CHAPTER III.

Applied Optics:--Eye-pieces; Achromatic Objectives; Condensers.

It is almost unnecessary to say that the eye-piece forms a most important part of applied optics in the microscope. It is an optical combination designed to bring the pencil of rays from the objective to assist in the formation of a real or virtual image before it arrives at the eye of the observer. Greater attention has been given of late years to the improvement of the eye-piece, since flatness of field much depends upon it. Opticians have therefore sought to make it both achromatic and compensatory.

There are several forms of eye-pieces in use, some of which partake of a special character, and these will receive attention in their proper places. It is, however, customary among English opticians to denote the value of their several eye-pieces by Roman capitals, A, B, C, D, and E. Continental opticians, on the other hand, have a preference for numerals, 1, 2, 3, 4, 5 and 6, or more, and by which they are recognised.

The eye-piece in more general use is that known as the _Huyghenian_ (Fig. 99); this came into use upwards of two centuries ago. It was constructed by Christian Huyghens, a Dutch philosopher and eminent man of science, secretary to William III.

It was made for the eye-piece of a telescope he constructed with his own hands, and it has been in constant use as the eye-piece of the microscope for nearly two centuries. It consists of two plano-convex lenses, with their plane surfaces turned towards the eye, and divided at a distance equal to half the sum of their focal lengths--in other words, at half the sum of the focal length of the eye-glass and of the distance from the field-glass at which an image from the object glass would be formed, a stop, or diaphragm, being placed between the two lenses for the reason about to be explained. Huyghens himself appears to have been quite unaware of the value of an eye-piece so cleverly constructed.

It was reserved for Boscovich to point out that, by this important arrangement, he had corrected a portion of the chromatic aberration incidental to the earlier form of eye-pieces. Let Fig. 100 represent the Huyghenian eye-piece of a microscope, _f f_ being the field-glass, and _e e_ the eye-glass, and _l m n_ the two extreme rays of each of the three pencils emanating from the centre and ends of the object, of which, but for the field-glass, a series of coloured images would be formed from _r r_ to _b b_; those near _r r_ being red, those near _b b_ blue, and the intermediate ones green, yellow, and so on, corresponding with the colours of the prismatic Spectrum.

The effect described, that of projecting the blue image beyond the red, over-correcting the object-glass as to colour, is purposely produced; it is also seen that the images _b b_ and _r r_ are curved in the wrong direction to be seen distinctly by the convex eye-lens; this then is a further defect of the compound microscope made up of two lenses. But the field-glass, at the same time that it bends the rays and converges them to foci at _b′ b′_ and _r′ r′_, also reverses the curvature of the images as here shown, giving them the form best adapted for distinct vision by the eye-glass _e e_. The field-glass has at the same time brought the blue and red images closer together, so that they produce an almost colourless image to the eye. The chromatic aberration of lenses has been clearly explained in a previous chapter. But let it be supposed that the object-glass had not been over-corrected, that it had been perfectly achromatic; the rays would then have appeared coloured as soon as they had passed the field-glass; the blue rays of the central pencil, for example, would converge at _b′′_, and the red rays at _r′′_, which is just the reverse of what is required of the eye-lens; for as its blue focus is also shorter than its red, it would require that the blue image should be at _r′′_, and the red at _b′′_. This effect is due to over-correction of the object-glass, which removes the blue foci _b b_ as much beyond the red foci _r r_ as the sum of the distances between the red and the blue foci of the field-lens and eye-lens; so that the separation _b r_ is exactly taken up in passing through those two lenses, and the several colours coincide, so far as focal distance is concerned, as the rays pass the eye-lens. So that while they coincide as to distance, they differ in another respect--the blue image is rendered smaller than the red by the greater refractive power of the field-glass upon the former. In tracing the pencil _l_, for instance, it will be noticed that, after passing the field-glass, two sets of lines are drawn, one whole and one dotted, the former representing the red, and the latter the blue rays. This accidental effect in the Huyghenian eye-piece was pointed out by Boscovich. The separation into colours of the field-glass is like the over-correction of the object-glass--and opens the way to its complete correction. If the differently-coloured rays were kept together till they reached the eye-glass, they would still be coloured, and present coloured images to the eye. The separating effected by the field-glass causes the blue rays to fall so much nearer the centre of the eye-glass, where, owing to its spherical figure, the refractive power is less than at the margin, so that spherical error of the eye-lens may be said to constitute a nearly equal balance to the chromatic dispersion of the field-lens, and the blue and red rays _l′_ and _l′′_ emerge nearly parallel, presenting a fairly good definition of a single point to the eye. The same may be said of the intermediate colours of the other pencils. The eye-glass thus constructed not only brings together the images _b′ b′_, _r′ r′_, but it likewise has the most important effect of rendering them flatter, and assisting in the correction of chromatic and spherical aberration.

The later form of the Huyghenian eye-piece is that of the late Sir George Airy, the field-glass of which is a meniscus with the convex side turned towards the objective, and the eye-lens a crossed convex with its flatter side towards the eye. Another negative eye-piece is that known as the _Kellner_, or orthoscopic eye-piece. It consists of a bi-convex field-glass and an achromatic doublet eye-lens. This magnifies ten times, but it in no way compares with the Huyghenian in value. Neither does it afford the same flatness of field.

The _Ramsden_, or positive eye-piece, is chiefly employed as a micrometer eye-piece for the measurement of the values of magnified images. The construction of this eye-piece is shown in Fig. 101, a divided scale being cut on a strip of glass in 1/100ths of an inch, every fifth of which is cut longer than the rest to facilitate the reading of the markings, and at the same time that of the image of the object, both being distinctly seen together, as in the accompanying reduced micro-photograph of blood corpuscles, Fig. 102.

The value of such measurements in reference to the real object, when once obtained; is constant for the same objective. It becomes apparent, then, that the value of the divisions seen in the eye-piece micrometer must be found with all the objectives used, and carefully tabulated.

It was Mr. Lister who first proposed to place on the stage of the microscope a divided scale of a certain value. Viewing the scale as a microscopic object, he observed how many of the divisions on the scale attached to the eye-piece corresponded with one or more of a magnified image. If, for instance, ten of those in the eye-piece correspond with one of those in the image, and if the divisions are known to be equal, then the image is ten times larger than the object, and the dimensions of the object ten times less than that indicated by the micrometer. If the divisions on the micrometer and on the magnified scale are not equal, it becomes a mere rule-of-three sum; but in general this trouble is taken by the maker of the instrument, who furnishes a table showing the value of each division of the micrometer for every object-glass with which it will be employed.

Mr. Jackson’s simple and cheap micrometer is represented in Fig. 103. It consists of a slip of glass placed in the focus of the eye-glass, with the divisions sufficiently fine to have the value of the ten-thousandth part of an inch with the quarter-inch object-glass, and the twenty-thousandth with the eighth; at the same time the half, or even the quarter of a division may be estimated, thus affording the means of attaining considerable accuracy, and may be used to supersede the more complicated and expensive screw-micrometer, being handier to use, and not liable to derangement in inexperienced hands.

The positive eye-piece affords the best view of the micrometer, the negative of the object. The former is quite free from distortion, even to the edges of the field; but the object is slightly coloured. The latter is free from colour, and is slightly distorted at the edges. In the centre of the field, however, to the extent of half its diameter, there is no perceptible distortion, and the clearness of the definition gives a precision to the measurement which is very satisfactory.

Short bold lines are ruled on a piece of glass, _a_, Fig. 103, to facilitate counting, the fifth is drawn longer, and the tenth still longer, as in the common rule. Very fine levigated plumbago is rubbed into the lines to render them visible; they are then covered with a piece of thin glass, cemented by Canada balsam, to prevent the plumbago from being wiped out. The slip of glass thus prepared is secured in a thin brass frame, so that it may slide freely into its place.

Slips are cut in the negative eye-piece on each side, so that the brass frame may be pressed across the field in the focus of the eye-glass, as at _m_; the cell of which should have a longer screw than usual, to admit of adjustment for different eyes. The brass frame is retained in its place by a spring within the tube of the eye-piece; and in using it the object is brought to the centre of the field by the stage movements; the coincidence between one side of it and one of the long lines is made with great accuracy by means of the small screw acting upon the slip of glass. The divisions are then read off as easily as the inches and tenths on a common rule. The operation, indeed, is nothing more than the laying of a rule across the body to be measured; and it matters not whether the object be transparent or opaque, mounted or unmounted, if its edges can be distinctly seen, its diameter can be taken.

Previously, however, to using the micrometer, the value of its divisions should be ascertained with each object-glass; the method of doing this is as follows:--

Place a slip of ruled glass on the stage; and having turned the eye-piece so that the lines on the two glasses are parallel, read off the number of divisions in the eye-piece which cover one on the stage. Repeat this process with different portions of the stage-micrometer, and if there be a difference, take the mean. Suppose the hundredth of an inch on the stage requires eighteen divisions in the eye-piece to cover it; it is plain that an inch would require eighteen hundred, and an object which occupied nine of these divisions would measure the two-hundredth of an inch. Take the instance supposed, and let the microscope be furnished with a draw-tube, marked on the side with inches and tenths. By drawing this out a short distance, the image of the stage micrometer will be expanded until one division is covered by twenty in the eye-piece. These will then have the value of two-thousandths of an inch, and the object which before measured nine will then measure ten; which, divided by 2,000, gives the decimal fraction ·005.

Enter in a table the length to which the tube is drawn out, and the number of divisions on the eye-piece micrometer equivalent to an inch on the stage; and any measurements afterwards taken with the same micrometer and object-glass may, by a short process of mental arithmetic, be reduced to the decimal parts of an inch, if not actually observed in them.

In ascertaining the value of the micrometer with a deep objective, if the hundredth of an inch on the stage occupies too much of the field, then the two-hundredth or five-hundredth should be used and the number of the divisions corresponding to that quantity be multiplied by two hundred or five hundred, as the case may be.

The micrometer should not be fitted into too deep an eye-piece, as it is essential to preserve good definition. A middle-power Kellner or Huyghenian is frequently employed; at all events, use the eye-piece of lower power rather than impair the image.

The eye-lens above the micrometer should not be of shorter focus than three-quarters of an inch, even with high-power objectives.

_The Ramsden Eye-piece._--The cobweb micrometer is the most efficient piece of apparatus yet brought into use for measuring the magnified image. It is made by stretching across the field of the eye-piece two extremely fine parallel wires or cobwebs, one or both of which can be separated by the action of a micrometer screw, the trap head of which is divided into a hundred or more equal parts, which successively pass by an index as the milled head is turned, shown in Fig. 104. A portion of the field of view is cut off at right angles to the filaments by a scale formed of a thin plate of brass having notches at its edges, the distances between which correspond to the threads of the screw, every fifth notch (as in the previous case) being made deeper than the rest, to make the work of enumeration easier. The number of entire divisions on the scale shows then how many complete turns of the screw have been made in the separation of the wires, while the number of index points on the milled head shows the value to the fraction of a turn, that may have been made in addition. A screw with one hundred threads to the inch is that usually employed; this gives to each division in the scale in the eye-piece the value of 1/100th of an inch. The edge of the milled head is also divided into the same number of parts.

In Watson’s Ramsden screw micrometer, Fig. 104, the micrometer scale (seen detached) is ruled on a circular piece of glass, and this, by unscrewing the top, is dropped into its place, and one of the wires, both being fixed, is set a little to the side of the field, the teeth of the screw being cut to 1/100ths, and the drum giving the fractional space between the teeth to 1/100ths, so that the 1/10000th of an inch can be read off. This micrometer eye-piece is constructed entirely of aluminium, a decided advantage, being so much lighter than brass to handle.

In the screw micrometer of other makers, other modifications are found. An iris diaphragm being placed below the web to suit the power of the eye-piece employed, a guiding line at right angles to the web is sometimes added. Care should be taken to see that when the movable web coincides exactly with the fixed web, the indicator on the graduated head stands at zero.

_The Compensating Eye-piece._--The very important improvements effected in the construction of the objective naturally led up to an equally useful change for the better in the eye-piece.

All objectives of wide aperture, from the curvature of their hemispherical front lenses, show a certain amount of colour defect in the extra-axial portion of the field, even if perfectly achromatic in the centre. Whether an image be directly projected by the objective, or whether it be examined with an aplanatic eye-piece, colour fringes may be detected, possibly in an increasing degree towards the periphery. This residual chromatic aberration has at length been very nearly eliminated by the aid of the compensating eye-piece.

The construction of compensating eye-pieces is somewhat remarkable, since they have an equivalent error in an opposite direction--that is, the image formed by the red rays is greater than that corresponding to the blue rays; consequently, eye-pieces so constructed serve to compensate for the unequal magnification produced by different coloured rays, and images appear free from colour up to the margin of the field.

Zeiss’s compensating eye-pieces are so arranged that the lower focal points of each series lie in the same plane when inserted in the body-tube of the microscope; no alteration of focus is therefore required on changing one eye-piece for another. This of itself is not only an advantage but also a saving of time, while the distance between the upper focal point of the objective and the lower one of the eye-piece, which is the determining element of magnification, remains constant.

The ordinary working eye-pieces, Huyghenian and others, commencing with a magnification of four diameters, are so constructed that they can be conveniently used, as we are accustomed to use them in England, with high powers, Zeiss’s Nos. 12 and 18 compensating eye-pieces being adapted for use with his lower power apochromatic lenses of 16 and 8 mm. The numbering of the eye-pieces is carried out on the plan originally proposed by Professor Abbe--that is, the number denotes how many times an eye-piece, when employed with a given tube-length, increases the initial magnifying power of the objective, and at the same time furnishes figures for their rational enumeration. It is on this basis that the German compensating eye-pieces have been arranged in series, and in agreement with their magnifying power and distinctive numberings of 2, 4, 6, 8, 12, 18. Of these several eye-pieces, 12 is found to be the most useful. The magnification obtained by combining a compensating eye-piece with any apochromatic objective is found by multiplying its number by the initial magnification of the objective, as given in the following proof:--An objective of 3·0 mm. focus, for example, gives in itself a magnification of 83·3 (calculated, for the conventional distance of vision, 250 mm.); eye-piece 12 therefore gives with this objective a magnification of 12 × 83·3 = 1000 diameters. The classification, however, of these eye-pieces, as furnished by Abbe, is dependent upon increase in the total magnifying power of the microscope obtained by means of the eye-piece as compared with that given by the objective alone. The numbering, then, denotes how many times an eye-piece increases the magnifying power of the objective when used with a given body-tube; the proper measure of the eye-piece magnification; and, at the same time, the figures for rational enumeration.

Compensating eye-pieces have been introduced for the correction of certain errors in high-power objectives--those made with hemispherical fronts. All such lenses, whether apochromatic or not, are greatly improved by the compensating eye-piece, but the dry objective and the lower powers are certainly deteriorated. The lower power compensating eye-pieces are Huyghenian, the higher are combinations, with no field-lens, and therefore in working act as a single or positive eye-piece. This is of importance to those who work with low powers--the older forms of objectives.

Messrs. Watson and Swift have adopted a new formula for their series of _achromatic eye-pieces_, whereby their magnification and flatness of field are improved. These also bear a constant ratio to the initial power of their objectives.

The compensating eye-pieces of these makers are constructed on the same principle as those of Zeiss’s for the correction of errors of colour in the marginal portion of the field, and consequently are in every way as effective as those of Continental manufacture. Figs. 106, 107, and 108 show in dotted outline the form and position of the several lenses combined in these eye-pieces.

_Projection Eye-pieces_ are chiefly used in micro-photography, and for screen demonstrations. The cap of this eye-piece is provided with a spiral adjustment for focussing, the diaphragm being placed in front of the eye-lens, an essential arrangement for obtaining an accurate focus. The ring seen below the cap, Fig. 108, is graduated so that the rotation for distance of screen may be carefully recorded.

Schmidt’s goniometer positive eye-piece, for measuring the angles of crystals, is so arranged as to be easily rotated within a large and accurately graduated circle. In the focus of the eye-piece a single cobweb is drawn across, and to the upper part is attached a vernier. The crystals being placed in the field of the microscope, care being taken that they lie _perfectly flat_, the vernier is brought to zero, and then the whole apparatus turned until the line is parallel with one face of the crystal; the frame-work bearing the cobweb, with the vernier, is now rotated until the cobweb becomes parallel with the next face of the crystal, and the number of degrees which it has traversed may then be accurately read off.

_Goniometer._--If a higher degree of precision is required, then, the double-refracting goniometer invented by the late Dr. Leeson must be substituted. With this goniometer (Fig. 109) the angles of crystals, whether microscopic or otherwise, can be measured. It has removed the earlier difficulties incident to similar instruments formerly in use. Among other advantages, it is capable of measuring opaque and even imperfect crystals, beside microscopic crystals and those in the interior of other transparent media. It is equally applicable to the largest crystals, and will measure angles without removing the crystal from a specimen, provided only the whole is placed on a suitable adjusting stage. The value of the goniometer depends on the application of a doubly refracting prism, either of Iceland spar or of quartz, cut of such a thickness as will partially separate the two images of the angle it is proposed to measure.

Dr. Leeson strongly insisted on the importance of the microscope in the examination of the planes of crystals subjected to measurement, as obliquity in many cases arises from not only conchoidal fractures, but also from imperfect laminæ elevating one portion of a plane, and yet allowing a very tolerable reflection when measured by the double refracting goniometer.

Microscopes for crystallographic and petrological research are now specially constructed for measuring the angles of crystals.

Erector eye-pieces and erecting prisms are employed for the purpose of causing the image presented to the eye to correspond with that of the object. They are also helpful in making minute dissections of structure; the loss of light, however, by sending it through two additional surfaces is a drawback, and impairs the sharpness of the image. Nachet designed an extremely ingenious arrangement whereby the inverted image became erect; he adapted a simple rectangular prism to the eye-piece. The obliquity which a prism gives to the visual rays when the microscope is used in the erect position, as for dissecting, is an advantage, as it brings the image to the eye at an angle very nearly corresponding to that of the inclined position in which the microscope is ordinarily used.

The Achromatic Objective.

_The Achromatic Objective_, of all the optical and mechanical adjuncts to the microscope, is in every way the most necessary, as well as the most important. The ideal of perfection aimed at by the optician is a combination of lenses that shall produce a perfect image--that is, one absolutely perfect in definition and almost free from colour. The method resorted to for the elimination of spherical and chromatic aberration in the lens has been fully explained in a former chapter. It will now be my endeavour to show the progressive stages of achromatism and evolution of the microscope throughout the present century.

It is almost as difficult to assign the date of the earliest application of achromatism to the microscope as to that of the inception and many modifications of the instrument in past ages; indeed, the question of priority in every step taken in its improvement has been the subject of controversy.

Among the earlier workers in the first decade of this century will be found the name of Bernardo Marzoni, who was curator of the Physical Laboratory of the Lyceum of Brescia. He, an amateur optician, it has come to light, in 1808 constructed an achromatic objective, and exhibited it at Milan in 1811, when he obtained the award of a silver medal for its merits, under the authority of the “Institute Reale delli Scienzo.” Through the good offices of the late Mr. John Mayall one of Marzoni’s objectives, which had been carefully preserved, was presented to the Royal Microscopical Society of London in 1890.[20] This objective is a cemented combination, with the plane side of the flint-lens presented to the object. This was an improvement of a practical kind, and of which Chevalier subsequently availed himself. In 1823 Selligue, a French optician, is credited with having first suggested the plan of combining two, three, or four plano-convex achromatic doublets of similar foci, one above the other, to increase the power and the aperture of the microscope. Fresnel, who reported upon this invention, preferred on the whole Adam’s arrangement, because it gave a larger field. Selligue subsequently improved his objective by placing a small diaphragm between the mirror and the object.

In this country, Tully was induced by Dr. Goring to work at the achromatic objective, and his first efforts were attended with a success quite equal to that of Chevalier’s. Lister on examining these lenses said:--“The French optician knows nothing of the value of aperture, but he has shown us that fine performance is not confined to triple objectives.” Amici, the amateur optician of Modena, visited this country in 1827 and brought his achromatic microscope and objectives, which were seen to give increase of aperture by combining doublets with triplets. The most lasting improvement in the achromatic objective was that of Joseph Jackson Lister, F.R.S., the father of Lord Lister, and one of the founders of the Royal Microscopical Society of London.

Lister’s discoveries at this period (1829) in the history of the optics of the microscope were of greater importance than they have been represented to be. That he was an enthusiast is manifest, for, being unable to find an optician to carry out his formula for grinding lenses, he at once set to work to grind his own, and in a short time was able to make a lens which was said to be the best of the day.

Lister, in a paper contributed to the proceedings of the Royal Society the same year, pointed out how the aberrations of one doublet could be neutralised by a second. He further demonstrated that the flint lens should be a plano-concave joined by a permanent cement to the convex crown-glass. The first condition, he states, “obviates the risk of error in centring the two curves, and the second diminishes by one half the loss of light from reflection, which is very great at the numerous surfaces of every combination.” These two conditions then--that the flint lens shall be plano-concave, and that it shall be joined by some cement (Canada balsam) to the convex--may be taken as the basis for the microscopic objective, provided they can be reconciled with the correction of spherical and chromatic aberration of a large pencil.

Andrew Ross was not slow to perceive the value of Lister’s suggestions and in 1831 he had constructed an object-glass on the lines laid down by Lister, Fig. 112; _a a′_ representing the anterior pair, _m_ the middle, and _p_ the posterior, the three sets combined forming the achromatic objective, consisting of three pairs of lenses, a double-convex crown-glass, and a plano-concave of flint.

Lister proposed other combinations, and himself made an object-glass consisting of a meniscus pair with a triple middle, and a back plano-convex doublet. This had a working distance of ·11 and proved to be so great a success that other opticians--Hugh Powell, 1834; James Smith, 1839--made objectives after the same formula.

The publication of Lister’s data proved of value in another direction: it stimulated opticians to apply themselves to the further improvement of the achromatic objective. Andrew Ross was one of the more earnest workers in giving effect to Lister’s principles and a short time afterwards found that a triple combination, with the lenses separated by short intervals, gave better results. In the accompanying diagram the changes made in the combination of the objective from 1831, and extending over a period of about twenty years from this date, are shown.

Each objective, from the 1/2-inch to the 1/12-inch, is seen to be built up of at least six or eight different fronts, the back combinations being a triplet formed of two double-convex lenses of crown glass with an intermediary double concave lens of flint-glass.

No sooner had Ross constructed 1/4-inch achromatic objectives on Lister’s formula than he discovered an error which had hitherto escaped attention, viz., that the thinnest cover-glass of an object produced a considerable amount of refractive disturbance. A marked difference was observed in the image when viewed with or without a cover-glass. This difficulty was first met by the addition of a draw-tube to the microscope body. But as this also impaired the image, Lister overcame the difficulty by mounting the front lens of the objective in a separate tube made to fit over a second tube carrying the two pairs of lenses. This arrangement led up to his invention of the _screw-collar adjustment_, the mechanism for applying which is shown in Fig. 114. The anterior lens _a_ at the end of the tube is enclosed in a brass-piece _b_ containing the combination; the tube _a_, holding the lens nearest the object, is then made to move up or down the cylinder _b_, thus varying the distance, according to the thickness of the glass covering the object, by turning the screw ring _c_, thus causing the one tube to slide over the other, and clamping them together when properly adjusted. An aperture is made in the tube _a_, within which is seen a mark engraved on the cylinder, on the edge of which are two marks, a longer and a shorter, engraved upon the tube. When the mark on the cylinder coincides with the longer mark on the tube, the adjustment is made for an uncovered object; and when the coincidence is with the shorter mark, the proper distance is obtained to balance the aberrations produced by a cover-glass the hundredth of an inch thick; such glass covers are now supplied. The adjustment should be tested experimentally by moving the milled edge which separates or closes the combinations, and at the same time using the fine adjusting screw of the microscope. The difficulty associated with the cover-glass of old has, by the introduction of the homogeneous immersion system, been very nearly eliminated. There still remains, however, a disturbing amount of residual colour aberration in the achromatic dry objective, and for the correction of which Zeiss proposed mounting the several lenses on a method somewhat different to that so long in use in this country. Fig. 115 shows an objective in which the screw-collar ring _b b_ is made to adjust the exact distance between the two back lenses placed at _a a_. The value of the screw-collar is not questioned. It is difficult to obtain at all times cover-glasses of a perfectly uniform thickness; they will vary, and therefore perfect definition must be obtained, as heretofore, by adjusting for each separate preparation while the object is under examination.

As early as 1842 the excellence of Andrew Ross’s achromatic objectives were acknowledged, and his formula for their construction was generally followed. No doubt many of these early objectives of his manufacture are still regarded as treasures. I possess a 1/2-inch and a 1/4-inch, which I believe to be comparable with any achromatic objectives of the same apertures of the present day. These I have always found most serviceable for histological work.

In 1850 Mr. Wenham produced an achromatic objective of considerable achromatic value. This consisted of a single hemispherical front combination, shown in the accompanying enlarged diagram, Fig. 116. Wenham’s formula seems to have been generally adopted by Continental opticians, who sold these lenses at a reduction of price. In Paris, Prazmowski and Hartnack--I have had one of Hartnack’s earliest immersions in use for many years--brought this form of objective to greater perfection, and in 1867 Powell and Lealand adopted the single front combination system in their early water-immersion objective, whereby the focal distance was said to be “practically a constant quantity, while reduction of aperture by making the front lens thinner ensures a much greater working distance without affecting the aberrations, since the first refraction takes place at the posterior or curved surface of the front lens, the removal of any portion of thickness at the anterior or plane surface simply cuts off zones of peripheral rays without altering the distance--any space being filled by the homogeneous immersion fluid, or by an extra thickness of cover-glass.”[21]

Great improvements were brought about by R. B. Tolles, of Boston, 1874, in the objective, as well as in the optical and mechanical parts of the microscope, most of which, however, must be ascribed to the criticisms and suggestions of amateur workers skilled in the exhibition of test-objects--the late Dr. Woodward of Washington, for example, whose series of photographs of the more difficult frustules of diatoms have rarely been surpassed. Such results were due to improvements made in the optical part of the microscope at his suggestion. He came to the conclusion, arrived at about the same time by mathematical scientists, that increase of power in the microscope was only possible in two directions, the qualitative and the quantitative.

It was now that microscopists turned to the late Professor Abbe for assistance in perfecting the objective in the dioptric direction. This, he pointed out, must be looked for in further improvements in the art of glass-making.

A series of experiments ultimately brought to light a mineral substance, _Fluorite_, which, when combined in the proper proportion, one part to two of German crown and flint glass, was found to have the qualities looked for, and to possess different relations of a dispersive and refractive power. From Professor Abbe’s researches, begun in 1876, we have had the aperture of the objective greatly enlarged, and the homogeneous system brought into general use.

Previous to this date the best made objective merely approximated to colour correction. Undoubtedly the chief object to be obtained was the removal or diminution of the secondary colour aberration. This, together with other residual errors Abbe pointed out in 1880, led to the improvement of the optical quality of the glass used in the manufacture of all optical instruments, the chief difficulties being surmounted in the Jena glass factory, whereby a complete revolution was effected in the microscopic objective. The apochromatic glasses of Zeiss, Powell, Beck, Ross, Watson, Swift, and other makers, in which the secondary spectrum has been totally eliminated, or only a negligible tertiary spectrum remains--that is to say, the objectives of these makers--are now corrected for three spectrum rays, and not two, as in the older objectives; and only those who look forward for making further discoveries in the intimate structure of bacilli or for resolving the finest diatom markings can be said to fully appreciate the importance and value of the investigations of the late Professor Abbe, and which have, so to speak, entirely changed old empirical views as to the value of high aperture, and demonstrated that high amplification, unless associated by proportionally high aperture, necessarily produces untrue images of minute structures. It was he also who introduced a practically perfect system of estimating apertures, known as the “numerical aperture notation,” by which not only can an accurate comparison be made of the relative apertures of any series of objectives, whether dry or immersion, but their resolving power under the various conditions of the kind of light employed. Their penetrating power and their illuminating power can now be estimated with mathematical exactness.

The practical advantages, then, secured by the adoption of the homogeneous system were, on the whole, greater than any before made or believed to be possible, and when taken into account in connection with the improvement of the eye-piece (also due to Abbe), almost perfect achromatism and homogeneity between objective, object, and eye-piece is secured, together with a sharp definition of the image over the whole visual field. These, with an increase of working distance between the object and the objective, and other important results, have been placed within the reach of the microscopist by men of science, and the outcome is the general adoption of the homogeneous system, termed by Carl Zeiss, a fellow-worker with Abbe, the[22] apochromatic system of constructing objectives.

Relative Merits of the English and German Objectives.

As to the relative merits of German-made objectives, no superiority can be claimed for them over those made by English opticians.

The Continental form of the 1/12-inch oil-immersion objective, shown in Fig. 118, on the scale of 6 to 1, consists of four systems of lenses, namely, the front, a deep hemispherical crown lens of high refractive index; the second front of the system, an achromatic lens of such a form that it gathers the light from the hemispherical front; the middle lens, a single meniscus; and the back an achromatised lens, the second front of the back being connected in such a way as to compensate for the spherical and chromatic aberrations of the front lens.

The first homogeneous immersion objective which came under my observation was manufactured in the well-known Jena workshop of Carl Zeiss, December, 1877. This had a very considerable increase of _numerical aperture_, upwards of 50 per cent.; a clear gain, as an oil angle of even 110° proved to be of greater value than an angle of 180° in air, while the resolving power of the objective was increased in like proportion. There does not at present appear to be a bar to the construction of objectives of yet higher power, with increase of aperture. The available course open in this direction is the further discovery of another vitreous material and a suitable immersion fluid with an index of 1·8 or 1·9, and glass with a corresponding index, so as to ensure homogeneity of the combination. Zeiss asserts that in the more difficult departments of microscopical research the apochromatic lenses will supplant the older objectives, yet there are many problems in microscopy awaiting solution which do not demand the highest attainable degree of perfection in the objective, and in the majority of cases the older achromatic objective is all that is needful, provided it is good of its kind. The achromatic objectives and eye-pieces of the older type have still an advantage, as, owing to their simpler construction, really good lenses of the class required can be purchased at considerably lower prices than the objectives of the new series. These, from being more complicated in construction, involve a greater amount of skilled manual labour.

The German glasses of to-day afford satisfactory evidence both of skill and workmanship displayed in their production. Their cost is greater, then, for the reason given, as will be seen on reference to Continental catalogues. The dry series of objectives cost somewhat less, a 1/2-inch (numerical aperture 0·30) can be had for £1 10s., and a 1/6-inch (numerical aperture 0·65) for £2. On the other hand, the apochromatic series rapidly increase in price as the numerical aperture approaches the limit of numerical aperture 0·40. The best of Zeiss’s series are the 12 mm. (1/2-inch) and the 3 mm. (1/8-inch), numerical aperture 1·4, both of which possess the optical capacity assigned to them. These objectives are undoubtedly the finest to be met with in the workshop of any optician. Achromatic objectives of Continental manufacture have been as much improved as those of English make by the introduction of the newer varieties of glass, as already explained, while a new nomenclature has sprung up in consequence. We now have semi-apochromatic and parachromatic. The German opticians have followed Zeiss’s lead, since almost the same series of objectives are given in the catalogues of Leitz, Reichert, and Seibert, while the quality of both dry and immersion objectives is found to be much the same. The low price of Reichert’s immersion objectives should be noted, as their performance is quite perfect. A 1/12-inch (numerical aperture 1·30) of Leitz’s, with which I have worked at _bacteria_, has given me much satisfaction; supplied by Watson and Baker at £5. A 1/12-inch dry objective by the same maker (numerical aperture 0·87) costs £3, and a water immersion 1/12-inch (numerical aperture 1·10) £3 5s. Leitz reminds me that it requires a good lens of from six to seven hundred magnifying power for the examination of bacteria. For this reason he has constructed a new form of lens, a 1/10-inch oil-immersion of 2·5 mm. focus, for the purpose of adding to the resources of bacteriology. This lens necessarily has a lower magnification than his former 1/12-inch oil-lens, but as it is less costly to manufacture it is sold at a smaller price. The before-mentioned 1/12-inch, with a No. 3 compensating eye-piece, gives a magnification of over seven hundred or eight hundred diameters. To secure the best results in using the higher powers of Leitz’s, from No. 5 upwards, a cover-glass of 0·17 mm. in thickness should be used, and care taken to make the length of the draw-tube equal to 170 mm. This length of tube should be adhered to in the use of this optician’s oil-immersion lenses. If the microscope be provided with a nose-piece, the draw-tube should be drawn out to 160 mm.; in its absence it should be set at 170 mm., a deviation of 10 mm. or more from the correct tube-length deteriorates from the value of Leitz’s oil-immersion objectives as of other opticians. It is suggested that the German apochromatic combination of three cemented lenses is that adopted by Steinheil long before, in the construction of his well-known hand-magnifier (see page 77, Fig. 51). Zeiss’s 3 mm. objective has a triple front, balanced by two triple backs--in all nine lenses--a somewhat amplified diagram of which is represented in Fig. 118. The formula for this combination was furnished by Tolles, of Boston, America, and it at once secured increase of aperture (the value of this optician’s many contributions to microscopy has since his death been generally acknowledged). The metrical equivalent focus assigned by Zeiss to his series of dry achromatic objectives is given in somewhat ambiguous terms, which tend to confuse rather than classify them; for instance, two lenses of the same aperture--24 mm. and 16 mm.--corresponding to the English 1-inch and 2/3-inch, each have assigned to them an aperture of 0·30; a 12 mm. and 8 mm., corresponding to the English 1/2-inch and 1/3-inch, have an aperture of 0·65; while a 6 mm. = 1/4-inch, and a 4 mm. = 1/4-inch and 1/6-inch, have each an aperture of 0·95.

Nachet exhibited at the Antwerp Exhibition a fine 1/10-inch oil-immersion, which was highly praised by the jurors.

It is necessary, to make the fact perfectly clear, that dry and immersion lenses having the same angular aperture have also a similar defining power. The pencil of rays, however, differs in intensity and density as the rays emerging from the cover-glass of the object into air are very considerably deflected, and the cone suffers a corresponding loss of brightness. On this important point, then, I believe it will prove of value to interpolate a clear and full exposition of the change brought about by the cover-glass.

It is not difficult, then, to perceive the importance of Amici’s discovery as to the value of a drop of water inserted between the object and the objective, and it now seems somewhat surprising it should have been so long neglected by opticians, since it is at once seen to diminish the reflection which takes place in the incidence of oblique light. The film of water not only gives increased aperture, but also greater cleanness and sharpness to the image. The film, then, as already shown, collects the straying away of peripheral rays of light, and sends them on to the eye-piece, and greatly assists in rendering the image more perfect, and materially aids in the removal of residuary secondary aberrations; while with air, or dry objectives, a certain amount of aberration takes place, sufficient to affect the pencils on their passage from the radiant to the medium of the front lens, adding a considerable ratio to the total spherical aberration with the objective, which, in the case of wide angles, increases disproportionately from the axis outwards. This can only be corrected by a rough method of balancing; that is, by introducing an excess of opposite aberration in the posterior lens. An uncorrected residuum, rapidly increasing with larger apertures, is then left, and this appears in the image amplified by the total power of the objective, so that with a non-homogeneous medium there is a maximum angular aperture which cannot be surpassed without undergoing a perceptible loss of definition, provided working distance is required. If we abolish the anterior aberration for all colours, by an immersion fluid which is equal to cover-glass in refractive and dispersive power, the difficulty is at once overcome. If, for instance, we have an objective of 140° in glass (= 1·25 N.A.) and water as the immersion fluid, the aberration in front would affect a pencil of 140°. Substituting a homogeneous medium, the same pencil, contracted to the equivalent angle in that medium of 112°, will be admitted to the front lens without any aberration, and may be made to emerge from the curved surface also without any disturbing aberration, but contracted to an angle varying from 70° to 90°. The first considerable spherical aberration of the pencil then occurs at the anterior surface of the _second_ lens, where the maximum obliquity of the rays is already considerably diminished.

Figs. 119 and 119_a_ will doubtless make this clearer. If the objective of 140° works with water (Fig. 119), there would be a cone of rays extending up to 70° on both sides of the axis, _and this large cone would be submitted to spherical aberration at the front surface a_. But with homogeneous immersion (Fig. 119_a_) the whole cone of 112° is admitted to the front lens without any aberration, there being no refraction at the plane surface; and as the spherical surface of the front lens is without notable spherical aberration, the incident pencil is brought from the focus F to the conjugate focus F′, and contracted to an angle of divergence of 70°-90° _without having undergone any spherical aberration at all_.

The problem of correcting a very wide-angled objective has thus been reduced by the homogeneous oil-immersion system, both in theory and practice.[23]

Abbe’s Test-plate.

Abbe designed the test-plate (Fig. 120) for testing the spherical and chromatic aberrations of objectives, and estimating the thickness of cover-glasses corresponding to the most perfect correction: six glasses, having the exact thickness marked on each, 0·09 to 0·24 mm., cemented in succession on a slip, their lower surface silvered and engraved with parallel lines, the contours of which form the test. These being coarsely ruled are easily resolved by the lowest powers; yet, from the extreme thinness of the silver, they form also a delicate test for objectives of the highest power and widest aperture. The test-plate in its original size is seen in Fig. 120, with one of the circles enlarged.

To examine an objective of large aperture, the discs must be focussed in succession, observing in each case the quality of the image in the centre of the field, and the variation produced by using, alternately, central and very oblique illumination.

When the objective is perfectly corrected for spherical aberration, the outlines of the lines in the centre of the field will be perfectly sharp by oblique illumination, and without any nebulous doubling or indistinctness of the edges. If, after exactly adjusting the objective for oblique light, central illumination is used, no alteration of the focus should be necessary to show the outlines with equal sharpness.

If an objective fulfils these conditions with any one of the discs, it is free from spherical aberration when used with cover-glasses of that thickness. On the other hand, if every disc shows nebulous doubling, or an indistinct appearance of the edges of the line with oblique illumination, or, if the objective requires a different focal adjustment to get equal sharpness with central as with oblique light, the spherical correction of the objective is more or less imperfect.

Nebulous doubling with oblique illumination indicates over-correction of the marginal zone; indistinctness of the edges without marked nebulosity indicates under-correction of the zone; an alteration of the focus for oblique and central illumination points to an absence of concurrent action of the separate zones, which may be due to either an average under or over correction, or to irregularity in the convergence of the rays.

COVER-GLASS GAUGE.

Zeiss has gone a step further to lay the microscopist’s ghost of the cover-glass. He invented a measurer (Fig. 121) whereby the precise determination of thickness of glass-covers can be obtained. This measurement is effected by a clip projecting from a circular box; the reading is given by an indicator moving over a divided circle on the lid of the box. The divisions seen cut round the circumference show 1/100ths of a millimeter. This ingenious gauge measures upwards of 5 mm.

This necessary and important digression has led me away from the consideration of the achromatic objective, and to which I shall now return.

English Immersion and Dry Objectives.

The homogeneous immersion system met with its earliest as well as its staunchest advocates among English opticians. Among its more energetic supporters were Messrs. Powell and Lealand, who were the first to construct a 1/8-inch immersion objective on a formula of their own, and which was found to resolve test-objects not before capable of resolution by their dry objectives. This encouraged them to make a 1/16-inch, acquired by Dr. Woodward for the Army Medical Department, Washington, and subsequently a 1/25-inch; neither of which surpassed their 1/8-inch in aperture, and a new formula was tried in the construction of their first oil-immersion objective. This had a duplex front, and two double backs; but even this did not quite accomplish what was expected of it, and another change was subsequently made; the anterior front combination became greater than a hemisphere--a balloon-lens. This at once gave an increase of aperture to a 1/12-inch objective of 1·43 numerical aperture. After some few more trials a more important change of the formula took place. The front lens was made of flint-glass, and the combination took the form represented in diagram (Fig. 122). This, on an enlarged scale, represents Powell’s 1/12-inch numerical aperture 1·50. It is a homogeneous apochromatic immersion of high quality and very flat field. It will be noticed that in this combination the four curves of the lenses are very deep compared with those of other opticians.

_Messrs. Ross_ have made many important improvements and changes in the construction of their several series of achromatic objectives; the calculations and formulæ for which were made exclusively for them by Dr. Schrœder. The list is too long to quote, but most of these lenses are of a high-class character, and work with admirable precision. Among the best of their objectives, I can commend a 1-inch of 30° and two oil-immersions, a 1/8-inch of 1·20 and a 1/12-inch of 1·25 numerical aperture, each of which bear the highest oculars equally well; a good test, as I have always maintained, of excellence. Their 1/10-inch has a somewhat larger aperture, and therefore shows a fine image of the podura scale. The finish of Ross’s several series of objectives fully maintains the high character and reputation of this old-established firm of opticians.

_Messrs. R. and J. Beck_ have bestowed great attention upon the improvement of their dry-objective series, much in demand for histological work, especially among the students of city hospitals, who usually commence their pathological work with the cheaper forms of objectives. In that case an inch objective of about 25° air angle, a 1/2-inch of not less than 40°, and a 1/4-inch or 1/5-inch magnifying from 50 to 250 diameters, is quite sufficient for most of their work. For bacteriological research, Messrs. Beck supply a 1/6-inch immersion taken from a series, having a high aperture and a better finish at a moderate price. Their 1/10-inch immersion has in my hands proved a serviceable power for bacteriological research; it requires a good sub-stage illuminating achromatic condenser to obtain the best results.

_Messrs. Watson and Sons_ have much enhanced their reputation by the marked improvement lately brought about in the manufacture of their whole series of objectives. This probably is chiefly due to the introduction of the _Jena_ glass into their manufacture, and which has enabled them to give increase of aperture to one series in particular, that of the para-chromatic, all of which in consequence are of very high quality. It is difficult to particularise their several objectives, the whole having special features in proportion to their magnifying powers, while much care seems to have been bestowed on them for the elimination of residual colour. A 1/8-inch with correction collar is comprised of a single deep and rather thick front lens, plano-concave flint, and double convex-crown for the middle and triple combination for the back, the latter consisting of two crown lenses cemented to a dense flint (Fig. 124) drawn to scale of 5-1, with lined portions intended to represent the flint, and white the crown glass lenses of the combination. The initial magnification of this objective is 83 diameters, and the numerical aperture ·94. This superior objective can be had for the small sum of £2. Another remarkably useful and cheap objective, their 1-inch numerical aperture 0·21, consists of two achromatic systems forming the front and back with the separation between them of about half an inch, and may also be especially recommended for students’ work.

In the accompanying diagram the lenses are drawn on too large a scale, and therefore the distance between the two combinations should be much greater.

Among the more useful of Watson’s series, the 1-inch, the 1/2-inch, and the 1/6-inch, together with the 1/8-inch dry-objective, and a 1/9-inch, will be found the most serviceable.

_Messrs. Baker_ have their own series of objectives, most of which are so very nearly allied to those of the continental opticians; and what has been said of Zeiss’s and Leitz’s objectives may be taken to apply also to Baker’s, who have an established reputation for their histological series, all of which are well suited for students’ and class-room work.

_Messrs. Swift and Son_ have a new series of objectives, semi-apochromatic and pan-aplanatic, most of which are excellent in quality and show increased flatness of field together with that of achromatism; the index of refraction in each series having been correctly determined together with exact radial focal distance, thus affording more available aperture. I may select for special commendation their 1/12-inch £5 5_s._ homogeneous immersion objective, which is in every way suitable for bacteriological work; its definition is very good, as is seen in a micro-photograph of podura scale, given further on. Their dry 1/6-inch can be had for £1 16_s._--a marvel of cheapness. Of their general series the most useful for histological work are the 1/2-inch, the 1/3-inch at £1 12_s._, and their 1/5-inch of numerical aperture 0·87 at £3.

_Mr. Pillischer_, of Bond Street, has manufactured many excellent objectives. A fine homogeneous oil-immersion 1/12-inch numerical aperture 1·25 is worthy of special notice; it will be found suitable for bacteriological work; it has fine definition with a considerable amount of penetration.

A more intelligent idea of the magnifying power of the objective combined with the eye-piece will be gained by consulting the table given below; precision in this respect has long been a desideratum with microscopists.

Magnifying Powers of Eye-Pieces and Objectives.

A TYPICAL AND INITIAL SELECTION OF POWERS OF EYE-PIECES CALCULATED FOR THE 10-INCH TUBE-LENGTH.

HUYGHENIAN EYE-PIECES.

NAME A B C D E F OF MAKER. 0 or No. 1 2 3 4 5 6

Baker 6 8 12 15 -- -- Diameters. Beck, R. & J. 4 8 15 20 25 not made. " Leitz 5 6 7 8 10 12 " " Powell & Lealand 5 7·5 10 20 40 " " Reichert 2·5 3·5 4 5 6·5 " " [24] Ross 3 8 12-1/2 20 25 40 " [25] Swift & Son 6 9 12 15 18 21 " Watson & Sons 4 6 8 10 12 15 " Zeiss 3 4 5·5 7 9 not made. "

COMPENSATING EYE-PIECES FOR USE WITH APOCHROMATIC OBJECTIVES.

Zeiss 2 4 8 12 18 27 Diameters.

This may be taken as a typical set, further treated of among Eye-pieces.

INITIAL POWERS OF OBJECTIVES CALCULATED FOR THE 10-INCH TUBE-LENGTH.

This is ascertained by dividing the distance of distinct vision 10 inches by the focus of the objective, thus--

Focus-inches 4 3 2 1-1/2 1 2/3 1/2 4/10 1/4 1/5 1/6 1/8 1/12 Initial magnifying power 2·5 3·3 5 7·5 10 15 20 25 40 50 60 80 120 diameters.

A reference to the above table will at once show that the nomenclature of objectives expresses at once the initial magnifying powers, but as makers have great difficulty in so calculating their formulæ so as to obtain the _exact_ power, these figures must be taken as approximate. Thus a 1/4-inch, which should magnify 40 diameters if true to its description, might actually magnify a little more or less.

The magnifying powers of Zeiss’s and other apochromatic objectives can be ascertained by dividing the focal length of the objective in millimeters into 250 mm. (the distance of distinct vision), thus

Focus millimetres 24 16 12 4 3 2 1·5 Initial magnifying power 10·5 15·5 21 63 83 125 167 diameters.

The total magnification, when any eye-piece is working in conjunction with an objective, is ascertained by multiplying the initial power of the objective by that of the eye-piece.

The above calculations are all for a 10-inch tube-length. Should, however, a shorter or longer length of body be employed, the magnification can at once be ascertained by a proportion sum. If the magnification be 180 with 10-inch tube-length, what would it be with a 6-inch body--10 : 6 :: 180 = 108 diameters.

Abbe designed three different forms of eye-pieces: 1, the searcher eye-piece; 2, the working eye-piece; and 3, the projecting eye-piece. The _Searcher_ is a negative form of low power. The working is both negative and positive, the positive form of which is constructed on a newer principle; while the projection is chiefly intended for microphotography, its field being small and its definition superlatively sharp. These are severally explained among eye-pieces.

High-Power Objectives.

_Points of Importance for securing the best results with High-power Objectives._--Always give to the body-tube of the microscope the length for which the objective is corrected, 0·160 mm. for the short continental tube, and 0·250 mm. for the English tube (10-inch). Employ both dry and immersion objectives mounted for correction, commencing with a numerical aperture of 0·75 (that is about 100° in air). If the graduation is not given in thickness of cover-glass apply to the maker to correct this omission.

With the homogeneous oil-immersion objective it is highly necessary to utilise all marginal pencils of light, to optically unite the upper lens of the condenser with the preparation as well as the front lens of the objective by means of a liquid having the same index of refraction or at least equal to that of the immersion. _Cedar Oil_ has been generally adopted for the purpose mentioned, the better way of using which is as follows: place a drop on the centre of the front objective, or on the top of the cover-glass, and then lower the objective by means of the coarse adjustment until it comes in contact with the oil, and carefully bring into focus by the fine adjustment. If the slide is held between the finger and thumb of one hand and moved from side to side, while the other hand is working the fine adjustment, there can be no danger of injuring either the objective or the specimen. Before putting the microscope away, take a fine camel-hair brush dipped in ether, alcohol, or methylated spirit, and carefully remove the oil from the objective and the glass cover of the object; a soft chamois leather or cambric pocket handkerchief will dry it off, or a piece of fine white blotting paper answers equally well. Should the lens come accidentally into contact with the Canada balsam, it must be very carefully removed either by ether or alcohol. The former is by far the safest, as alcohol, if not very carefully used, quickly dissolves out the balsam and loosens the cover-glass of the object.

Achromatic Condensers.

_The Achromatic Condenser_ can no longer be classed among the _accessories_ of the microscope, since it is an absolutely indispensable part of its optical arrangements. Its value, then, cannot be overrated, and the corrections of the lenses which enter into the construction of the condenser should be made as perfect as they can be made--in fact, as nearly approaching that of the objective as it is possible to make them. It may therefore be of interest to know something of the rise and progress of the achromatic condenser. In my first chapter I have noticed the earlier attempts made by Dr. Wollaston, whose experiments led him to fit to the underside of the stage of his microscope a short tube, in which a plano-convex lens of about three-quarters of an inch focal length was made to slide up and down (afterwards moved up and down by two knobs); to improve definition he placed a stop between the mirror and the lens. The stop was found to act better when placed between the lens and the object. From this improvement Dr. Wollaston enunciated that “the intensity of illumination will depend upon the diameter of the illuminating lens and the proportion of the image to the perforation, and may be regulated according to the wish of the observer.” Dujardin in France and Tully in England were at work in the same direction. The former a year or two later on contrived an instrument, which he termed an _eclairage_, to remedy the defects of Wollaston’s, and for illuminating objects with achromatic light. This was submitted for approval to Sir David Brewster, who, when the use of the achromatic condenser was first broached, used these encouraging words:--“I have no hesitation in saying that the apparatus for illumination requires to be as perfect as the apparatus for vision, and on this account I would recommend that the illuminating lens should be perfectly free from chromatic and spherical aberration, and that the greatest care be taken to exclude all extraneous light both from the object and eye of the observer.” This far-seeing observer in optical science has borne good fruit, and the outcome of his views is seen in the great development and improvement of the achromatic condenser. In 1839 Andrew Ross made his first useful form of condenser, and gave rules for the illumination of objects in an article written for the “Penny Cyclopædia.” These, epitomised, read as follows: 1. That the illuminating cone should equal the aperture of the objective, and no more. 2. With daylight, a white cloud being in focus, the object has to be placed nearly at the apex of the cone. The object is seen better sometimes above and sometimes below the apex of the cone. 3. With lamplight a bull’s-eye lens is to be used, to parallelise the rays, so that they may be similar to those coming from the white cloud. It has been seen that Mr. Lister foreshadowed the sub-stage condenser.

The early form of Ross’s condenser consists of two small brass tubes made to slide one in the other. To the outer one is attached a flat brass plate which slides underneath the stage of the microscope, and by means of a screw the adjustment of the axis of the illuminator is effected. The upper portion of the apparatus carries the achromatic combination, which by a rack and pinion movement is brought nearer to, or removed further from the object on the stage. The several parts of the illuminator unscrew, so that the lenses may be used either combined for high powers, or separated for low powers.

Messrs. Smith & Beck greatly improved upon Ross’s condenser by adding another achromatic lens to the combination, three being employed when used with high-power objectives and two or even one with the lower, the adjustment and focussing being made by rack and pinion arrangement beneath the stage. Some further changes for the better were made in the condenser by Powell, and in 1850 an amateur microscopist, Mr. Gillett, fully grasping the value of controlling the cone of rays passing into the microscope, devised a new form of condenser, in connection with which a revolving series of diaphragms of different values were made to pass between the achromatic lenses and the source of light.

Andrew Ross constructed the first condenser on Gillett’s principle, and this proved to be one of the most successful pieces of apparatus contrived. _Gillett’s Condenser_ consists of an achromatic lens _c_, about equal to an object-glass of one quarter of an inch focal length, with an aperture of 80°. This lens is screwed into the top of a brass tube, and intersecting which, at an angle of about 25°, is a circular rotating brass plate _a b_, provided with a conical diaphragm, having a series of circular apertures of different sizes _h g_, each of which in succession, as the diaphragm is rotated, proportionally limits the light transmitted through the illuminating lens. The circular plate in which the conical diaphragm is fixed is provided with a spring and catch _e f_, the latter indicating when an aperture is central with the illuminating lens, also the number of the aperture as marked on the graduated circular plate. Three of these apertures have central discs for circularly oblique illumination, allowing only the passage of a hollow cone of light to illuminate the object. The illuminator above described is placed in the secondary stage _i i_, which is situated below the general stage of the microscope, and consists of a cylindrical tube having a rotatory motion, also a rectangular adjustment, which is effected by means of two screws _l m_, one in front, and the other on the left side of its frame. This tube receives and supports all the various illuminating and polarising apparatus, and other auxiliaries.

_Directions for using Gillett’s Condenser._--In the adjustment of the compound body of the microscope for using with Gillett’s illuminator, one or two important points should be observed--first, centricity; and secondly, the fittest compensation of the light to be employed. With regard to the first, place the illuminator in the cylindrical tube, and press upwards the sliding bar _k_ in its place, until checked by the stop; move the microscope body either vertically or inclined for convenient use; and, with the rack and pinion which regulates the sliding bar, bring the illuminating lens to a level with the upper surface of the object-stage; then move the arm which holds the microscope body to the right, until it meets the stop, whereby its central position is attained; adjust the reflecting mirror so as to throw light up the illuminator, and place upon the mirror a piece of clean white paper to obtain a uniform disc of light. Then put on the low eye-piece, and a low power (the half-inch), as more convenient for the mere adjustment of the instrument; place a transparent object on the stage, adjust the microscope-tube, until vision is obtained of the object; then remove the object, and take off the cap of the eye-piece, and in its place fix on the eye-glass called the “centring eye-glass,”[26] which will be found greatly to facilitate the adjustment now under consideration, namely, the centring of the compound body of the microscope with the illuminating apparatus of whatever description. The centring-glass, being thus affixed to the top of the eye-piece, is adjusted by its sliding-tube (without disturbing the microscope-tube) until the images of the diaphragms in the object-glass and centring lens are distinctly seen. The illuminator should now be moved by means of the left-hand screw on the secondary stage while looking through the microscope, to enable the observer to recognize the diaphragm belonging to the illuminator, and by means of the two adjusting screws to place this diaphragm central with the others: thus the first condition, that of centricity, will be accomplished. Remove the white paper from the mirror, and also the centring-glass, and replace the cap on the eye-piece, also the object on the stage, of which distinct vision should then be obtained by the rack and pinion, or fine screw adjustment, should it have become deranged.

The re-publication of the original directions is given with the view of showing what a clear conception Gillett had of the value of his invention. The careful directions given for centring must be regarded with interest, although nearly superseded by the centring screw arrangement in connection with the sub-stage. The best results, he goes on to say, will be secured by using the plain mirror and focussing the window-bar on the object, while a white-cloud illuminator will afford as much light as may be required. It is a mistake to suppose that direct light is more critical than indirect. As a rule, the student is given to over-illuminate the object. These questions will, however, be discussed further on.

Very many modifications of Gillett’s condenser have, since 1850, become known to microscopists. Ross’s present improved form (Fig. 127) is made to drop into the sub-stage of the microscope, and when adjusted, is an extremely efficient instrument. The optical part is similar to a 4/10-inch objective. It has two sets of revolving diaphragms, with apertures and stops for showing surface markings in a perfect manner.

Abbe’s Condenser.

The essential feature of this condenser is its short focus, which collects the light reflected by the mirror, so as to form a cone of rays of very large aperture, having its focus in the plane of the object.

The full aperture of the illuminating cone should only be used when finely granular and deeply stained particles (protoplasm, bacteria, &c.) are being examined with objectives of large aperture. In all cases the cone must be suitably reduced, either by an iris, or other form of diaphragm (_central illumination_). By placing the diaphragm excentrically, by means of rack-work attached to the carrier, the central rays are excluded and a certain extra-axial portion of the illuminating pencil falls upon the object (_oblique illumination_). When the diaphragm is thus excentrically placed, this oblique pencil can be directed from all sides by rotating the carrier round the optic axis. The central stop diaphragm shuts off all the axial and transmits only the marginal rays, thus producing _dark-ground illumination_. The iris diaphragm (Fig. 128) is so shaped that the edge of its smallest opening closely approximates the object-slide on the stage.

The Abbe condenser is the most popular form in use, for all purposes. Owing to the large aperture of the cone of light which it projects, it can be employed with the highest powers; by removing the top lens it can also be used with low powers. Dark ground illumination may be obtained with it up to a 1/4-inch objective.

The condenser is made in two forms of 1·2 and 1·4 numerical aperture by Messrs. Watson. The lenses are mounted in aluminium. Fig. 130 is in more general use, but by workers with high powers Fig. 131 is preferred, as it ensures the most oblique illumination with objectives of largest aperture. It is preferred for photo-micrographic purposes.

_Watson’s Achromatic Condenser_ (Fig. 132), 1·0 numerical aperture, shown in section, although originally designed for use with the micro-spectroscope, is equally efficient for ordinary purposes. This condenser transmits a larger aplanatic cone of light than Abbe’s. It may therefore be employed with higher power objectives, and by removing the top lens it is just as useful a condenser for lower powers. Being constructed with lenses of an unusually large size, it is well adapted for use with the micro-spectroscope. It is certainly one of the best all-round condensers in use. The new Schott glass enters into the construction of the lenses, and these are mounted in aluminium.

Many microscopists consider on the whole that Powell’s sub-stage apochromatic condenser with collar correction (Fig. 133) surpasses that of Abbe. The mechanical arrangement of Powell’s is very simple: the correction collar is similar to that of an ordinary objective, it has a steeper spiral slot and only half a revolution of movement; a long arc is fixed to the collar so that it may conveniently be reached by the finger. It is so constructed as to turn easily and smoothly at the slightest touch. The collar moves only the back lens of the combination, leaving the mount rigid. The diaphragms are regulated by A and B.

The object of the correctional movement is to increase the maximum aplanatic aperture of the condenser by separating the lenses. If the back of a wide-angled objective be examined when an object is illuminated by the full aperture of the condenser, the edge of the flame being in focus, it will be noticed that the illuminated portion of the back lens will be oval and pointed instead of circular. Also that when the condenser is racked up, although the external shape of the illuminated portion becomes more circular, two dark patches will appear on either side of the centre, showing the operation of the spherical aberration of the condenser. If under these circumstances the lenses are separated by means of the collar adjustment, the black spots will be closed up, and a circular and evenly-illuminated disc of illumination of a larger size will result. The wheel of diaphragms, or a series of graduated diaphragm discs to drop into a holder, is intended for critical work; the diaphragm can always be recorded, and the identical illuminating cone reproduced.

Hence we have a simple method of graduating apertures between any two contiguous diaphragms; if, for example, we place the lever to the left, so that the lens may be separated as far as possible, and use a No. 6 diaphragm, and if, on examining the object, it is thought that the illuminating cone is not large enough, and if when No. 7 is turned on it is found too much, we can go back to No. 6, and by turning the lever 60° towards the right, closing the lenses and increasing the power a little, we shall obtain an aperture somewhere between Nos. 6 and 7 diaphragm. Thus we can by means of the correction collar graduate the aperture with the facility as with an iris, and we can record any particular aperture with a degree of accuracy foreign to the iris. It must be admitted, however, that the cone of light transmitted by the condenser is a very small one.

Powell also supplies an apochromatic oil-immersion condenser, numerical aperture 1·40, but without collar correction; Fig. 134 shows the sliding tube lowered by arm A and cell B withdrawn for changing stops, which can be done without altering the focus of the condenser. Fig. 134_a_ shows the cell B closed and raised by arm A close to the back lens of optical combination. In Fig. 134_b_ six of the principal stops are shown. Powell’s dry apochromatic condenser, of nearly 0·9 aplanatic cone, is also very good; but the high price of all is a bar to their more general use. The speciality of these is the conversion of axis light into condensed oblique incident light by the refraction of the condenser.

Messrs. R. & J. Beck have various forms of achromatic condensers, some of which partake of a somewhat elaborate arrangement; others are simple and inexpensive, to suit the students’ microscope; as when the light of the concave mirror proves insufficient for any object requiring intense transmitted light, an achromatic condenser must be adapted to even the students’ form of microscope. The latest form of condenser (Fig. 135) is fitted with revolving stops and iris diaphragm, and other appliances for obtaining satisfactory results.

_Beck’s Compound Illuminating Apparatus_ (Fig. 136).--It is useful in working with the microscope to be enabled to rapidly change the illumination, and for this reason this compound form of condenser has been constructed. It consists of an upper portion A, a wide-angle condenser, the aperture of which can be reduced at will by an iris diaphragm, moved by the lever B. This can be used for all other purposes. Below this diaphragm is a plate C, which can be swung back out of position at will, as shown in outline. Into a cell in this plate the stops D can be dropped, and the condenser can be used for dark field illumination, or for high powers as an oblique illuminator. A large-size polarising prism E, fastens to the plate C, and can be removed when not required. In this way any of the various modes of illumination may be separately or conjointly obtained.

Their condenser (Fig. 137) has a large aperture, and facilities for rotating the series of diaphragms. It is available for either dry or immersion objectives up to 1·3 numerical aperture on diatoms, and wet or dry histological objects. The spherical form of the front is worked by a milled-head that rotates a series of lenses and diaphragms. It also avoids the inconvenience of having the connecting fluid drawn away by capillary attraction, as would be the case if mounted on a flat surface. It is also less in the way of the sub-stage movements.

_The Parachromatic Condenser_ of Messrs. Watson (Fig. 138) was made to meet a demand for a condenser giving a large solid cone of illumination free from colour. The optical part of this condenser consists of a full hemispherical front lens, and the middle and back combinations of such forms as to produce the necessary corrections. The Jena phosphate crown and silicate flints are used in its manufacture, and to these are due its special qualities. The total aperture of the condenser is 1·0, and it yields an aplanatic aperture of ·90 numerical aperture. The magnifying power is 2/7ths of an inch. From this it will be seen that it is especially suitable for use with high-power objectives.

It can also be employed without the front lens, when the magnifying power is 4/10ths of an inch, and the numerical aperture ·35. It is mounted in an exceedingly convenient manner, the iris diaphragm being fitted in such a way as to be absolutely central with the optical system.

The arc through which the handle controlling the iris travels is divided, and indicates the aperture at which the condenser may be working at any time. An important feature in this condenser is that it is almost wholly free from colour. As a rule condensers of the same form are found difficult to work with, because of the small diameter of the field or back lens. This difficulty has been successfully overcome by increasing the size of this lens, and the whole of which is fully utilised.

Most London opticians have their own especial form of achromatic condenser, designed for and fitted to their several stands and objectives, varying from a small price to the more expensively-fitted accessories.

Messrs. Swift’s illuminating apparatus (Fig. 139) is conveniently supplied with numerous useful appliances. The optical combination A is computed to be used as an effective spot lens from a 3-inch objective up to a sixth. C C are two small milled heads by means of which the optical combination A is centred to the axis of the objective. The revolving diaphragm E has four apertures for the purpose of receiving central stops, oblique light discs, and selenite films. D is a frame carrying two revolving cells, into one of which a mica film is placed, which can be revolved with ease over either of the selenites below, whereby changes of colour can be obtained in experimenting with polarised light. The darts and P A’s indicate the position of the positive axis of the mica and selenite films, and by this means results can be recorded, etc. Either of the revolving cells can be thrown into the centre of the condenser, and there stopped by means of a spring catch; when so arranged the mica film, &c., may be revolved in its place by turning the cell D, as both cells are geared together with fine racked teeth. F is a polarising prism mounted on an eccentric arm, rendered central when in use, or thrown out, as seen, when out of use. G is the rack dove-tail slide for indicating and focussing the condenser on the object. The advantages associated with this condenser consist in having the polarising prism, selenite films, dark-ground, and oblique light stops, so that they may be brought close under the optical combination.

Baker’s Nelson Condenser, shown in Fig. 140, is intended for use with their medium instruments. It has, however, many pieces of apparatus essential to those of a higher class. It is applicable, indeed, to all instruments having sufficient depth beneath the stage to receive it. It comprises an achromatic combination of 90° aperture, available with all powers up to 1/8-inch tinted glass for neutralising the yellow rays of artificial light, focussing adjustment, dark-ground illuminator, large diaphragm with rotating tube to carry oblique light stops, small wheel of apertures, polarising prism with two selenite films, clear aperture, and oblique light-shutter for low powers.

Baker’s Students’ Condenser (Fig. 141) is designed to take the place of Abbe’s, and costs much less. It transmits a larger aplanatic cone of light, and can be used either with high or low powers by removing the front lens. It is equally useful for photo-micrographic work.

Mr. J. Mayall’s semi-cylinder or prism for oblique illumination (Fig. 142) is a convenient form, as it permits of the semi-cylinder being tilted and placed excentrically; in this manner, without immersion contact, and by suitable adjustment, a dry object can be viewed with any colour of monochromatic light. If placed in immersion contact with the slide, the utmost obliquity of incident light can be obtained. Objects in fluid may be placed on the plane-surface of the semi-cylinder, and illuminated by ordinary transmitted light, or rendered “self-luminous” in a dark field, as with the hemispherical illuminator or Wenham’s immersion paraboloid. A concave mirror with a double arm is quite sufficient to direct the illuminating pencil. This semi-cylinder was originally made by Tolles, of Boston, for measuring apertures, but, at Mr. Mayall’s suggestion, Messrs. Ross mounted it as an illuminator.

The spiral slot should be fixed close beneath the larger lens of the condenser, and when properly arranged will be found a convenient mode of obtaining oblique light.

_The Webster-Collins Universal Condenser_ (Fig. 143) is so well known that it scarcely calls for any lengthy description. It is an inexpensive form of condenser, designed in the first instance for use with the students’ microscope. It is fitted into the sub-stage; has an iris diaphragm as well as a series of revolving diaphragms moved by a milled head screw arrangement.

Oblique Illumination.

_Wenham’s Parabolic Condenser._--Mr. Wenham’s many useful additions to the microscope and its accessories demand especial notice. When mention is made of the various immersion condensers (illuminators, as he preferred to call them), his original right-angled prism, his truncated hemispherical lens, his immersion paraboloid, and his reflex illuminator, in which rays beyond the angle of total reflexion are utilised by reflex action from cover-glass on to the surface of the object, every one of these well-devised inventions will always be spoken of in terms of praise. All in their turn conferred a great service upon the microscope, and enabled the student to clear up difficulties that stood in the way of developing structure when achromatic lenses and dry-objectives were considered perfect. The superior illumination of the object was wholly due to, and effected by, _reflected_ rays from the object to the aperture of the objective, and obviously, reflex action could only take place with dry-objectives. This reflex action must be regarded as Mr. Wenham’s special discovery. It must be observed, however, that it is not the same as the more modern achromatic appliances used for throwing _direct_ rays upon the object, and which proved the existence of apertures capable of direct transmission up to 27° measured in the body of the front lens.

The most practical of Mr. Wenham’s inventions is probably the hemispherical lens, since adopted by Messrs. Ross in connection with their excellent Zentmayer stand, and which has proved eminently serviceable. But the fact is that devices of the kind for obtaining direct oblique light require a thin stage, and therefore most of those who possess the earlier-made microscope stand would doubtless hail the appearance of any appliance which will convert axial light into oblique light; as by so doing the possessors of such instruments, in which the stage is generally of considerable thickness, would enjoy the pleasure of seeing the best resolution it is possible to get with their dry-objectives.[27]

_Wenham’s Parabolic Reflector._--This will be better understood by reference to Fig. 145, which represents it in section A B C, and shows that the rays of light _r r′ r′′_, entering perpendicularly at its surface C, and then reflected by its parabolic surface A B to a focus at F, can form no part of the largest pencil of light admitted by the object-glass and represented by G F H; but an object placed at F will interrupt the rays and be strongly illuminated. A stop at S prevents any light from passing through direct from the mirror.

In the microscope the _parabolic reflector_ fits into the cylindrical fitting under the stage, and the adjustment of its focus upon the object is made by giving it a spiral motion when fitted in--that is, carefully pushing it up or down at the same time that it is turned round by the milled edge B B. It must then be focussed by the rack and pinion motion. As the rays of light must be parallel when they enter it, a _flat mirror_, which in this case should be added to the instrument, is generally used; daylight will then require only direct reflection, but the rays from an artificial source will have to be made parallel by placing a side condenser between the light and the mirror, about 1-3/4 inch from the former and 4-1/2 inches from the latter. Nearly the whole surface of the mirror should be equally illuminated; this may be tested by temporarily placing upon it a card or piece of white paper. Parallel rays can also be obtained from the concave mirror, if the light is placed about 2-1/2 inches from it. Dark-ground illumination is not suitable for very transparent objects--that is, unless there is a considerable difference in their index of refraction, or they are pervaded by air-cells.

One very remarkable example of this may be seen in the tracheal system of insects. If any of the transparent larvæ of the various kinds of gnat be mounted in gelatine and glycerine jelly, slightly warmed but not enough to kill the insect outright, about the third day the fluids circulating in the body will be absorbed and replaced by air. Illuminated by the parabolic condenser, and viewed with a binocular microscope, and a low power, the gnat-larva becomes a superb object. The body of the insect is but faintly visible, and in its place is displayed a marvellous tracheal skeleton, with the tubes standing out in perspective, shining brilliantly, like a structure of burnished silver. Unfortunately, such objects are not permanent, for when the whole of the water dries up, the tracheal tubes either collapse or become refilled with fluid.

As to the blackness of field, and luminosity of the object, this depends upon excess of light from the paraboloid received beyond the angle of aperture of the object-glass. It is found in practice that more and more of the inner annulus of rays from the paraboloid has to be stopped off, until at last, with high-angled objectives, it is scarcely possible to obtain a black field.

The light, on the whole, most suitable for this method of illumination is lamp, the rays of which should in all cases be rendered more parallel by means of a large plano-convex lens, or condenser.

_Wenham’s Immersion Condenser._--Mr. Wenham, in the year 1856, described various forms of oblique illuminators, one of which was an immersion; a simple right-angled prism, connected by a fluid medium of oil of cloves. This, however, was abandoned for a nearly hemispherical lens connected with the slide, and although an improvement, did not touch the point of excellence Mr. Wenham was looking for. Ultimately he adopted a semi-circular disc of glass of the exact form and size represented in the drawing, Fig. 146, having a quarter-inch radius, with a well-polished rounded edge, the sides being grasped by a simple kind of open clip attached to the sub-stage, the fluid medium used for connecting the upper surface with the slide being either water, glycerine, or oil; an increase of oblique illumination being obtained by swinging the ordinary mirror sideways. By means of an illuminator of the kind difficult objects mounted in balsam are resolved. This simple piece of glass collects and concentrates light in a marvellous manner, and is by no means a bad substitute for some of the more costly forms of achromatic condenser. It can be used either in fluid contact with the slide, or dry, as an ordinary condenser.

Mr. Wenham subsequently contrived a small truncated glass paraboloid, for use in fluid contact with the slide; water, glycerine, oil, or other substance being employed as a contact medium. The rays of light in this illuminator, being internally reflected from a convex surface of glass, impinge obliquely on the under surface of the slide, and are transmitted by the fluid uniting medium, and internally reflected from the upper surface of the cover-glass to the objective. To use the reflex illuminator efficiently it must be racked up to a level with the stage. The centre of rotation is then set true by a dot on the fitting, seen with a low power, a drop of water is then placed on the top, and upon this the slide is laid. Minute objects _on the slide_ must be found either by the aid of a low power, by their greater brilliancy, or by rotating the illuminator; the effect on the podura scale is superb, the whole scale appearing dotted with bright blue spots in a zig-zag direction. Objects for this illuminator should be especially selected and mounted.

The Amici Prism, originally designed for oblique illumination, consists of a flattened triangular glass prism, the two narrower sides of which are slightly convex, while the third or broadest side forms the reflecting surface. When properly used, it is capable of transmitting a very oblique pencil of light. The prism is either mounted, as in Fig. 147, for slipping into the fitting of the sub-stage, or on an independent stand, as arranged for Powell’s microscope, page 85, Fig. 56.

Method of Employing the Achromatic Condenser to the Greatest Advantage.

_Its Illumination._--Good daylight is the best for general work. The microscope should be placed near a window with a northern aspect. Direct sunlight should never be utilised; the best light is that reflected from a white cloud. A good paraffin lamp is the most serviceable artificial source of light, and it is quite under control. As an illuminant more often brought into requisition in the smoky atmosphere of towns, the paraffin lamp is on the whole the handiest and the most useful. If gas-light can be brought into use as suggested for micro-photography, with the incandescent mantle, it will be found to be the purest and best form of artificial illumination for the microscope. Among paraffin lamps those constructed by Baker and Swift are all that can be desired.[28]

As the chimneys of these lamps are made of metal, and blackened, no reflected light disturbs the eye. Care must be taken to have the wick evenly trimmed; the metal chimney has a glazed front, giving exit to the rays of light, the flat of the flame being used with low powers, and the image of the flame being reflected by a plane mirror to give equal illumination of the whole field. In working with high powers, the lamp is turned with the flame edge-wise, and at the same time the mirror must be dispensed with. By working, as it is termed, directly on the edge of the flame, the illumination is greatly increased, and a band of light can be concentrated on any part of the preparation it is desired to make a careful study of.

To obtain the best results, time and care must be given to the illumination of the object. The lamp and microscope having been placed in position, a low power is first used and the smallest diaphragm. On looking through the microscope it will probably be observed that the image of the diaphragm is not in the centre of the field; by moving the centring screw of the condenser this may be adjusted. The low power is then replaced by a high power, the largest diaphragm used, and the bacteria or diatom brought into focus. The diaphragm must now be replaced by one of medium size, and by racking the condenser up and down, a point will be arrived at when the image of the edge of the flame appears as an intensely bright band of light. If this is not exactly in the centre of the field the centring screw of the condenser must again be adjusted. With regard to the use of diaphragms, various sizes should be tried while focussing with the fine adjustment, at the same time using the correction colour; in this way we obtain the sharpest possible image. When the condenser has been accurately centred, it will still be necessary to focus it for each individual specimen, so as to correct for difference in the thickness of slides and the layers of mounting medium. Correction for different thickness of cover-glasses must be made by the aid of the collar adjustment in the following way: a high-power eye-piece is substituted for the ordinary eye-piece, and the faults in the image will thereby be intensified. By moving the collar completely round, first in one direction and then in the other, while carefully observing the effect of the image, it will be seen to become obviously worse whichever way the collar is turned. The collar must then be turned through gradually diminishing distances until an intermediate point is reached at which the best image results with the high-power eye-piece, and on replacing this by the low-power eye-piece the sharpest possible image will be obtained.

_Effect of the Sub-stage Condenser._--The sub-stage condenser gives the most powerful illumination when it has been racked up until it almost touches the specimen. It produces a cone of rays of very short focus, and the apex of the cone should correspond with the particular bacterium or group of bacterias under observation. The effect of the condenser without a diaphragm is to obliterate what Koch has termed a _structure picture_. If the component parts of a tissue section were colourless and of the same refractive power as the medium in which the section is mounted, nothing would be visible under the microscope. As, however, the cells and their nuclei and the tissues do not differ in this respect, the rays which pass through them are diffracted, and an image of lines and shadows is developed. If in such a tissue there were minute coloured objects, and if it were possible to mount the tissue in a medium of exactly the same refractive power, the tissue being then invisible, the detection of the coloured objects would be much facilitated. This is exactly what is required in dealing with bacteria which has been stained with aniline dyes, and the desired result can be obtained by the use of the sub-stage condenser.

If we use the full aperture of the condenser the greatly converged rays play on the component parts of the tissue, light enters from all sides, the shadows disappear, and the structure picture is lost. If now a diaphragm is inserted, so that we are practically only dealing with parallel rays, the structure picture reappears. As the diaphragm is gradually increased in size the structure picture gradually becomes less and less distinct, while the colour picture, the image of the stained bacteria, becomes more and more intense. When, therefore, bacteria in the living condition and unstained tissues are examined, a diaphragm must be used, and when the attention is to be concentrated upon the stained bacteria in a section or in a cover-glass preparation the diaphragm must be removed and the field flooded with light--(Crookshank).

The wide-angle condenser, it will be understood, consists of a combination of lenses, which concentrate all the light entering them to a small point, and the condenser must be so accurately focussed that this brilliant cone of light, when it emerges from the upper lens of the condenser, falls upon the object from all directions, forming a wide-angle cone of light, at the apex of which the object must be placed (see Fig. 149). That is to say, the object is illuminated by a cone of rays passing through it in all directions.

There are, however, objects which require a fully illuminated field, when the lamp should be turned round and the Herschel lens condenser (shown in section, Fig. 148) should be used to collect the light and throw it upon the mirror. For moderate powers, as a four-tenth or one-fifth, the condenser should be used a little below the focus to give an even illumination over the whole field. Moreover, as to the use of the condenser for defining general objects, it must be borne in mind that to show different kinds of structure different apertures in the iris diaphragm are necessary, and that whereas some objects show their structure better with a large angle of light cut down in intensity by the use of blue glass, others show better with a small pencil of direct rays. For the resolution of diatoms it is often necessary to use oblique light only, and for this purpose diaphragms with central patches are used, the iris diaphragm being opened to its full extent. An annular ring of oblique light emerges from the condenser upon the object, and it is in this manner also that dark-ground illumination is obtained with moderate and low powers.

THE DIAPHRAGM.

The early form of diaphragm in use was that shown in Fig. 150.

It consists simply of a circular brass plate with a series of circular openings of different sizes, arranged to revolve upon another plate by a central pin or axis, the last being also provided with an opening as large as the largest in the diaphragm-plate, and corresponding in situation to the axis of the microscope body. The holes in the diaphragm-plate are centred and retained in place by a bent spring in the second plate, which rubs against the edge of the diaphragm-plate and catches in a notch. The blank space shuts off the light from the mirror when condensed light is about to be used. It is usually made to fit in under the stage of the microscope. This has been almost superseded by the iris diaphragm, originally designed by Wales, of America. It was made by this optician for his working students’ microscope. An early form of the iris diaphragm is seen in Fig. 151. By pressing upon the lever handle at the side the aperture gradually closes up, and without for a moment losing sight of the object under examination.

The Mirror.

The mode in which an object is illuminated is, in the words of the late Andrew Ross, “second only in importance to the excellence of the glass through which it is seen.” To ensure good illumination the mirror should be in direct co-ordination with the objective and eye-piece; it must be regarded as a part of the same system, and tending by a combined series of acts to a perfect result. Illumination of the object is recognised as of three kinds or qualities--reflected, transmitted, and refracted light. For the illumination of transparent objects, transmitted light is brought into use; for opaque objects, reflected light is needed.

The mirror should be about 2-1/2 or 3 inches in diameter, and it must not be fixed, but made to slide up and down the stem under the stage, so that the rays of light emanating from it may be brought to a focus. The utility of the mirror is so obvious that it is occasionally passed over in silence by writers. To myself it appears to be an important accessory of the microscope, and I shall therefore proceed to combine theory with practice in what I have to say with regard to the mirror.

The microscope mirror should be the segment of a true sphere, and its centre that of a true curvature. If the mirror has a true circular boundary, the central point on line A (Fig. 152) of the reflecting surface, is the pole of the same. The line A C is known as its principal axis, and any other straight line through C, which meets the mirror, is its secondary axis. When the incident axis is perfectly parallel to the principal axis, the reflected rays converge to a point F, its principal focus. So much for the theory of the mirror. Now we come to its practical use.

Simple as the mirror of the microscope may appear to be, if the curve of the surface is not perfect, it will yield a secondary reflection or double pencil of rays. The plane mirror will occasionally be found to emit more than one reflection of the lamp-flame; this we find may be corrected by rotating the mirror in its cell. Many years ago I proposed to meet a difficulty of the kind by arranging a rectangular prism on a separate stand, shown in Fig. 153, consisting of a prism A B, mounted in gimbal C, D, and E, secured to a brass tube G, fitted to the stem, and thus made to take the place of the mirror.

The direct method of employing the mirror, that more generally resorted to, is by reflecting rays from the concave surface; the plane surface is preferred when the condenser is used. Whichever is employed, it should not be forgotten that the _optic axis_ must be preserved throughout, and so brought to the centre of the open tube of the microscope. Another method is to interpose a bull’s-eye lens, and in this way supply the mirror with a beam of parallel rays of light. The plane side of the bull’s-eye lens should be turned towards the lamp, so that lamp, bull’s-eye, sub-stage condenser, and objective, are brought into an exact line, the bull’s-eye being set at right-angles to the line. A piece of thin white paper held across the bottom of the sub-stage will serve to show whether the rays of light are fairly parallel. The next care is to focus the object on the stage, and then the sub-stage condenser on the slide; further correction should be made by means of the centring screws of the sub-stage, or by moving the bull’s-eye lens or lamp slightly, thus perfecting the arrangements for working with parallel rays of light.

Accessories of the Microscope.

The accessories and appliances of the microscope have become so very numerous, that any attempt to describe them and explain the uses to which they are put would demand more space than I find myself in a position to bestow upon them. I must therefore confine my remarks to those accessories in more general use.

Having described the method of employing transmitted light, I have a few words to add with regard to the illumination of opaque objects by reflected light. A very early and efficient form of opaque illumination is the well-known _Lieberkühn_. This has not been entirely surpassed by more recent inventions. The concave speculum termed a Lieberkühn, so named after its celebrated inventor, directly reflects down upon the object the light received either from the mirror or bull’s-eye lens. It consists of a silver cap, which slides over the objective (Fig. 154), _a_ indicating the lower part of the compound body, and _b_ the objective over which slides the Lieberkühn, _c_; the rays of light are collected to a focus upon the object at _d_. The object may either be mounted on a slip of glass, or held by the stage-forceps, _f_; if very small, or transparent, it may be gummed to the dark well, _e_, or mounted on a Beck’s opaque disc-revolver.

This holder will be found useful for the examination of opaque or other objects that cannot be conveniently held by the stage forceps, the specimen being temporarily attached to it by gum or gold size. The holder is intended to rotate, so that every portion of the object can be brought into view. In this way it will be found useful in the study of insects, foraminifera, &c.

With the Lieberkühn, however, the illumination of opaque objects must be more or less one-sided, and therefore, the silver side-reflector has superseded it for general use (Fig. 157). To ensure a more perfect illumination of the object, the bull’s-eye lens should also be used. Mr. Sorby devised a reflector to fit over the objective. It consists of a semi-circular cap; is, in short, a modification of the parabolic reflector. The light from the mirror can, by slightly varying its inclination, be brought into use with this reflector.

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.

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.

For the purpose, screw on a low-power objective to the end of the draw-tube (Fig. 184).[31] As the light requires to be passed through the crystals at a considerable angle, a wide-angled condenser should be employed, but it need not be achromatic. The objective most suitable is a 4/10-inch, of ·64 numerical aperture, but a 1/4-inch of ·71 numerical aperture, or a 1/3-inch of ·65 numerical aperture, will answer the purpose equally well. As the whole of the back lens of the objective should be visible through the analysing Nicol prism, the back lens of the objective must not be too large; thus a 1/2-inch of ·65 numerical aperture will not be so effective. The analysing prism may be placed either where it is in the drawing, below the stage, or above the eye-piece. It works equally well above the objective, the position it ordinarily occupies in the microscope.

For the draw-tube a 2-inch objective and a B Huyghenian eye-piece answers very well. Before screwing the objective on to the end of the draw-tube centre the light in the usual manner, the Nicol’s being turned so as to give a light field, then screw the objective on to the end of the aperture, and put the crystal on the stage, rack down the body so that the objective on the nose-piece nearly touches the crystal, then focus with the draw-tube only. The sub-stage condenser should be racked up close to the underside of the crystal.

Opticians, however, have more recently furnished a special form of microscope (_The Petrological Microscope_, Fig. 79, p. 112), for the use of those students who may desire to prosecute so fascinating a study, and determine the optic axial angles of crystals.

Fuess[32] lately introduced a new form of microscope for polarising and viewing biaxial crystals, which he believes to be needed, as in the ordinary microscope the opening of the polariser is scarcely a third of that of the condenser; moreover, it is not absolutely necessary that the polariser and analyser should be Nicol’s prisms. This fact was discovered by myself many years ago. Fuess utilises a bundle of thin glass plates, as in the older Nuremberg polariscope. The frame holding plates can be readily adjusted at the proper polarising angle, the analyser being the ordinary small Nicol, screwed above the objective. The illuminator is an Abbe’s triple condenser, of numerical aperture 1·40, which can be adjusted in the ordinary way. The front lens of this should have a diameter of 11·12 mm. and the lower lens of 30 mm. This increase in the condenser fully compensates for the loss of light by the bundle of glass plates, and also enables thick sections of crystals to be examined in convergent polarised light. The ocular used should have a large field; the A Huyghenian answers best. A suggestion to return to the original Nuremberg polariser is very opportune, as _Iceland spar is becoming scarce_.

Mr. A. Mickel accidentally discovered that an opalescent mirror can be converted into an excellent and inexpensive substitute for the Nicol-prism polariser.

Rotation of Plane of Polarisation.

When a plate of quartz (rock-crystal), even of considerable thickness, cut perpendicular to the axis, is interposed between the polariser and analyser, colour is exhibited, the tints changing as the analyser is rotated; and similar effects of colour are produced by employing, instead of quartz, a solution of sugar enclosed in a tube with plain glass ends.

The action thus exerted by quartz and sugar is called _rotation of the plane of polarisation_, a name which sufficiently expresses the observed phenomena. In the case of ordinary quartz, and solutions of sugar-candy, it is necessary to rotate the analyser in the direction of watch-hands as seen by the observer, and the rotation of the plane of polarisation is said to be _right-handed_. In the case of what is called _left-handed_ quartz, and of solutions of non-crystallisable sugar, the rotation of the plane of polarisation is in the opposite direction, and the observer must rotate the analyser against watch-hands.

_Quartz_ belongs to the uniaxial system of crystals, and accordingly exhibits one series of rings only, and no perfect central black cross.

On revolving the tourmaline the colour gradually changes, and passes through all the colours of the spectrum. It can be cut to exhibit either right-handed polarisation or left-handed polarisation and also to exhibit straight lines.

_Calc Spar._--A uniaxial crystal showing only one system of rings, and a black cross, changing into a white cross on revolving the tourmaline.

_Topaz._--A biaxial crystal exhibiting only one system of rings with one fringe, owing to the wide separation of the axes. The fringe and colours change on revolving the tourmaline.

_Borax._--A biaxial crystal; the colours are seen to be more intense than in topaz, but the rings not so complete--only one set of rings can be seen, owing to their wide separation.

_Rochelle Salt._--A biaxial crystal; the colours are more widely spread out than the former, and only one set of rings seen at the same time.

_Carbonate of Lead._--A biaxial crystal; axes not so far separated, and both systems of rings are more widely spread than those of potassium nitrate.

_Aragonite._--A biaxial crystal; axes widely separated, but both systems of rings seen at the same time. A fine crystal for displaying the biaxial system.

It was long believed that all crystals had only one axis of double refraction; but Brewster found that the greater number of crystals which occur in the mineral kingdom have _two axes_ of double refraction, or rather axes around which double refraction takes place; in the axes themselves there is no double refraction.

Potassium nitrate crystallises in six-sided prisms with angles of about 120°. It has two axes of double refraction. These axes are each inclined about 2-1/2° to the axes of the prism, and 5° to each other. If, therefore, a small piece be split off a prism of potassium nitrate with a knife driven by a sharp blow of a hammer, and the two surfaces polished perpendicular to the axes of the prism, so as to leave the thickness of the sixth or eighth of an inch, and then a ray of polarised light be transmitted along the axes of the prism, the double system of rings will be clearly visible.

When the line connecting the two axes of the crystal is inclined 45° to the plane of primitive polarisation, a cross is seen on revolving the potassium nitrate; it gradually assumes the form of two hyperbolic curves, as in Fig. 185. But if the tourmaline be again revolved through half a quadrant, the black cross will be replaced by white spaces, as in the second figure. These systems of rings have, generally speaking, the same colours as those of thin plates, or as those of a system of rings revolving around one axis. The orders of the colours commence at the centres of each system; but at a certain distance, which corresponds to the sixth ring, the rings, instead of returning and encircling each pole, encircle the two poles as an ellipse does its two foci. If the thickness of the plate of _nitre_ be diminished or increased, the rings are diminished or increased according to the thickness of the crystal.

Small specimens of various salts may be crystallised and mounted in Canada balsam for viewing under the stage of the microscope; by arresting crystallisation at certain stages, a greater variety of forms and colours will be obtained: we may enumerate salicine, asparagine, acetate of copper, phospho-borate of soda, sugar, carbonate of lime, potassium chlorate, oxalic acid, and all the oxalates found in urine, with the other salts from the same fluid, a few of which are shown in Plate VIII.

The late Dr. Herapath described a salt of quinine, remarkable for its polarising properties. The crystals of this salt, when examined by reflected light, have a brilliant emerald-green colour, with almost a metallic lustre; they appear like portions of the elytræ of the cantharides beetle, and are also very similar to murexide in appearance. When examined by transmitted light, they scarcely possess any colour, there is only a slightly olive-green tinge; but if two crystals, crossing at right-angles, be examined, the spot where they intersect appears perfectly black, even if the crystals are not more than one five-hundredth of an inch in thickness. If the light be in the slightest degree polarised--as by reflection from a cloud, or by the blue sky, or from the glass surface of the mirror of the microscope placed at the polarising angle 65° 45′--these little prisms and films assume complementary colours: one appears green, and the other pink, and the part at which they cross is chocolate or deep chestnut-brown, instead of black. Dr. Herapath succeeded in making artificial tourmalines large enough to surmount the eye-piece of the microscope; so that all experiments with those crystals upon polarised light may be made without the tourmaline or Nicol’s prism. The finest rosette crystals are made as follows:--To a moderately strong solution of _Cinchonidine_ add a drop or two of Herapath’s test-fluid.[33] A few drops of this is placed on the centre of a glass slide, and put aside until the first crystals are observed to be formed near the margin. The slide should now be placed upon the stage of the microscope, and the progress of formation of the crystals closely watched. When these are seen to be large enough, and it is deemed necessary to stop their further development, the slide must be quickly transferred to the palm of the hand, the warmth of which will be found sufficient to stop further crystallisation. These crystals attract moisture, deliquesce, and should therefore be kept in a perfectly dry place.

To render these crystals evident, it merely remains to bring the glass-slide upon the field of the microscope, with the selenite stage and single tourmaline, or Nicol’s prism, beneath it; instantly the crystals assume the two complementary colours of the stage: red and green, supposing that the pink stage is employed; or blue and yellow, provided the blue selenite is made use of. All those crystals at right angles to the plane of the tourmaline produce that tint which an analysing-plate of tourmaline would produce when at right angles to the polarising-plate; whilst those at 90° to these educe the complementary tint, as the analysing-plate would also have done if revolved through an arc of 90°.

This test is a delicate one for quinine (Fig. 186, _a_ and _b_); not only do these peculiar crystals act in the way just related, but they may be easily proved to possess the optical properties of that remarkable salt, the sulphate of iodo-quinine.

To test for quinidine, it is merely necessary to allow a drop of acid solution to evaporate to dryness upon the slide, and to examine the crystalline mass by two tourmalines, crossed at right angles, and without the stage. Immediately little circular discs of white, with a well-defined black cross, start into existence, should quinidine be present even in very minute traces. These crystals are represented in Fig. 187.

If the selenite stage be employed in the examination of this object, one of the most gorgeous appearances in the whole domain of the polarising microscope is displayed: the black cross disappears, and is replaced by one consisting of two colours, and divided into a cross having a red and green fringe, whilst the four intermediate sectors are a gorgeous orange-yellow. These appearances alter on the revolution of the analysing-plate of tourmaline; when the blue stage is employed, the cross assumes a blue or yellow tint, varying according to the position of the analysing plate. These phenomena are analogous to those exhibited by certain circular crystals of boracic acid, and to circular discs of salicine (prepared by fusion), the difference being that the salts of quinidine have more intense depolarising powers than either of the other substances; the mode of preparation, however, excludes these from consideration. Quinine prepared in the same manner as quinidine has a very different mode of crystallisation; but it occasionally presents circular corneous plates, also exhibiting the black cross and white sectors, but not with one-tenth part of the brilliancy, which of course enables us readily to discriminate the two.

Urinary salts are more readily seen under polarised light than by white light. Ice doubly refracts, while water singly refracts. Ice takes the rhomboidic form; and snow in its crystalline forms may be regarded as the skeleton crystals of this system (Fig. 189). A sheet of clear ice, of about one inch thick, and slowly formed in still weather, shows circular rings with a cross by polarised light.

It is probable that the conditions of snow formation are more complex than might be imagined, familiar as we are with the conditions relating to the crystallisation of water on the earth’s surface. A great variety of animal, vegetable, and other substances possess a doubly refracting or depolarising structure, as: a quill cut and laid out flat on glass; the cornea of a sheep’s eye; skin, hair, a thin section of a finger-nail; sections of bone, teeth, horn, silk, cotton, whalebone; stems of plants containing silica or flint; barley, wheat, &c. The larger-grained starches form splendid objects; _tous-les-mois_, the largest, may be taken as a type of all others. This presents a black cross, the arms of which meet at the hilum (Fig. 190). On rotating the analyser, the black cross disappears, and at 90° is replaced by a white cross; another, but much fainter, black cross is seen between the arms of the white cross, no colour being perceptible. But if a thin plate of selenite be interposed between the starch-grains and the polariser, a series of delicate colours appear, all of which change on revolving the analyser, becoming complementary at every quadrant of the circle. West and East India arrow-root, sago, tapioca, and many other starch-grains, present a similar appearance; but in proportion as the grains are smaller, so are their markings and colourings less distinct.

Molecular Rotation.

For the purpose of studying the various interesting phenomena of molecular rotation, a few necessary pieces of apparatus must be added to the microscope. First, an ordinary iron three-armed retort stand, to the lower arm of which must be attached either a polarising prism or a bundle of glass plates inclined at the polarising angle; in the upper an analysing prism. The fluid to be examined should be contained in a narrow glass tube about eight inches in height, and this must be attached to the middle arm. If the prisms be crossed before inserting a fluid possessing rotatory power, the light passing through the analyser will be coloured. If a solution of sugar be employed, and the light which passes through the second prism is seen to be red, but on rotating the analyser towards the right the colour changes to yellow, and passes through green to violet, it may be concluded that the rotation is right-handed. If, on the contrary, the analyser requires to be turned towards the left hand, we conclude that the polarisation is left-handed. These phenomena are wholly distinct from those accompanying the action of doubly refracting substances upon plane polarised light. It is not easy to explain in a limited space the course to be followed in ascertaining the amount of rotation produced by different substances. Monochromatic light should be used. If we are about to examine a sugar solution with the prisms crossed, the index attached to the analyser must first be made to point to zero. The sugar is then introduced, when it will be necessary to rotate the analyser 23° to the right, in order that the light may be extinguished. This is the amount of rotation for that particular fluid at a given density and that height of column. As the arc varies with increase or decrease of density and height of the fluid, it is needful to reduce it to a unit of height and density. The following formula is that given by Biot:--P = quantity of matter in a unit of solution; _d_ = sp. gr.; _l_ = length of column; _a_ = arc of rotation; _m_ = molecular rotation.

Then _m_ = _a_/(_l p d_).

The application of the polarising apparatus to the microscope is of much value in determining minute structure. It may also be defined as an instrument of analysis; a test of difference in density between any two or more parts of the same substance. All structures, therefore, belonging either to the animal, vegetable, or mineral kingdom, in which the power of unequal or double refraction is suspected to be present, are those that should especially be re-investigated by polarised light. Some of the most delicate of the elementary tissues of animal structure, the ultimate fibrillæ of muscles, &c., are amongst the most interesting subjects that might be studied with advantage under this method of investigation. The chemist may perform the most dexterous analysis; the crystallographer may examine crystals by the nicest determination of their forms and cleavage; the anatomist or botanist may use the dissecting knife and microscope with the most exquisite skill; but there are still structures in the mineral, vegetable, and animal kingdoms which will defy all such modes of examination, and will yield only to the magical analysis of polarised light.

Formation and Polarisation of Crystals.

The inorganic kingdom will afford to the microscopist a never-ending number of objects of unsurpassed beauty and interest. The phenomena of crystallisation in its varied combinations can be made a useful and instructive occupation. Although ignorant of the means whereby the great majority of minerals and crystals have been formed in the vast laboratory of Nature, we can, nevertheless, imitate in a small degree Nature’s handiworks by crystallising out a large number of substances, and watch their numerous transformations in the smallest appreciable quantities, when aided by the microscope.

Among natural crystals we look for the material for the formation of our lenses, while the varieties of granites present us with the earliest crystallised condition of the earth’s crust as it cooled down, the structure of which is beautifully exhibited under polarised light. In Plate VIII. various crystalline and other bodies are displayed. In No. 158 is a section of new red sandstone; 159 of quartz; and 160 of granite. Special reference is made to others in the following list of salts and other substances which form a beautiful series of objects for study under polarised light:--

SALTS.

Alum. Asparagine. Aspartic Acid. Plate VIII. No. 168. Bitartrate of Ammonia. Boracic Acid. Borax. No. 164. Carbonate of Lime. " Soda. Chlorate of Potash. Chloride of Barium. " Cobalt. " Copper and Ammonia. " Sodium. Cholesterine. Chromate of Potash. Cinchonine. Cinchonidine. Citric Acid. Hippuric Acid. Iodide of Mercury. " Potassium. " Quinine. Iodo-disulphate of Quinine. Kreatine. No. 166. Murexide. Nitrate of Bismuth. " Barytes. " Brucine. " Copper. " Potash. " Strontian. " Uranium. Oxalate of Ammonia. " Chromium. " Chromium and Potash. " Lime. " Soda. Indurated Sandstone, Howth. Indurated Sandstone, Bromsgrove. Gibraltar Rock. Granite, various localities. No. 160. Hornblend Schist. Labrador Spar. Norway Rock. Quartz Rock, various. No. 159. " in Bog Iron Ore. Quartzite, Mont Blanc. Sandstone. No. 158. Satin Spar. Selenites, various colours. Tin Ore, with Tourmalin. Oxalic Acid. Oxalurate of Ammonia. Permanganate of Potash. Phosphate of Lead and Soda. Platino-cyanide of Magnesia. Plumose Quinidine. Prussiate of Potash, red and yellow. Quinidine. Santonine. Salicine. Salignine. No. 162. Sulphate of Cadmium. " Copper. No. 161. " Copper and Potash. " Iron. No. 163. " Iron and Cobalt. No. 165. " Magnesia. " Nickel and Potash. " Soda. " Zinc. Sugar. Tartaric Acid. Thionurate of Ammonia. Triple Phosphate. Urate of Ammonia. " Soda. Urea, and most urinary deposits. Uric Acid.

MINERALS.

Agates, various. Asbestiform Serpentine. Avanturine. Carbonate of Lime. Carrara Marble.

ANIMAL STRUCTURES.

Cat’s Tongue. No. 174. Grayling Scale. No. 176. Holothuria, Spicules of. Nos. 171-2. Prawn Shell. No. 175.

VEGETABLE CRYSTALLINE SUBSTANCES.

CUTICLE of Leaf of Correa Cardinalis. " " Deutzia scabra. No. 173. " " Elæagnus. " " Onosma taurica. Equisetum. No. 170. Fibro cells from orchid. No. 169. " Oncidium bicallosum. Scalariform Vessels from Fern. Scyllium Caniculum. No. 177. SILICIOUS CUTICLES, various. Starches, various. No. 167.

The formation of artificial crystal may be readily effected, and the process watched, under the microscope, by simply placing a drop of saturated solution of any salt upon a previously warmed slip of glass.

Interesting results will be obtained by combining two or more chemical salts in the following manner. To a nearly saturated solution of the sulphate of copper and sulphate of magnesia add a drop on the glass-slide, and dry quickly. To effect this, heat the slide so as to fuse the salts in its water of crystallisation, and there remains an amorphous film on the hot glass. Put the slide aside and allow it to cool slowly; it will gradually absorb a certain amount of moisture from the air, and begin to throw out crystals. If now placed under the microscope, numerous points will be seen to start out here and there. The starting points may be produced at pleasure by touching the film with a fine needle point, so as to admit of a slight amount of moisture being absorbed by the mass of salt. Development is at once suspended by applying gentle heat; cover the specimen with balsam and thin glass. The balsam should completely cover the edges of the thin glass circle, otherwise moisture will probably insinuate itself, and destroy the form of the crystals.

Mr. Thomas succeeded in crystallising “the salts of the magnetic metals” at very high temperatures, with very curious results. In Plate VIII. are seen crystals of sulphate of iron and cobalt, No. 163; and of nickel and potash, No. 165, obtained in the following manner:--Add to a concentrated solution of iron a small quantity of sugar, to prevent oxidation. Put a drop of the solution on a glass slide, and drive out the water of crystallisation as quickly as possible by the aid of a spirit lamp; then with a Bunsen’s burner bring the plate to a high temperature. Immediately a remarkable change is seen to take place in the form of the crystal, and if properly managed the “foliation” represented in the plate will be fairly exhibited. The slide must not be allowed to cool down too rapidly or the crystals will probably absorb moisture from the atmosphere, and in so doing the crystals alter their forms. Immerse them in balsam, and cover in the usual way before quite cold.

_Sublimation of Alkaloids._--The late Dr. Guy, F.R.S., directed the attention of microscopists to the fact that the crystalline shape of bodies belonging to the inorganic world might be of service in medical jurisprudence. Subsequently, Dr. A. Helwig, of Mayence, investigated this subject, and found the plan applicable not only to inorganic but also to organic substances, and especially to poisonous alkaloids. By using a white porcelain saucer Dr. Guy was able to watch the process of crystallisation more minutely, and to regulate it more exactly. He was, in fact, able to obtain characteristic crusts composed of crystals of strychnine weighing not more than 1/3000th or 1/5000th of a grain. Morphia affords equally characteristic results. For the examination of these, Dr. Guy recommended the use of a binocular microscope with an inch object-glass. But it is not to crystalline forms alone that one need trust; the whole behaviour of a substance as it melts and is converted into vapour is eminently characteristic, and when once deposited on the microscopical slide, under the object-glass, the application of re-agents may give still more satisfactory results. The re-agents, however, which are here to be applied are not of the kind ordinarily employed. Colour-tests under the microscope are, comparatively speaking, useless; those that give rise to peculiar crystalline forms are rather to be sought after. For instance, the crystals produced by the action of carbozotic acid on morphia are by themselves almost perfectly characteristic. These experiments should not, however, be undertaken for medico-legal purposes by one unskilled in their conduct, for the effects of the reagents themselves might be mistaken by the uninitiated for the result of their action on the substance under examination. For the special method of procedure, see Dr. W. Guy, “On the Sublimation of the Alkaloids.”[34]

The Micro-spectroscope.

Spectrum analysis has, from its first introduction by Kirschoff in 1859, maintained its fascination over men of science throughout the civilised world. Microscopists, astronomers, and chemists have assigned to the spectroscope a highly important position amongst scientific instruments of research. At quite an early period of its history it appeared to ourselves to promise an extension of the work of the microscope in pathology and microscopy, and second only to that of astronomy and chemistry. The chief hindrances to the use of the spectroscope were, in the early days, of a twofold nature; a widespread, but quite erroneous view of the serious difficulties of employing the instrument, and the want of a first aid to its use.

So valuable a means of research has this process of analysis proved to be, that the discoveries made by the spectroscope appear marvellous. The spectroscope was first made known as a refined instrument for the analysis of light by two Germans, a physicist and a chemist, Kirschoff and Bunsen. In 1860, the latter succeeded in detecting and separating two new alkaline bodies from all other bodies from the waters obtained from the Durkeim springs, less than 0·0002 part of a milligramme of which can be detected by spectrum analysis. It is to the labours of Huggins, Norman Lockyer and others that we are indebted for the wonderful discoveries made in astronomy; and chiefly so to Brewster, Herschel, and Talbot, for showing that certain metals give off light of a high degree of refrangibility; that distinct bands are situated at a distance beyond the last visible violet ray ten times as great as the length of the whole visible spectrum from red to violet.

With regard to the discoveries made in connection with physiological research, we are indebted to F. Hoppe, who in 1862 first described the absorption bands of human blood. His results were confirmed by the investigations of Professor Sir George Gabriel Stokes, who, by adding certain reducing agents to the blood, found that he could change scarlet blood into purple--“purple cruorine”--and in this way the place occupied by the absorption band in the spectrum could be made to change. He reduced the hæmoglobin by robbing the blood of its oxygen. Thus, by Stokes’ and other methods, we have since arrived at extremely valuable results, and the explanation of the difference in colour between arterial and venous blood; and it has also enabled us to show wherein the breathing power of the red corpuscles resides, and further explains phenomena which before his investigations were inexplicable.

The spectroscope seems likely to be of almost as great use in medicine as it has already proved to be in solar and terrestrial chemistry, if we may form an opinion from the large amount of literature which has appeared on the subject. The inception of this magical instrument arose on the instance of a discovery made by Dr. Wollaston in 1802, who, on making a slit in the shutter of his room, instead of a round hole, the spectrum of sunlight, instead of being composed of a number of coloured discs, was now a band of pure colours, each colour being free from admixture with the next to it. Moreover, he found that this colour band was not continuous, as Newton described it, but interrupted here and there by _fine black lines_.

In 1814, Fräunhofer,[35] a German optician, discovered these lines quite independently, and mapped out 576 of them, calling the more prominent of them A, B, C, D, E, F, G, H, which lines he used as marks of comparison. He also found that the distances of these lines from each other may vary according to the nature of the substance composing the prism; thus, their relative distances are not the same in prisms of flint-glass, crown-glass, and bisulphide of carbon, but they always occupy the same position relatively to the colours of the spectrum. Kirschoff and Angström had mapped out in 1880 no less a number than 2,000 Fräunhofer lines, a portion of which are correctly shown in the accompanying chart (Fig. 191).

In 1830, Simms, a London optician, made an improvement in the construction of the spectroscope by placing a lens in front of the prism, so arranged that the slit was in the focus of the lens. This lens turns the light, after it has passed through the slit, into a cylindrical beam before entering the prism. Another lens, also introduced by him, receives the circular beam emerging from the prism, and compels it to throw an image of the slit, which may be magnified at pleasure for each ray. The lens between the prism and the slit is termed the _collimating_ lens. Thus the following are the essential parts of a chemical spectroscope:--(1) a slit, the edges of which are two knife-edges of steel very truly ground, and exactly parallel to each other, and in a direction parallel to the refracting edge of the prism, to admit a pencil of rays. (2) A collimating lens; a convex lens with the slit at its principal focus, which renders the rays parallel before entering the prism. (3) A prism of dense glass, in which the rays are refracted and dispersed. (4) An observing telescope constructed like an astronomical refractor of small size, and placed so that the rays shall traverse it after emerging from the prism. Such are the essentials of a one-prism chemical spectroscope.

The form of instrument in use with the microscope is the “_direct vision_” spectroscope, consisting of two prisms of flint-glass, placed between three of crown-glass cemented together by Canada balsam; the spectrum being viewed directly by the eye. The earliest constructed form of micro-spectroscope is shown in Fig. 192, the Browning-Huggins.

It was, however, Mr. Sorby who suggested that the prism should be made of dense flint-glass and of such a form that it could be used in two different positions, and that in one it should give twice the dispersion that it would in the other, but that the angle made by the incident and emergent rays should be the same in both positions.

Figs. 193 and 193_a_ represent prisms of the kind arranged to use in two different positions, i and i′ being the same angle as I and I′.

For most absorption-bands, particularly if faint, the prism should be used in the first position, in which it gives the least dispersion; when greater dispersion is required, so as to separate some particular lines more widely, or to show the spectra of the metals, or Fräunhofer’s lines in the solar spectrum, then the prism must be used as in Fig. 193_a_. This answers well for liquids or transparent objects, but it is, of course, not applicable to opaque objects.

To combine both purposes, some form of direct vision-prisms that maybe applied to the body of the microscope is required. Fig. 194 represents an arrangement of direct vision-prisms, invented by Herschel. The line R R′ shows the path of a ray of light through the prisms, where it would be seen that the emergent ray R′ is parallel and coincident with the incident ray R.

Another very compact combination is shown in Fig. 194_a_. Any number of these prisms (P P P) may be used, according to the amount of dispersion required. They are mounted in a similar way to a Nicol’s prism, and are applied directly over the eye-piece of the microscope. The slit S S is placed in the focus of the first glass (F) if a negative, or below the second glass if a positive eye-piece be employed. One edge of the slit is movable, and, in using the instrument, the slit is first opened wide, so that a clear view of the object is obtained. The part of the object of which the spectrum is to be examined is then made to coincide with the fixed edge of the slit, and the movable edge is screwed up, until a brilliant coloured spectrum is produced. The absorption-bands will then be readily found by slightly altering the focus. This contrivance answers perfectly for opaque objects, without any preparation; and, when desirable, the same prism can be placed below the stage, and a micrometer used in the eye-piece of the microscope, thus avoiding a multiplicity of apparatus.

A later and better form of instrument is the Sorby-Browning eye-piece (Fig. 195), shown in section (Fig. 196) ready for inserting into the body-tube of the microscope, the prism of which is contained in a small tube, removable at pleasure. Below the prism is an achromatic eye-piece, having an adjustable slit between the two lenses, the upper lens being furnished with a screw motion to focus the slit. A side slit, capable of adjustment, admits, when required, a second beam of light from any object whose spectrum it is desired to compare with that of the object placed on the stage of the microscope. This second beam of light strikes against a very small prism, suitably placed inside the apparatus, and is reflected up through the compound prism, forming a spectrum in the same field with that obtained from the object on the stage.

A is a brass tube, carrying the compound direct vision prism; B, a milled head, with screw motion to adjust the focus of the achromatic eye lens C, seen in the sectional view as a triple combination of prisms. Another screw at right angles to C, which from its position cannot be well shown in the figure, regulates the slit horizontally. This screw has a larger head, and when once recognised cannot be mistaken for the other. D D is a clip and ledge for holding a small tube, so that the spectrum given by its contents may be compared with one from an object on the stage. E is a round hole for a square-headed screw, opening and shutting a slit, admitting the quantity of light required to form the second spectrum. A light entering the round hole near E strikes against the right-angled prism, which is placed inside the apparatus, and is reflected up through the slit belonging to the compound prism. If any incandescent object be placed in a suitable position with reference to the round hole, its spectrum will be obtained. F shows the position of the field lens of the eye-piece. The tube is made to fit the microscope to which the instrument is applied. To use this instrument insert F as an eye-piece in the microscope tube, taking care that the slit at the top of the eye-piece is in the same direction as the slit below the prism. Screw on to the microscope the object-glass required, and place the object whose spectrum is to be viewed on the stage. Illuminate with the stage mirror if it be transparent; with mirror, Lieberkühn, and dark well, by side reflector, or bull’s-eye condenser if opaque. Remove A, and open the slit by means of the milled-head, not shown in figure, but which is at right angles to D D. When the slit is sufficiently open the rest of the apparatus acts as an ordinary eye-piece, and any object can be focussed in the usual way. Having focussed the object, replace A, and gradually close the slit till a good spectrum is obtained. The spectrum will be much improved by throwing the object a little out of focus.

Every part of the spectrum differs a little from adjacent parts in refrangibility, and delicate bands or lines can only be brought out by accurately focussing that particular part of the spectrum. This can be done by the milled-head B. Disappointment will occur in any attempt at delicate investigation if the directions given be not carefully followed out.

Opposite E a small mirror is attached. It is like the mirror below the stage of a microscope, and is mounted in a similar manner. By means of this mirror light may be reflected into the eye-piece, and in this way two spectra may be procured from one lamp.

Method of using the Micro-Spectroscope.

A beginner with the micro-spectroscope should first make himself fully acquainted with the spectroscope by holding it up to the sky and noting the effects of opening and regulating the slit, by rotating the screw C, Figs. 195 and 197. The lines will be well seen on closing down the opening. This screw diminishes the length of the slit, when the spectrum is seen as a narrow ribbon of prismatic colours. The screw E regulates the admission of light through the aperture above D. The better objects with which to commence the study of the absorption bands are, aniline dye, much diluted, madder, permanganate of potash, and blood. As each colour varies in refrangibility, the focus must be adjusted by the screw E. When it is desired to view the spectrum of a very minute object, the prisms should be removed by withdrawing the tube containing them, the slit set open, and the object brought into the centre of the field; the vertical and horizontal slits must then be partially closed up, and the prisms replaced, when a suitable objective is employed to examine the spectrum. For ordinary observations a magnifying power of an inch and a half or two inches will be suitable, but for small quantities of material a higher power must be employed, when a single blood corpuscle can be made to show its characteristic absorption band. After having obtained the best image of any object on stage, throw it slightly out of focus, and substitute the micro-spectroscopic eye-piece for the Huyghenian. Opaque objects should be examined by reflected light, by means of the bull’s-eye condenser, or side reflector. Mr. Sorby uses a binocular microscope, which enables him to regulate the focussing and throwing out of focus of the object.

In examining crystals or other small objects, a small cardboard diaphragm should be placed beneath them; and when examining the spectra of liquids in cells, slip a small cap with a perforation of 1/10-inch in diameter over the tube containing the 1/2-inch or 2-inch objective. Substances which give absorption bands or lines in the red are best seen by artificial light, while those which show bands in the violet are better seen by daylight. By following rules of the kind we are less likely to mix the bands of the absorption spectrum with the Fräunhofer lines. For example, if the edge of a band happens to coincide with a Fräunhofer line, the observer is apt to imagine that the band is better defined and more abruptly shaded on one side than it really is.

_Cells and Tubes._--These are either supplied ready-made by the optician, or can be formed out of small pieces of barometer tubing, with the edges ground down and cemented on ordinary glass slips. In Fig. 198 is seen the several kinds of cells and tubes usually employed, while the little flat tubes commonly in use as bouquet holders will be found of use, with the side stage reflecting spectrum as comparison tubes; being of different diameters they allow of two or more depths of colour in the fluid intended for examination.

In the case of many other fluids the sloping form of cell (Fig. 198) will be useful, as different shades of fluids can be examined without removal from the stage of the microscope. The deeper cells are cut from a piece of barometer tubing of about half to an inch long, one end being cemented to a piece of flatted glass, and the other covered over temporarily or permanently with a thin piece of glass on the top, held in its place by capillary attraction, thus admitting of the tube being turned upside down.

_Re-agents required._--A diluted solution of ammonia, citric acid, double tartrate of potash and soda (the last being used to prevent the precipitation of oxide of iron), and the double sulphate of the protoxide of iron and ammonia (employed to deoxidise blood, etc.). In some special cases, dilute hydrochloric acid, purified boric acid, and sulphate of soda are required.

The character of stains of blood varies with age and with the nature of the substance with which it happens to be combined. This is important to remember in connection with _Jurisprudence_, when the micro-spectroscope is brought into use for the detection of blood stains. The spectrum used in important cases of the kind should have a compound prism, with enough, but not too great dispersive power, otherwise the bands become, as it were, diluted, and less distinct.

If the blood stain is quite recent, the colouring matter will be hæmoglobin only. This easily dissolves out in water, and when sufficiently diluted gives the spectrum of oxy-hæmoglobin, which on the addition of ammonia, together with a small quantity of the double tartrate, a small piece of ferrous salt, and stirring carefully without the admission of air, changes the spectrum of reduced hæmoglobin. When stirred again, so as to expose the solution as much as possible to air, the two bands reappear; on gradually adding citric acid in small quantities the colour begins to change, and the bands are seen to gradually fade away; if there should have been much blood present, a band appears in the red; the further addition of ammonia makes all clear again, but does not restore the original bands, because the hæmoglobin has been permanently changed into hæmatin. This reaction alone distinguishes blood from most other colouring matters, since other substances after being changed by acids are restored by alkalies to their original state. There are many other curious facts connected with the spectroscopic analysis of blood, which are fully explained and illustrated by Dr. Maemunn in his book on “The Use of the Spectroscope in Medicine,” and also in Dr. Thudicum’s[36] reports and charts, which are the most complete. Sir George Stokes, F.R.S., was one of the first to show the essential value of the spectral phenomena of hematine, and who proved, after Hoppe had first drawn attention to the fact, that this colouring matter is capable of existing in two states of oxidation, and that a very different spectrum is produced according as the substance, which he termed _cruorine_, is in a more or less oxidised condition. The chart appended to his paper[37] affords an imperfect representation of the changes seen in the spectrum.

Proto-sulphate of iron, or proto-chloride of tin, causes the reduction of the colouring-matter, but, on exposure to air, oxygen is absorbed, and the solution again exhibits the spectrum characteristic of the more oxidised state. The different substances obtained from blood colouring-matter produce different bands. Thus, _hæmatin_ gives rise to a band in the red spectrum D; _hæmato-globulin_ produces two bands, the second twice the breadth of the first in the yellow portion of the spectrum between the lines D and E, No. 1. The absorption-bands differ according to the strength of the solution employed, and the medium in which the blood-salt is dissolved; but an exceedingly minute proportion dissolved in water is sufficient to bring out very distinct bands. B represents the red end of the spectrum and G the green as it approaches the violet end.

_Mapping the Spectra._--In the sectional view given of the micro-spectroscope (Fig. 196), the internal construction of the instrument is shown, and the arrangement made for throwing a bright point on to the surface of the upper prism is clearly seen. The mapping out is accomplished by means of a photographic scale fixed as a standard spectrum (Fig. 198), in the position of A A, illuminated by the small mirror at R, and focussed by a small lens at C, so that on looking into the instrument one can see the spectrum accurately divided into one hundred equal parts, and scale readings can be made at once; the only precaution needed is to be sure the D (or the sodium line, if D cannot be got) always stands at the same number on the scale. To map absorption spectra on this scale we have to lay down a line, as many millimetres long as there are divisions in the scale, and mark the position of the bands on this line. Mr. Browning supplies scales printed off ready for use. But the mapping out of spectra, as Mr. Sorby pointed out, requires some consideration; since the number of divisions depends on the thickness of the interference-plate, it becomes necessary to decide what number should be adopted. Ten it was thought would be most suitable; but, on trial, it appeared to be too few for practical work. Twenty is too many, since it then becomes extremely difficult to count them. Twelve is as many as can well be counted; it is a number easily remembered, is sufficiently accurate, and has other practical advantages. With twelve divisions the sodium-line 0 comes very accurately at 3-1/2; thus, by adjusting the plate so that a bright sodium-light is brought into the centre of the band, when the Nicol’s prisms are also crossed accurately at 3-1/2, parallelism is secured, together with a wider field of observation. The general character of the scale will be best understood from the following figure, in which the bands are numbered, and given below the principal Fräunhofer lines. The centre of the bands is black, and they are shaded off gradually at each side, so that the shaded part is about equal to the intermediate bright spaces. Taking, then, the centres of the black bands as 1, 2, 3, &c., the centres of the spaces are 1-1/2, 2-1/2, 3-1/2, &c., the lower edges of each 3/4, 1-3/4, &c., and the upper 1-1/4, 2-1/4, &c., we can easily divide these quarters into eighths by the eye: and this is as near as is required in the subject before us, and corresponds as nearly as possible to 1/100th part of the whole spectrum, visible under ordinary circumstances by gaslight and daylight. Absorption-bands at the red end are best seen by lamp-light, and those at the blue end by daylight.

On this scale the position of some of the principal lines of the solar spectrum is about as follows:--

A 3/4 B 1-1/2 C 2-3/8 D 3-1/2 E 5-11/16 b 6-3/16 F 7-1/2 G 10-5/8

At first plates of selenite, which are easily prepared, were used, because they can be split to nearly the requisite thickness with parallel faces; but their depolarising power varied much with temperature. Even the ordinary atmospheric changes alter the position of the bands. However, quartz cut parallel to the principal axis of the crystal is but slightly affected, and is not open to the same objection; but this is prepared with some difficulty. The sides should be perfectly parallel, the thickness about ·043-inch, and gradually polished down with rouge until the sodium-line is seen in its proper place. This must be done with care, since a difference of 1/10000-inch in thickness would make it almost worthless.

The two Nicol’s prisms and the intervening plate are mounted in a tube, and attached to a piece of brass in such a manner that the centre of the aperture exactly corresponds to the centre of any of the cells used in the experiments, and must be made to correspond with equal care, so that any of them, or this apparatus in particular, may be placed on the stage and in proper position without further adjustment, whereby both time and trouble are saved.

Absorption Spectrum of Chromule.

In 1869 I published in the Journal of the Royal Microscopical Society[38] a paper on results obtained by the spectrum analysis of the colouring-matter of plants and flowers, some of which were of considerable interest in many respects. My examinations extended to several hundred different specimens, from which I was led to conclude that the chromule of flowers is, for the most part, due to the chemical action of the actinic rays of light over the protoplasm of the plant, more so than to that of soil. But as certain roots of plants, as those of the alkanet, yield their colouring-matter to oil, and in a much smaller degree to spirit or water, it follows then that conclusions of any kind can only be drawn after a long and careful study of the question. Some of the results obtained were, however, of some interest at the time, that, for example, seen in three different solutions of the chlorophyll of _Cinchona succirubra_, one of three solutions in alcohol, scarcely coloured, having in fact only a faint tinge of green colour, and the spectrum of which much astonished me at the time. It gave four well-marked absorption-bands; one deep sharp line _in the red_; another, rather narrower, in the orange, coincident with D, or the sodium-line; one in the green, about _b_, coincident with the Thallium green band; and a fourth on the blue line F, nearly as broad as that in the red. The ethereal solution gave different results. It showed only three bands of absorption, nearly the same as in the last case (though all of them fainter); but the fourth in the blue was not apparent, the whole of that end of the spectrum being absorbed a little beyond the green line _b_. This solution was _deep emerald-green_, and even dilution did not alter the phenomena. The _acid_ alcoholic solution was as deeply green as the last, but gave only the sharp broad absorption-band in the red, and two very faint ghostly bands in the position described above of the D and _b_ lines respectively.

Further additional researches on the chlorophyll of plants furnished curious results, the chlorophyll being dissolved out by alcohol, digested for some hours, and without heat; some plants being fresh, and others dried. Five classes of phenomena exhibited themselves, but _all_ agreed in having the red absorption-band broad, sharp, and well defined, some having this one band only, the Lilac being of this type.

There are two classes in which two absorption-bands occur. One has the red and the orange bands, of which the Fuchsia, Guelder-rose, and Tansy are examples; another, in which the red and the green bands are alone co-existent. Ivy is the type of the class, and it is immaterial whether we take last year’s leaves or those of the early spring; the results are the same.

The fourth class consists of the two former spectra superposed. Three lines occur, the red, the orange, and the green bands, at C, D, and _b_, as before. This is by far the largest class, and I have thirty or forty examples of it. _Œnothera biennis_, Laurestinus, &c., are types with the ethereal solution of the leaves of Red Bark.

The fifth class consists of those having properties similar to the alcoholic solution of Red Bark described. But I only found eight of these, and not all equal in colour power, namely: Berberry, Sloe, Tea, Hyoscyamus, Digitalis, Senna, and Red Bark. The results obtained appeared at the time to be well worth following up to a more practical conclusion than that arrived at. It should be noted that in the preparation of vegetable colouring matters for the micro-spectroscope, care must be taken to employ only a small quantity of spirits of wine to filter the solution, and evaporate it at once to dryness at a very gentle heat, otherwise if we attempt to keep the colouring matters in a fluid state they quickly decompose. It is necessary also to employ various re-agents in developing characteristic spectra. The most valuable re-agent is sulphite of soda. This admits of the division of colours into groups.

It is better to use a dilute alcoholic solution for the extraction of colour from plants, and to observe the spectrum in a column of about three-quarters of an inch in height. By this means it is quite possible to ascertain that the spectrum of chlorophyll presents seven distinct absorption bands.

For further information on this interesting subject I must refer the reader to Mr. Sorby’s paper “On a Definite Method of Qualitative Analysis of Vegetable and Animal Colouring Matter by means of the Spectrum Microscope,” “Proc. Roy. Soc.,” No. 92, 1867.