designed that at its lowest position the flame is only three inches from the table. Here the microscopist is furnished with a lamp which will accomplish all he may require with regard to illumination.

Watson’s lamp (Fig. 161) has a metal chimney, and is somewhat simpler in structure than those already referred to. For the student, the simpler and cheaper form will answer every purpose. A glass holder for carrying various tinted slips of coloured glass to act as a screen or modifier of the light is much employed, and assists in determining fine structures (Fig. 162).

Nose-pieces and Objective Changers.

A convenient appendage to the microscope is the rotating nose-piece, invented by Mr. Charles Brooke, F.R.S., and intended to carry two or more objectives, whereby a saving of time is effected, and the trouble of repeatedly screwing and unscrewing is avoided. In the application of the nose-piece attention should be given to centring. Messrs. Baker’s objective changer is intended to facilitate the placing and replacing the nose-piece in position. This adaptation consists of a milled head, acting on three jaws, having a universal screw thread, a decided improvement on the screw. Zeiss has adopted a tube-sliding objective changer with centring adjustments. Messrs. Watson met the difficulty of centring by making the nose-piece a part of the body-tube of their microscopes (Fig. 163). This, when adapted to the shorter body of the students’ microscope, fully compensates for want of length.

Their triple nose-piece is constructed with much care, and when in use is found very effective. It is manufactured of that very light metal aluminium, and which minimises the strain produced by the heavier brass nose-piece.

_Finders._--The finder affords a necessary and useful means of registering the position of any particular object, so that it may be readily found again at any subsequent period. In the work of examination the finder will save time when making a special research, extending over a considerable surface.

That the finder has been of use may be surmised from the number invented and figured in the “Journal of the Royal Microscopical Society.” By far the most useful form is that of graduating the plates of the mechanical stage, dividing a certain portion into 100 parts. Powell and Lealand have adopted this system in their No. 1 stands, while Baker and Watson have added a graduated scale on silver to 1/100th mm. as a finder, and also a stage micrometer in 1/10th and 1/100th of a millimetre, together with a Maltwood finder for lodging the position of any desired portion of a specimen under examination.

The _Maltwood_ finder (Fig. 165) can be used with any microscope, and without a mechanical stage. This useful finder continues to occupy a permanent place among the accessories of the microscope. It consists of a glass slide, 3 × 1-1/4 inches, on which is photographed a scale occupying a square inch; this is divided by horizontal and vertical lines into 2,500 squares, each of which contains two numbers marking its “latitude,” or place in the vertical series, and its “longitude,” or place in the horizontal series. The scale is in each instance an exact distance from the bottom and left-hand end of the glass slide; and the slide, when in use, should rest upon the ledge of the stage of the microscope, and be made to abut against a stop, a simple pin, about an inch and a half from the centre of the stage.

Dr. Pantacsek’s finder appears to have some advantage over Maltwood’s, but it cannot be used with the same facility, and therefore will not displace an old favourite. _The Amyot_ finder I have long had in use; it is efficient and inexpensive--can indeed, if misplaced or lost, be replaced by the aid of the square and compasses.

_The Okeden_ finder consists of two graduated scales, one _vertical_, attached to the fixed stage-plate, the other _horizontal_, attached to an arm carried by the intermediate plate; the first of these scales enables the worker to “set” the vertically-sliding plate to any determinate position in relation to the fixed plate, while the second gives the power of setting the horizontally-sliding plate by that of the intermediate.

_Micrometers._--It is of the utmost importance to have a means of measuring with accuracy the objects, or part of objects, under observation. The most efficient piece of apparatus for the purpose is the micrometer eye-piece, the earlier form of which, Jackson’s, has been described under the heading _Eye-pieces_ (p. 144). In the case of micrometers, as in that of most other accessories, every optician has his own adaptation and method of employing the same.

For the measurement of bacteria, a stage micrometer should be used with a camera lucida. The stage micrometer consists of a slip of thin glass ruled with a scale consisting of tenths and hundredths of a millimetre. The image is projected on to a piece of paper placed on the table, and the drawing made, and the object to be measured can be readily compared with the scale.

In the Ramsden micrometer eye-piece, as previously explained, two fine wires are stretched across the field of an eye-piece, one of which can be moved by a micrometer screw. In the field there is also a scale with teeth, and the interval between them corresponds to that of the threads of the screw.

The circumference of the brass head is usually divided into one hundred parts, and a screw with one hundred threads to the inch is used. The bacterium to be measured is brought into a position in which an edge appears to be in contact with the fixed wire, and the micrometer screw is turned until the travelling wire appears to be in contact with the other edge. The scale in the field and scale on the milled head, together, give the number of complete turns of the screw and the value of a fraction of a turn in separating the wires.

In the micrometer eye-piece constructed by Zeiss, the eye-piece with a glass plate with crossed lines is carried across the field by means of a micrometer screw. Each division on the edge of a drum corresponds to ·01 mm. Complete revolutions of the drum are counted by means of a figured scale in the visual field.

In the micrometer used with Zeiss’s _apochromatic_ objectives and compensating eye-pieces the divisions are so computed, that, with a tube-length of 160 mm., the value of one interval represents, with each objective, just as many micra (·0001 mm.) as there are millimetres in its focal length. A value of tables is therefore not required for these eye-pieces, since the focus of the lenses indicates their micrometer values within 5 per cent.

The _Camera Lucida_ will prove an extremely useful adjunct to the micrometer, and a large number of contrivances have been devised for its employment. There are those which project the image on to the surface of a sheet of paper provided for the drawing, and those which project the pencil and paper into the field of the microscope. The former method is that usually adopted. To draw an object, with either a Wollaston camera lucida or a neutral tint reflector, such as that of Beale’s, both of which are made to slide on and take the place of the cap of the eye-piece, as shown in Fig. 168, with its flat side uppermost, the whole instrument must be raised until the edge of the prism is exactly 10 inches from a piece of paper placed upon the table; with the latter the instrument retains its vertical position, and the image of the object is thrown on the paper placed in front of the stand. The light must be so regulated that no more than is really necessary is upon the object, whilst a full light should be thrown upon the paper. Only one eye is to be used; and if one half of the pupil be directed over the edge of the prism, the object will appear upon the paper, and can be traced on it by a pencil, the point of which will also be seen. Should any blueness be visible in the field, the prism is pushed too far on, and should be drawn back till the colour disappears.

The position in which the microscope must be placed is shown in the accompanying illustration (Fig. 169).

Beale’s neutral tint reflector (Fig. 170) is much in use, and its advantages are utility, simplicity, and inexpensiveness.

The Abbe model of camera lucida has been brought into use because the projected image can be better illuminated, and is consequently so much brighter. This form is now made in aluminium by Messrs. Watson & Sons. In place of the image being traced by projection on paper, the reverse is the case, both the paper and pencil are projected into the field of view. The mirror reflects the paper on to the silvered surface of a prism placed over the eye-lens of the eye-piece of the microscope, and it is thereby conveyed to the eye. There is a central opening in the silvering through which microscopic vision is obtained. It is fitted in a new manner by means of a cloth-lined adapter, fitting over the outside of the microscope tube; this saves all trouble in centring and ensures concentricity. Where the instrument has capped eye-pieces, the camera lucida must be adapted to the eye-piece, the cap being removed. The apparatus can be disconnected from the fitting adapter by means of a sliding pin, and readily replaced, or can be lifted over out of the way, as shown in the drawing. Being made almost entirely in aluminium it is very much lighter than other forms of apparatus, and does not cause vibration. It can be used with the microscope _at any angle_, the only necessity being that the paper on which the sketch is made should be kept at the same angle as the instrument.

Micro-Photography.

Micro-photography or photo-micrography, as it is indifferently termed, has, to a very considerable extent, superseded the use of the camera lucida for the delineation of images seen under the microscope. I may claim to be among the first workers with the microscope (1841) to prove beyond a doubt that the camera could be made to render invaluable aid to the microscopist, whereby a great saving of time might be effected, and a drawing obtained with greater accuracy than that of the pencil of the draughtsman.

It was about 1864-5 that Dr. Woodward’s earlier micro-photographs were first seen in London. His skill in the manipulation of the microscope had been long known. His first series of photographs of test diatoms created, I remember, quite a sensation; they have probably never been surpassed. These were taken by sun-light, magnesium, and electric-light. I was the recipient of a series taken at a later date (1870), and which, bound in quarto volume, are almost as perfect in definition as any of a later date taken by oil-immersion objectives.

The objectives used by Dr. Woodward, throughout, were a 1/8-inch of Wales’s (new series), and a 1/16-inch immersion, of Powell & Lealand’s, especially produced for work with the camera. The magnification varied from 800 to 3,000 diameters, a frustule of _Grammatophora Marina_ magnified 2,500, and a scale of podura, marked 3,000 in my collection, are equal in definition to those taken by a high-angle 1/12-inch oil-immersion. Pathological specimens taken with lower powers are equally instructive, a section of epithelial cancer showing both nuclei and cells with distinctness.

Dr. Maddox in 1864 was also experimentally engaged in the improvement of the processes of photography for the purpose of promoting the work of microscopists. His labours were attended with great success. To him we are indebted for the gelatine dry-plate process, which gave a remarkable impetus to photography in general. Dr. Maddox has, for a period extending over forty years, diligently and successfully cultivated and promoted micro-photography. Among other workers to whom we are indebted for improvements in micro-photography I may mention Wenham, Draper, Shadbolt, Highley, Koch, Sternberg, Pringle, Leitz, and Pfeiffer.

Dr. Koch justly claims the credit of having extended the application of micro-photography to the delineation of bacteria. A series of instructive micro-photographs were published by him in 1877.

The importance of the camera has become more manifest as the work of the bacteriologist has progressed. Koch strongly advocated micro-photography on the ground that illustrations, especially of bacteria, should be as true to nature as possible. Dr. Edgar Crookshank holds the same opinion, and in support of his views we have numerous illustrations of the bacteria given in his valuable “Text-book of Bacteriology.” But he does not disguise the truth that there are difficulties to be encountered, the first of which is owing to the fact that the smallest and most interesting bacteria can only be made visible in animal tissues by _staining_. This drawback has been very nearly overcome by the use of eosin-collodion. With this medium, and by shutting off portions of the spectrum by coloured glasses, Koch succeeded in obtaining photographs of bacteria, which were stained with blue and red aniline dyes. This method, however, introduced a disturbing element of another kind. Owing to the longer exposure required, the results were wanting in definition, attributable, it was thought, to vibrations of the apparatus produced by passing traffic, or by assistants moving about over the floor of the laboratory.

Koch nevertheless showed, at the great meeting of the International Medical Association in London, 1881, a series of micro-photographs of bacteria and tissue sections, which were the admiration of all who saw them. To meet a difficulty occasioned by the aniline dyes, Koch recommended that the preparations should be stained brown; other experimenters found that preparations stained either yellow or yellowish-brown gave good photographic representations; but it is by no means an easy matter to find a good differential stain of bacteria in the tissues, as even Bismarck brown is not entirely successful. Other bacteriologists have encountered similar difficulties at the outset. Hauser succeeded in showing the value of micro-photography in the production of pictures of _impression_ preparations and colonies of bacteria in nutrient-gelatine. But to give the general effect, as well as faithfully reproduce the minute details in these preparations of bacteria by the aid of the pencil, would in most cases create insurmountable difficulties, except in the hand of the most accomplished draughtsman. Hauser employed Gerlach’s apparatus, and Schleusser’s dry-plates, and obtained his illumination by means of a small incandescent lamp, which gave a strong white light. The preparations so photographed were for the most part stained brown, and mounted in the ordinary way in Canada balsam.

In 1884, Van Ermengen succeeded in photographing preparations of comma-bacilli stained with fuchsine and methyl violet. These pictures afforded the first practical illustration of the value of iso-chromatic plates in micro-photography, and their introduction marks a distinct era in the progress of micro-photography. The iso-chromatic, or more properly the ortho-chromatic, dry-plate process was introduced because in photography blue or violet comes out almost or quite white, while other colours, yellow and red, are represented by a sombre shade or even by black. This is due to the want of equality of strength between the luminous and the actinic or chemical rays of light. In other words, the violet and blue rays are more chemically active than any other portion of the spectrum. It was found, then, that if plates were coloured yellow with turmeric, the blue and violet rays were intercepted, and their actinism proportionately reduced.

“In 1881, the so-called iso-chromatic plates were introduced. The emulsion of bromide of silver and gelatine was stained with eosin, and it was claimed that colours could be represented with their relative intensity; chlorophyll and other stains have also been tried, and by such methods the ordinary gelatine dry-plates can be so treated that they will reproduce various colours, according to their relative light intensity, and thus be rendered _iso_-, or what is now known as ortho-chromatic.”

Apparatus and Material.

_Apparatus and Material_ used in micro-photography have, from time to time, been greatly varied by different workers, some preferring to use the microscope in the vertical position with the camera superimposed or fitted on the eye-piece of the microscope tube; others, again, prefer that both the microscope and the camera should be arrayed horizontally. In another form the ordinary microscope is dispensed with and the objective stage and mirror are adapted to the front of the camera, together with a suitable arrangement for holding the object. Lastly, the camera is lain aside, and an operating-room rendered impervious to light, takes its place, and the image is projected and focussed upon a ground glass screen held in its place by a separate support. This method has been made practical since the introduction into microscopy by Zeiss of the _projection_ eye-piece. It is well known that micro-photographs can be produced by employing these projection eye-pieces, as well as for screen illustrations in the lecture-room.

With regard to the position of the microscope and camera, the horizontal affords greater stability than the vertical, and is on this account to be preferred. The simplest apparatus consists of a camera fixed upon a base board, four or five feet in length, upon which the microscope can be clamped, and which also carries the lamp and bull’s-eye lens (Fig. 172). This arrangement I have found economical and useful. No more elaborate arrangement is actually necessary. Sunlight is no doubt the best, but a good paraffin lamp is a handy and available illuminant.

With the former, and rapid plates, a short exposure of three or four seconds, even when high powers are used, is found sufficient; whereas, with the paraffin lamp it will vary from three to ten minutes.

Walmsley gives the following table for exposures with the lamp:--

1-1/2-inch objective 3 to 45 seconds.
2/3-inch " 7 to 90 "
4/10-inch " 1/2 to 3 minutes.
1/5-inch " 2 to 7 "
1/10-inch " 4 to 10 "

For micro-photography the following practical rules must be observed. The sub-stage condenser may be dispensed with when low powers are used, as well as the mirror, and the lamp so placed that the image of the flat of the flame appears accurately adjusted in the centre of the field of the microscope. The bull’s-eye lens is so interposed, that the image of the flame disappears, and the whole field becomes equally illuminated with high powers; the sub-stage achromatic condenser must be used, and a greater intensity of illumination is obtained by placing the lamp-flame edgeways. It is advisable to begin the practice of micro-photography with low powers, and a trial experiment should be made with some well-known object as the blow-fly’s tongue.

Dr. Crookshank is of opinion that, in the case of micro-organisms when their biological characters are studied under low powers of the microscope, photographs are preferable, because they give a more faithful representation of the object. A micro-organism, even under the highest powers of the microscope, is so minute an object, that to represent it in a drawing requires a very delicate touch, and it is only too easy to make a _picture_ which gives an erroneous impression to those who have not seen the original. Photography enables the scientific worker to record rapid changes, and it is quite possible as the art advances we may find the film more sensitive than the human retina, and that it will bring out details in bacteria which would be otherwise unrecognised. The result, therefore, of experience is that in research laboratories it will come into more general use as a faithful and graphic method. I cannot better bring these observations to a close than by giving a quotation from Dr. Piersoll’s practical method of obtaining micro-photographs.

The three essential conditions to ensure success in micro-photography are:--(1) Satisfactory apparatus; (2) good illumination; (3) suitable preparations. With high amplifications (1,000 diameters and over), the conditions are greatly changed by the approach to the limit both of the shortness of the focus of the objective and of the length of the camera which can be advantageously used; for the first experience leads to the adoption of the 1/12-inch, for the second four feet is the limit, since a given high amplification, say 2,000 diameters, can be more satisfactorily and more conveniently obtained with a superior 1/12-inch connection with suitable optical means to increase the initial magnifying power of the objective, than with an unaided 1/25-inch lens, and the plate removed to a greater distance. Until quite recently the various amplifiers offered the best means of increasing the power of an objective, but the introduction of the _projection-oculars_ of Zeiss is an accessory piece of apparatus, far superior to any older device. These projection-oculars resemble ordinary microscopical oculars or eye-pieces only in general form and name, being optically a projection-objective in connection with a collecting lens. The new oil-immersion apochromatic lenses, in combination with these projection-oculars, form undoubtedly the more efficient equipment for high-power work; it is as true for high-power photography as for microscopical observation in general, that the best results are obtained with fine and necessarily expensive, optical appliances. If for the satisfactory study of the intimate structure of a cell, or of a micro-organism, the most improved immersion lenses are necessary, it is to be expected that, for the successful photography of the same, tools at least as good are needed. Sunlight certainly affords the most satisfactory illumination whereby good micro-photographs can be obtained, as well as for recording microscopical images. That by good lamp-light fair impressions of objects under extreme magnification can be secured is encouraging, but the negatives produced by such illumination seldom, if ever, possess the characteristics of a really good sunlight negative, where the sharpest details are combined with an exquisite softness and harmony of half-tones.

If the mirror of the microscope be of good size, it will only be necessary to make an arm on which to support the removed mirror outside some southerly exposed window, since it is desirable to have a greater distance between the mirror and the stage than would be possible were the mirror attached in its usual place. Where the microscope mirror is too small to be satisfactorily used, a rectangular wood-framed looking-glass is readily mounted, with the aid of a few strips of wood, so as to turn about both axes.

The rays from the plane side of the mirror should pass through a condensing lens (of 8-10-inch focus, if possible), so placed that they are brought to a focus before reaching the plane of the object. The exact position of the condensing lens is a matter of experience; usually, however, the most favourable illumination is obtained at that point where the field is brilliantly and _uniformly_ illuminated, just before the rays form the image source of light; the nearer the focus the less disturbance from diffraction rings. Ordinary objectives will require the employment of monochromatic light--produced either by a deep blue solution of ammonia-sulphate of copper, or by the green glass screen--since the optical and actinic foci do not usually perfectly coincide. Powers up to the 3/4-inch will require no further condenser; with the 1/4 or 1/6-inch objectives, the low power (1 or 3/4-inch) serves with advantage as an achromatic condenser, when attached to the sub-stage. The Abbe condenser, although so important for fine microscopical investigation, is not adapted to photography unless a very wide cone of light is desired, which, for the majority of preparations, is some advantage; a low-power objective, used as a condenser, is found to be more satisfactory than the Abbe with a small diaphragm.[30]

The greatest delicacy in manipulation is necessary, as in working with a 1/12-inch objective a turn too much of the fine adjustment will cause the image to vanish. With fine preparations of bacteria it is not easy to trace the image, and hence the advantage of commencing with a well-marked object, as that of the fly’s tongue. The development and fixation of the image must be proceeded with as in the ordinary photographic process. In the text-books of photography full accounts of failures will be found, their causes and prevention. Numerous papers and suggestions for micro-photographic work will also be found scattered throughout the “Journal of the Royal Microscopical Society.”

The _Projection Eye-piece_ has become an essential part of micro-photography, and it is so arranged that it may be employed with advantage with objectives of either the apochromatic or ordinary series for photographic purposes, projecting an exquisitely sharp image of the object on the plate. A diaphragm between the lenses limits the field, and a sharp image of it should appear on the screen when the eye-piece is adjusted. The adjustment may be effected by revolving the eye-piece cap in a spiral slot, so that the eye or top lens is either brought closer or removed farther away from the diaphragm, as may be required, and divisions and a reader are usually provided for registering positions. Such eye-pieces are made to fit any size microscope body.

Initial magnifying powers:--
English length of tube 10-in. 3 and 6.
Continental " " 6-in. 2 and 4.

The microscope and camera (Fig. 173) are here seen to be part of the same instrument. The bellows of the camera have an extension varying from 6 in. to 30 in. The board on which the microscope and limelight jet are fixed is made to turn out of the line of the camera to facilitate adjusting the instrument and radiant, either limelight, electric light or paraffin lamp; when this is done the board carrying the same is turned back to a stop which brings the microscope into a central position with the focussing screen. An adjustment is supplied at the side of the camera, geared to the slow movement, for finely focussing the object upon the screen. A light-excluding connection is fitted to the front of the camera and microscope; immediately behind this, in the bellows, is an exposing shutter which is manipulated by means of a small milled head. Two focussing screens are usually supplied, one grey, and one patent plate, together with a double dark slide.

Mr. Andrew Pringle’s vertical micro-photographic apparatus is an excellent form; it consists of a heavy base and brass support, carrying a quarter-plate camera, grey and plain glass focussing screen, double dark back, camera extending to 24 inches, and turning aside as shown in Fig 173. It is light-tight in all its connections.

To secure uniform results in micro-photography, only thin preparations, which lie as nearly as possible in one plane, can be relied upon for good and perfect negatives.

An electric arc lamp specially designed for micro-photographic work, wherever the electric current is available, is that known as “the Ross-Hepworth projection arc lamp.” The advantage gained by this form of lamp is not only on account of the ease with which it may be employed, but also on account of its superior power and quality. It is of primary importance that the lamp employed to convert the electricity into light should be of a good and reliable pattern. It is not essential that it should be automatic in its working--many experienced micro-photographers preferring a simple hand-feed lamp to the one of a more complicated kind, being so much less difficult to keep in order. A good hand-feed microscope-lamp has the advantage of greater simplicity and portability.

The argand gas-light arranged for me many years ago for micro-photography may be employed with advantage. It is clean, and always ready for use when brought down to the table attached by a piece of india-rubber tubing. The incandescent form of burner enhances its value, since the light is thereby rendered whiter. The arrangement is shown in the diagrammatic drawing, Fig. 175.

Over the argand burner B, is a pale-blue glass chimney, resting on a wire gauze, stage A; this secures a uniform current of air. The colour of the flame may be still more influenced by a disc of neutral tint, or other coloured glass, inserted into the circular opening at E, in a half-cylinder of metal, G, used to cut off all extraneous light; can be rotated on the stage by the ivory nob at H, a metallic reflector I, attached to the standard rod, on being brought parallel to F serves to concentrate the light and send it on to the bull’s-eye, and through it to the mirror, or directly to the photo-microscopic camera.

By removing the shield G, and bringing the shade M over the burner, it is at once converted into a useful microscopical lamp, for all ordinary purposes. The screw R clamps the lamp-flame at any height, while the support N carries a water-bath O, or a plate P, both of which will be found useful in preparing and mounting objects.

A special incandescent gas-lamp is made by Messrs. R. & J. Beck.

Polarisation of Light.

Common light moves in two planes at right angles to each other, while polarised light moves in one plane only. Common light may be turned into polarised light either by transmission or reflection; in the first instance, one of the planes of common light is got rid of by reflection; in the other, by absorption. Huyghens was one of the first physicists to notice that a ray of light has not the same properties in every part of its circumference, and he compared it to a magnet or a collection of magnets; and supposed that the minute particles of which it was said to be composed had different poles, which, when acted on in certain ways, arranged themselves in particular positions; and thence the term _polarisation_, a term having neither reference to cause nor effect. It is to Malus, however, who, in 1808, discovered polarisation by reflection, that we are indebted for the series of splendid phenomena which have since that period been developed; phenomena of such surpassing beauty as to exceed most ordinary objects presented to the eye under the microscope.

Certainly no more misleading name could well have been found to describe the causation, in one particular direction, of small displacements in the medium, through which the light waves are made to pass.

The effect of “polarising” light is simply to alter the directions of the vibrations of light, and allow of certain waves to pass which are vibrating in one direction only, vertical, horizontal, or oblique, as the case may be. The most efficient agent discovered for the polarisation of light is that of Iceland spar, cut and mounted as a “Nicol” prism.

By cutting crystals of Iceland spar into two parts, at a particular angle, and cementing them together again in the reverse way, Nicol succeeded in showing that one of the two polarising pencils could be totally deflected to one side, while the other is directly transmitted through the Nicol prism, and thereby the beam of light becomes at once “polarised” in one plane only. No apparent difference can be seen in the prism on holding it up to the light, except it be in a very slight loss of brightness; but if another similarly heated crystal be held before, and made to revolve around, a quarter of the circle just where the two cross each other, total darkness results. This phenomenon alternately recurs at every quadrature of the circle. A pair of Nicol prisms, when appropriately mounted, constitute “a polarising apparatus” for the microscope, one being fitted into the sub-stage, and the other either immediately above the objective or eye-piece, where it can be easily rotated, the object to be examined being placed on the stage of the microscope, that is, between the polarising and analysing prisms.

Tuffen West, del. Edmund Evans.

PLATE VIII.]

The significance of polarised light centres in the fact that it affords a wider insight into the structure of crystals, minerals, and a number of other substances, and which could not otherwise be obtained without its aid. Its usefulness is multifold, as even glass itself, when not properly annealed, exhibits points of fracture, by a display of Newton’s rings. The knowledge thus acquired is turned to account by glass manufacturers.

_Double refraction._--When an incident ray of light is refracted into a crystal of any other than the cubic system, or into compressed or _unannealed glass_, it gives rise to two refracted rays which take different paths; this phenomenon is termed _double refraction_. Attention was called to this in 1670, by Bartolin, who first observed it in Iceland spar; and the laws for this substance were accurately determined by Huyghens.

Iceland spar or calc spar is a form of crystallized carbonate of lime. It is composed of fifty-six parts of lime and forty-four parts of carbonic acid, and is usually found in rhombohedral forms of crystallization.

To observe the phenomenon of double refraction, a rhomb of Iceland spar may be laid on a page of a printed book, when all the letters seen through it will appear double; the depth of the blackness of the letters is seen to be considerably less than that of the originals, except where the two images overlap.

In order to state the laws of the phenomena with precision, it is necessary to attend to the crystalline form of Iceland spar, which has equal obtuse angles. If a line be drawn through one of these corners, making equal angles with the three edges which meet there, it, or any line parallel to it, is called the _axis_ of the crystal; the axis being, properly speaking, not a definite _line_ but a definite _direction_.

The angles of the crystals are the same in all specimens. If the crystal is of such proportions that these three edges spoken of are equal, as in the smaller crystal (Fig. 176), the axis is the direction of one of its diagonals, as represented.

Any plane containing (or parallel to) the axis is called the _principal plane_ of the crystal.

In the next diagram, Fig. 177, the line appears double, as _a b_ and _c d_, or the dot, as _e_ and _f_. Or allow a ray of light, _g h_, to fall thus on the crystal, it will in its passage through be separated into two rays, _h f_, _h e_; and on coming to the opposite surface of the crystal, will pass out at _e f_ in the direction of _i k_, parallel to _g h_. The plane _l m n o_ is designated the principal section of the crystal, and the line drawn from the solid angle _l_ to the angle _o_ is where the axis of the crystal will be found; this is its optic axis. Now when a ray of light passes along this axis, it is undivided, and there is only one image; but in all other directions there are two images.

Mr. Nicol, of Edinburgh first succeeded in making a rhomb of Iceland spar into a _single-image prism_. His method of splitting up the crystal into two equal parts was as follows:--

A rhomb of Iceland spar of one-fourth of an inch in length, and about four-eighths of an inch in breadth and thickness, is divided into two equal portions in a plane, passing through the acute lateral angle, and nearly touching the obtuse side angle. The sectional plane of each of these halves must be carefully polished, and the two portions cemented firmly together with Canada balsam, so as to form a rhomb similar to that before division; by this management the ordinary and extraordinary rays are so separated that only one is transmitted: the cause of this great divergence of the rays is considered to be owing to the action of the Canada balsam, the refractive index of which (1·549) is that between the ordinary (1·6543) and the extraordinary (1·4833) refraction of calcareous spar, and which will change the direction of both rays in an opposite manner before they enter the posterior half of the combination. The direction of rays passing through such a prism is indicated by the arrow, Fig. 178.

Polarised light cannot be distinguished from common light, as already said, by the naked eye; and for all experimental purposes in polarisation, two pieces of apparatus must be employed, one to produce polarisation, and the other to show or an analyse it. The former is called the _polariser_, the latter the _analyser_; and every apparatus that serves for one of these purposes will also serve for the other.

Polarising Apparatus for Students’ Microscope.

In all cases there are two positions, differing by 180°, which give a minimum of light, and the two positions intermediate between these give a maximum of light. The extent of the changes thus observed is a measure of the completeness of the polarisation of light.

The two prisms mounted as shown in Figs. 179 and 179_a_ constitute the apparatus adapted to the microscope. The polariser slips into place below the stage, and the analyser, with the prism fixed in a tube, is screwed in above the objective.

The definition is considered by some experimenters as somewhat better if the analyser be used above the eye-piece, and is certainly more easily rotated.

_Method of employing the Polarising Prism_ (Fig. 179).--After having adapted it to slide into a groove on the under-surface of the stage, where it is secured and kept in place by the small milled-head screw, the other prism (Fig. 179_a_) is screwed on above the object-glass, and thus passes directly into the body of the microscope. The light from the mirror having been reflected through them the axes of the two prisms must be made to coincide; this is done by regulating the milled-head screw until, by revolving the _polarising_ prism, the field of view is entirely darkened twice during its revolution. If very minute salts or crystals are submitted for examination then it will be found preferable to place the analyser above the eye-piece, as in Fig. 180. Thus the _polariscope_ is seen to consist of two parts; one for _polarising_, the other for _analysing_ or testing the light. There is no essential difference between the two parts, except what convenience or economy may lead us to adopt; and either part, therefore, may be used as polariser or analyser; but whichever is used as the polariser, the other becomes the analyser.

Opticians have their own methods of adapting the polariser and analyser to their several microscopes. Watson’s special form of apparatus is represented in Fig. 181, the polariser being adapted to the sub-stage, and the analyser to screw into the objective.

_Tourmaline._--A semi-transparent mineral, of a neutral or bluish tint, called tourmaline, when cut into thin slices (about 1/20-inch thick) with their faces parallel to their axes exhibit the same phenomena as the Nicol prism. The only objection to which is that the transmitted polarised beam is more or less coloured. The tourmaline to be preferred stops the most light when its axis is at right-angles to that of the polariser, and yet admits the most when in the same plane. Make choice of a tourmaline as perfect as possible; size is of less importance when intended for use with the microscope.

Transmission of rays through tourmaline is only one of several ways in which light can be polarised. When a beam of light is reflected from a polished surface of glass, wood, ivory, leather, or any other non-metallic substance, at an angle of 50° to 60° with the normal, it is more or less polarised, and in like manner a reflector composed of any of these substances may be employed as an analyser. In so using it, it should be rotated about an axis parallel to the incident rays which are to be tested, and the observation consists in noting whether this rotation produces changes in the amount of reflected light.

For every reflected substance there is a particular angle of incidence, which gives a maximum of polarisation in reflected light. It is called the _polarising angle_ for the substance, and its tangent is always equal to the index of refraction of the substance; or, what amounts to the same thing, it is that particular angle of incidence which is the complement of the angle of refraction, so that the refracted rays are at right angles. This important law was discovered experimentally by Sir David Brewster.

Tourmaline, like Iceland spar, is a negative uniaxial crystal; and its use as a polariser depends on the property which it possesses of absorbing the ordinary much more rapidly than the extraordinary ray, so that a thickness which is tolerably transparent to the latter is almost completely opaque to the former. Its pale cobalt blue colour enhances the beauty of certain crystal and mineral substances, but like Iceland spar, the paler and more perfect crystals are becoming scarce.

_Selenite_ is another mineral of value in polarisation experiments. It is a native crystalline hydrated sulphate of lime. A beautiful fibrous variety called _satin-gypsum_ is found in Derbyshire. The form of the crystal most frequently met with is that of an oblique rectangular prism, with ten rhomboidal faces, two of which are much larger than the rest. It is usually split up into thin laminæ parallel to their lateral faces; each film should have a thickness of from one-twentieth to one-sixtieth of an inch. In the two rectangular directions these films allow perpendicular rays of polarised light to traverse them unchanged, termed their _neutral axes_. In two other directions, however, which form respectively angles of 45° with the neutral axes, these films have the property of double refraction, a direction known as the _depolarising axis_.

The thickness of the film of selenite determines the particular tint. If, therefore, we use a film of irregular thickness, different colours are presented by the different thicknesses. These facts admit of very curious and beautiful illustration, when used under the object placed on the stage of the microscope. The films employed should be mounted between two glasses for protection. Some persons employ a large film, mounted in this way between the plates of glass, with a raised edge, to act as a stage for supporting the object, it is then called the “selenite stage.” The best film for the microscope is that which gives blue, and its complementary colour yellow. The late Mr. Darker constructed a selenite stage for the purpose (Fig. 182). With this a mixture of colours will be brought about, by superimposing three films, one on the other. By slight variations in their positions, produced by means of an endless-screw motion, all the colours of the spectrum can be shown. When objects are thus exhibited, it should be borne in mind that all negative tints, as they are termed, are diminished, and all positive tints increased; the effect of which is to mask the true character of the phenomena.

For a certain thickness of selenite the ellipse will become a circle, and we have thus what is called _circularly-polarised_ light, which is characterised by the property that rotation of the analyser produces no change of intensity. Circularly-polarised light is not, however, identical with ordinary light; for the interposition of an additional thickness of selenite converts it into elliptically (or in a particular case into plane) polarised light.

It is necessary, for the exhibition of colour in our experiments, that the plate of selenite should be very thin, otherwise the retardation of one component vibration as compared with the other will be greater by several complete periods for violet than for red, so that the ellipses will be identical for several different colours, and the total non-suppressed light will be sensibly white in all positions of the analyser.

Two thick plates may, however, be so combined as to produce the effect of one thin plate. For example, two selenite plates of nearly equal thickness may be laid one upon the other, so that the direction of greatest elasticity in the one shall be parallel to that of least elasticity in the other. The resultant effect in this case will be that due to the difference of their thicknesses. Two plates so laid are said to be _crossed_.

The following experiments will well serve to illustrate some of the more striking phenomena of double refraction, and will also be a useful introduction to its practical application. Take a plate of brass (Fig. 183) three inches by one, perforated with a series of holes from about one-sixteenth to one-fourth of an inch in diameter; the size of the smallest should be in accordance with the power of the objective, and the separating power of the double refraction.

_Experiment_ 1.--Place the brass plate so that the smallest hole shall be in the centre of the stage of the microscope; employ a low power (1-1/2 or 2 inches) objective, and adjust the focus as for the ordinary microscopic object; place the double image prism over the eye-piece, and two distinct images will be seen; by revolving the prism, the images will describe a circle, the circumference of which will cut the centre of the field of view; one of which is the ordinary, the other the extraordinary ray. By moving the slide from left to right the larger orifices will appear in the field, the images seen will not be completely separated, but will overlap, as represented in the figure.

_Experiment_ 2.--Insert the Nicol’s prism into its place under the stage, still retaining the double image prism over the eye-piece; then, by examining the object, there will appear in some positions two images, in others only one image; it will be seen, that at 90° this ray will be cut off, and that which was first observed will become visible; at 180°, or one-half the circle, an alternate change will take place; at 270°, another change; and at 360°, the completion of the circle, the first image will reappear.

Before proceeding to make the next experiment, the position of the Nicol’s prism should be adjusted, and its angles brought parallel with the square of the stage. The true relative position of the selenite should also be determined by noticing the natural flaws in the film, which should run parallel with each other, and be adjusted at an angle of about 46° with the square bars of the stage.

_Experiment_ 3.--If we now take the plate of selenite thus prepared, and place it under the piece of brass on the stage, we shall see, instead of the alternate black and white images, two coloured images composed of the constituents of white light, which will alternately change by revolving the eye-piece at every quarter of the circle; then, by passing along the brass, the images will overlap; and at the point at which they do so, white light will be produced. If, by accident, the prism be placed at an angle of 45° from the square part of the stage, no particular colour will be perceived, and it will then illustrate the phenomena of the neutral axis of the selenite, because when placed in the relative position no depolarisation takes place. The phenomena of polarised light may be further illustrated by the addition of a second double image prism, and a film of selenite adapted between the two. The systems of coloured rings in crystals cut perpendicularly to the principal axis of the crystal are best seen by employing the lowest object-glass.

_Biaxial Crystals._--To show perfectly the beautiful series of _rings and brushes_ which biaxial crystals exhibit, it becomes necessary to convert the microscope, for the time being, into (so to speak) a wide-angled telescope.

Inner draw-tube.

Objective in draw-tube.

Analysing Prism.

Objective.

Specimen under Examination.

Sub-stage Condenser.

Polarising Prism, fixed in sub-stage below.

Fig. 184.--Diagrammatic arrangement of the Polarising Microscope.