Method of using the Micro-Spectroscope.

Fig. 191.—Fräunhofer’s Spectrum Lines.

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 byfine black lines.

In 1814, Fräunhofer,35a 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 linesfrom 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 thecollimatinglens. 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 inFig. 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.

Fig. 192.—The Browning-Huggins Micro-spectroscope.

Fig. 193.Fig. 193a.

Fig. 193.

Fig. 193a.

Figs. 193 and 193arepresent prisms of the kind arranged to use in two different positions, i and i′ being the same angle asIandI′.

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 inFig. 193a. 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. 194represents an arrangement of direct vision-prisms, invented by Herschel. The lineR R′ shows the path of a ray of light through the prisms, where it would be seen that the emergent rayR′ is parallel and coincident with the incident rayR.

Fig. 194.Fig. 194a.

Fig. 194.

Fig. 194a.

Another very compact combination is shown inFig. 194a. Any number of these prisms (P P P) may be used, according to the amountof 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 slitS Sis 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.

Fig. 195.—The Sorby-Browning Micro-spectroscopic Eye-piece.

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.

Fig. 196.—Sectional view of bright-line Spectroscope; the letters also apply to the standard spectrum scale (Fig. 198).

Ais a brass tube, carrying the compound direct vision prism;B, a milled head, with screw motion to adjust the focus of the achromatic eye lensC, seen in the sectional view as a triple combination of prisms. Another screw at right angles toC, 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 Dis 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.Eis 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 nearEstrikes 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.Fshows 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 insertFas 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 tothe 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. RemoveA, and open the slit by means of the milled-head, not shown in figure, but which is at right angles toD 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, replaceA, 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.

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

Sectional View.

Figs. 197 and 197a.—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-headB. Disappointment will occur in any attempt at delicate investigation if the directions given be not carefully followed out.

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

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 screwC, 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 screwEregulates the admission of light through the aperture aboveD. 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 screwE. 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 of1⁄10-inch in diameter over the tube containing the ½-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 bandsin 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.

Standard Spectrum Scale.Cells for use with Spectroscope.Fig. 198.

Standard Spectrum Scale.Cells for use with Spectroscope.

Standard Spectrum Scale.

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. InFig. 198is 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 withJurisprudence, 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’s36reports 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 thesubstance, which he termedcruorine, is in a more or less oxidised condition. The chart appended to his paper37affords an imperfect representation of the changes seen in the spectrum.

No. 1.—Arterial Blood, Scarlet Cruorine.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.

No. 1.—Arterial Blood, Scarlet Cruorine.

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,hæmatingives rise to a band in the red spectrumD;hæmato-globulinproduces two bands, the second twice the breadth of the first in the yellow portion of the spectrum between the linesDandE, 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.Brepresents the red end of the spectrum andGthe 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 ofA A, illuminated bythe small mirror atR, and focussed by a small lens atC, 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 theD(or the sodium line, ifDcannot 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 3½; 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 3½, 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 1½, 2½, 3½, &c., the lower edges of each ¾, 1¾, &c., and the upper 1¼, 2¼, &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 to1⁄100th part of the wholespectrum, 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.

(Red end.)(Bue end.)Fig. 200.—

(Red end.)(Bue end.)

Fig. 200.—

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

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 of1⁄10000-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.

In 1869 I published in the Journal of the Royal Microscopical Society38a 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 ascertain 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 ofCinchona 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 linein the red; another, rather narrower, in the orange, coincident withD, or the sodium-line; one in the green, aboutb, coincident with the Thallium green band; and a fourth on the blue lineF, 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 lineb. This solution wasdeep emerald-green, and even dilution did not alter the phenomena. Theacidalcoholic 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 theDandblines 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, butallagreed 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, atC,D,andb, 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.

In this chapter it will be my aim to discuss the best practical methods of employing the microscope and its appliances to the greatest advantage. First, the student should select a quiet room for working in, with, if possible, a northern aspect, free from all tremor occasioned by passing vehicles. The table selected for use should be firm, and provided with drawers, in which his several appliances can be kept ready to hand. The microscope must be placed at such an inclination as will enable him to work in comfort, and without putting strain on the muscles of the neck or fatiguing the eyes. The next important point is that of light. Daylight, in some respects, is an advantage; this should come from a white cloud on a bright day, but as a rule more satisfactory results will be obtained by using a well-made lamp, as this can be controlled with ease, and used at a proper height and distance from the microscope. To have a good form of lamp is as much to be desired for the student as for those engaged in the more advanced work of microscopy.

Whatever the source of light we must on no account over-illuminate. The object having been placed on the stage of the microscope, the body should be racked down to within a quarter or half an inch of the specimen, and then, while looking through the eye-piece, should be slowly withdrawn until a sharp image comes into view. The fine adjustment may now be used for the more delicate focussing of the several parts of the field.

Accurate adjustment of focus is required when using a ¼-inch objective; details of the object, as striæ, being brought into view when a stronger light is thrown obliquely upon them from the mirror. If a 1-inch objective is used the light often proves to be in excess ofwhat is required, and this must be regulated by the aid of the diaphragm.

The iris diaphragm, made to drop into the under-stage, is more generally employed, as when racked up to the object it affords every necessary graduation of illumination.

Fig. 201.—Bull’s-eye Lens.

To illuminate opaque objects the light should be thrown upon them from above by the bull’s-eye lens (Fig. 201). The focus of such a lens and the lamp placed at four inches from it, is about three inches for daylight, or two inches for artificial light. A large object may be placed upon the stage of the microscope at once, but smaller objects are either laid on a glass slide or held in the stage forceps.

When illuminating objects from above all light from the mirror, or that which might enter the objective from below the stage, should be carefully excluded.Dark-field illuminationis a means of seeing a transparent object as an opaque one. The principle, however, is that all the light shall be thrown from below the object, but so obliquely that it cannot enter the object-glass unless interrupted by the object; this is best accomplished byWenham’s Parabola.

Glassof any kind requires occasional cleaning; a piece of soft washed chamois leather should be used for this purpose. The fronts of the objectives may be carefully wiped, but notunscrewedor tampered with; a short thick-set camel’s hair brush may be passed down to the back lens, and all dust removed without doing any harm. If the objective is animmersion, carefully remove the fluid from the front lens, as even distilled water will leave a stain behind. For removing oil see special directions given at page 171.

When cleaning theeye-pieces, which should be done occasionally, the cells containing the glasses must be unscrewed and replaced one at a time, so that they may not be made to change places.

Any dirt upon theeye-piecesmay be detected by turning them round whilst looking through the instrument; but if theobject-glassesare not clean, or are injured, it will, for the most part, only be seen by the object appearing misty.

Theobject-glasses, when in use but not on the microscope, should be stood upon the table with the screw downwards, to prevent dust getting into the lenses, and they should always be put into their brass cases when done with. A large bell-glass shade will be found the most useful cover for keeping dust from the instrument when not in use.

When looking through the eye-piece be sure to place the eye in close proximation to the cap, otherwise the whole field will not be perfectly visible; it should appear as an equally well-illuminated circular disc. If the eyelashes are reflected from the eye-glass, the observer is looking upon the eye-piece, and not through it.

The Mirror.—The working focal distance of the mirror is that which brings the images of the window-bars sharply out upon the object resting upon the stage. In other words, the focus of the mirror is that which brings parallel rays to a correct focus on the object-glass. If employing artificial light, then the flame of the lamp should be distinguishable; a slight change in the inclination of the mirror will throw the image of the lamp-flame out of the field.

The strongest light is reflected from the concave side of the mirror, that from the flat side is more diffuse and less intense. Oblique light can be obtained by turning the mirror on one side and then adjusting it so as to illuminate the field from that position. All the necessary mechanism of the microscope is easily and quickly learned. The object-glasses or objectives are, as previously explained, designated according to the focal distance of a single lens of the same magnifying power. Thus a 2-inch objective is understood to be a combination which has the magnifying power of a single lens whose focal point is two inches from the object, and so on with reference to other powers. By the aid of different eye-pieces an extensive range of magnifying power can be obtained; for example, the 2-inch objective with a deep eye-piece will give the same amplification as the quarter objective with the ordinary eye-piece. Indeed, for certain observations, the combination of a wide-angledlow-power objective, with a deep eye-piece, orcompensating eye-piece, is considered to have an advantage.

It has been already explained that two objectives, one of much greater power than the other, but both having only the same numerical aperture, will show only the same amount ofdetail; the higher power on a larger scale. That is, supposing with a ¼-inch objective of 1·0 numerical aperture certain structure is resolved, then a1⁄8-inch substituted with exactly the same numerical aperture, but with double the magnification, no moreresolving powerwill be found in the latter objective than in the former. For this reason a doubt has been expressed as to whether high-power objectives—especially the more expensive oil-immersions, made to transmit large pencils of light through their larger apertures—are so well adapted for ordinary research as the best series of dry achromatic objectives, or even, in some instances, the medium aperture lenses; undoubtedly, for histological (physiological and pathological) work, the latter will be found to meet the students’ requirements quite as well as the former.

The student or amateur will do well to commence with moderate or medium powers, a 2-inch, a 1-inch, a ½-inch, a4⁄10-inch, or ¼-inch. These, together with the A and B eye-pieces, will give a range of magnification from 30 to 250 diameters.

Penetrationin the objective is a quality for consideration, as the adjustment of high powers is a work of delicacy, and in some cases their penetration is impaired by the arrangement made to obtain finer definition. The value, however, of penetration in an objective is always considered to be of more or less importance. It is a quality whereby, under certain conditions, a more perfect insight into structure is obtained. As a rule, the objective having the largest working distance possesses the better penetration. Theoretically, the penetration of an objective decreases as the square of the angular aperture increases. For this reason the medical student will be justified in choosing the objectives I have named, since these will be better adapted to his work and pursuits. The penetration of the objective is a relative quality assessed at a different value by workers whose aims are widely different. But for the observation of living organisms, the cyclosis within the cell of the closterium or valisneria, for instance, preference will undoubtedly be in favour of the objective with good penetration.

Resolving Power.—This is a quality highly prized by the bacteriologist. In the case of the high-angled apochromatic oil-immersion, with its compensating eye-piece, its resolution is found to be of very considerable advantage, because of its capacity to receive and recombine all the diffraction spectra that lie beyond the range of the older achromatic objective, with its smaller angular aperture. The actual loss of resolving power consequent upon the contraction of aperture from 180° to 128½° is ten per cent., if not more. Resolution depends, then, upon the quality and quantity of the light admitted, the power of collecting the greatest number of rays, and the perfection of centring. In other words, upon the co-ordination of the illuminating system of the microscope—mirror, achromatic condenser, objective and eye-piece. If diatoms are employed as test-objects, it should not be forgotten that there are great differences, even in the same species, in the distances their lines are apart. For this reason ruled lines of known value, as Nobert’s lines, are to be preferred. The following example will suffice to show the value of a dry1⁄8-inch objective of 120° in defining the rulings of a 19-band plate, which is equivalent to the1⁄67000th of an inch. This objective, with careful illumination, showed them all; but when cut down by a diaphragm to 110°, the eighteenth line was not separable; further cut down to 100° the seventeenth was the limit, to 80° the fourteenth, and to 60° the tenth was barely reached.

Flatness of Field.—This quality in the objective has, by the introduction of the immersion system, lost much of the importance formerly attached to it. Some writers assume it to be an “optical impossibility.” The compensating eye-piece has had the effect of contracting the visual field, consequently the peripheral imperfections of the objective are of a less disturbing character. It has, however, not been made perfectly clear whether the highest perfection of the two primary qualities of a good objective,defining power and resolving power, can be always obtained in one and the same combination of lenses.

Doubtless,definingpower can be more satisfactorily determined by the examination of a suitable object, and the perfection of the image obtained; to assist in securing which, a solid axial cone of light equal to about three-fourths of the aperture of the objective must be employed.

To sum up, then, “the focal power of all objectives depends in their perfectdefinition, a property on which their converging power depends, and in turn their magnifying action is dependent; again, focal power is the curvature imprinted by the lens on a plane wave, and is reciprocal of the true focal length. It is appropriately expressed in terms of the proper unit of focal curvature, thedioptric; a unit of curvature.”39

Fig. 202.—Seiler’s Test Slide.

It may be taken as an axiom with microscopists that “neither the penetrating power nor the high-power defining objective is alone sufficient for every kind of work. The larger the details of ultimate structure, the narrower the aperture—and the converse; the minuter the dimensions of elementary structure, the wider must be the aperture of the objective.” Every worker with the microscope must have satisfied himself of the truth of this statement, when engaged in the study of the movements of living organisms, or defining the intimate structure of the minuter diatoms, or of the podura scale.

Test for Illumination.—Dr. C. Seiler recommends the human blood corpuscle as the best test of good illumination. He prepares the object in the following manner: Take for the purpose a clean glass slide of the ordinary kind, and place near its extreme edge a drop of fresh blood drawn by pricking the finger with a needle. Then take another slide of the same size, with ground edges, and bring one end in contact with the drop of blood, as shown inFig. 202, at an angle of 45°; then draw it evenly and quickly across the underslide, and the result will be to spread out the corpuscles evenly throughout. Blood discs being lenticular bodies, with depressed centres, act like so many little glass-lenses, and show diffraction rings if the light is not properly adjusted.40

Errors of Interpretation.—To be in a position to draw an accurate conclusion of the nature and properties of the object under examinationis a matter of great importance to the microscopist. The viewing of objects by transmitted light is of quite an exceptional character, rather calculated to mislead the judgment as well as the eye. It requires, therefore, an unusual amount of care to avoid falling into errors of interpretation. Among test objects the precise nature of the structural elements of the Diatomaceæ have given rise to great divergence of opinion. Then, again, the minute scales of the podura Springtails, one of the Collembola, and their congenersLepisma saccharina, the structure of which is equally debatable. Mr. R. Beck, in an instructive paper published in the “Transactions of the Royal Microscopical Society,” says that the scales of the Lepisma can be made to put on an appearance which bears little resemblance to their actual structure.

Fig. 203.—Portions of Scales of Lepisma.

In the more abundant kind of scales the prominent markings appear as a series of double lines. These run parallel and at considerable intervals from end to end of the scale, whilst other lines, generally much fainter, radiate from the quill, and take the same direction as the outline of the scale when near the fixed or quill end; but there is, in addition, an interrupted appearance at the sides of the scale, which is very different from the mere union, or “cross-hatchings,” of the two sets of lines (Fig. 203, Nos. 1 and 2, the upper portions).

The scales themselves are formed of some truly transparent substance, for water instantly and almost entirely obliterates their markings, but they reappear unaltered as the moisture leaves them; therefore the fact of their being visible at all, under any circumstances, is due to the refraction of light by superficial irregularities, and the following experiment establishes this fact, whilst it determines at the same time the structure of each side of the scale, which it is otherwise impossible to do from the appearance of the markings in their unaltered state:—

“Remove some of the scales by pressing a clean and dry slide against the body of the insect, and cover them with a piece of thin glass, which may be prevented from moving by a little gum at each corner. No. 3 may then be taken as an exaggerated section of the various parts.A Bis the glass slide, with a scale,C, closely adherent to it, andDthe thin glass-cover. If a very small drop of water be placed at the edge of the thin glass, it will run under by capillary attraction; but when it reaches the scale,C, it will run first between it and the glass slide,A B, because the attraction there will be greater, and consequently the markings on that side of the scale which is in contact with the slide will be obliterated, while those on the other side will, for some time at least, remain unaltered: when such is the case, the strongly marked vertical lines disappear, and the radiating ones become continuous. (SeeNo. 1, the lower left-hand portion.) To try the same experiment with the other, or inner surface of the scales, it is only requisite to transfer them, by pressing the first piece of glass, by which they were taken from the insect, upon another piece, and then the same process as before may be repeated with the scales that have adhered to the second slide, the radiating lines will now disappear, and the vertical ones become continuous. (SeeNo. 2, left portion.) These results, therefore, show that the interrupted appearance is produced by two sets of uninterrupted lines on different surfaces, the lines in each instance being caused by corrugations or folds on the external surfaces of the scales. Nos. 1 and 2 are parts of a camera lucida drawing of a scale which happened to have opposite surfaces obliterated in different parts. No. 4 shows parts of a small scale in a dry and natural state; at the upper part the interrupted appearance is not much unlike that seen at the sides of the larger scales; but lower down, where lines of equal strengthcross nearly at right angles, the lines are entirely lost in a series of dots, and exactly the same appearance is shown in No. 5 to be produced by the two scales at a part where they overlie each other, although each one separately shows only parallel vertical lines.”

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