Chapter III (4)

_In Sub-stage_: P, polarising prism; C, sub-stage condenser on stage; M, mineral or crystal. On nose-piece: O^1, objective, 4/10-inch; A, analysing prism.

_In Draw-tube_: O^2, 2 or 3 inch Objective; H, Huyghenian eye-piece.]

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.

Fig. 186.--In this figure heraldic lines are adopted to denote colour. The dotted parts indicate _yellow_, the straight lines _red_, the horizontal lines _blue_, and the diagonal, or oblique lines, _green_. The arrows show the plane of the tourmaline, _a_, blue stage; _b_, red stage of selenite employed.]

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.

_a_, Uric acid; _b_, Oxalate of lime, octahedral crystals of; _c_, Oxalate of lime allowed to dry, forming a black cube; _d_, Oxalate of lime as it occasionally appears, termed the dumb-bell crystal.]

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.

Figs. 197 and 197_a_.--The Beck-Sorby Micro-spectroscope Eye-piece, drawn on a scale of one half size.]

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 for use with Spectroscope.

Fig. 198.]

_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.

No. 2.--Venous Blood, Purple Cruorine.

No. 3.--Blood treated with Acetic Acid.

No. 4.--Solution of Hæmatin.

Fig. 199.--Sir George Stokes’ Chart of the Absorption Bands of Blood.]

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.

0 1 2 3 4 5 6 7 8 9 10 11 12

(Red end.) (Bue end.)

A B C D E _b_ F G

Fig. 200.--
]

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.