Practical Microscopy: Manipulation, and Mode of Using the Microscope.
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to within a quarter or half an inch of the specimen, and then, while looking through the eye-piece, should be slowly withdrawn until a sharp image comes into view.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 of what 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 illumination is 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 by Wenham’s Parabola.
Glass of 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 not unscrewed or 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 an immersion, 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 the eye-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 the eye-pieces may be detected by turning them round whilst looking through the instrument; but if the object-glasses are not clean, or are injured, it will, for the most part, only be seen by the object appearing misty.
The object-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-angled low-power objective, with a deep eye-piece, or compensating 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 of detail; 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 a 1⁄8-inch substituted with exactly the same numerical aperture, but with double the magnification, no more resolving power will 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, a 4⁄10-inch, or ¼-inch. These, together with the A and B eye-pieces, will give a range of magnification from 30 to 250 diameters.
Penetration in 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 dry 1⁄8-inch objective of 120° in defining the rulings of a 19-band plate, which is equivalent to the 1⁄67000th 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, defining power 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 perfect definition, 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, the dioptric; 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 in [Fig. 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 examination is 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 congeners Lepisma 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 B is the glass slide, with a scale, C, closely adherent to it, and D the 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. (See No. 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. (See No. 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 strength cross 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.”
Fig. 204.—Outer Membrane of Upper Plane of Red Beads thrown by each alternate hole of grating; on lowering the focus white interspaces turn into blue beads.
Fig. 204a.—Outer Membrane of Lower Plane of Beads thrown from remaining holes of grating; on raising the focus white interspaces turn into red beads.
Objective used, Zeiss’s apochromatic 1⁄12-inch oil-immersion, numerical aperture 1·40, magnifying power 1,750 diameters.
A well-known skilled observer of test objects[41] says: “Practically the resolving power of our achromatic objectives on lined objects reached their maximum in the late Dr. Woodward’s hands. Amphipleura pellucida was then, as now, the finest known regular structure of the diatoms. There appeared then nothing more to be gained in resolution when one of the apochromatic 1⁄12-inch objectives of Zeiss, with its entire absence of colour, passed into my hands, and I soon became convinced that it possessed the power of separating the different layers of structure in the valve, beyond the grasp of the dry-objective. The result of this increase of power enabled me to split up, as it were, the one plate of silex forming the valve of Pleurosigma formosum into three layers, and which had never before appeared to be possible; proving, in fact, that magnification without corresponding aperture is of little or no account.”
“The intimate structure of these test objects,” says Mr. Smith, “is built up on one plan, each being composed of two or more layers, (1) a valve with two layers, as in Pleurosigma balticum; (2) two layers with a grating and secondary markings placed diagonally, as in Pleurosigma formosum; (3) with two layers of a net-like structure, as in Pleurosigma angulatum, the fineness of the striæ or gratings of which measure the 1⁄50000th of an inch. Five other diatoms afford evidence of this compound structure. The presence of beads or hemispheres in one of the focal planes, and depressions or pits in another, are emphasised in the micro-photograph itself; reduced portions of the valve are represented in Figs. 204 and 204a.”
A portion of a diatom valve, Pleurosigma angulatum, micro-photographed on a higher scale of magnification, 4,500 diameters, is given further on.
Fig. 205.—Sections of an old-fashioned Glass Tumbler, from photographs by the late Mr. R. Beck.
Errors of interpretation arise either from the small cones of illumination afforded by the dry-objective, or the oblique illumination formerly resorted to for the resolution of these difficult test objects, and several of the lights and shadows resulting from the refractive power of the object itself. But the most common error is that produced by the reversal of the lights and shadows resulting from the refractive powers of the object itself. To make this clear, I reproduce two reduced photographs of a small section of an old-fashioned glass tumbler, covered externally with numerous hemispheres, illuminated by transmitted light ([Fig. 205]).
This illustration well emphasises the difficulty there is in determining structure under precisely similar conditions to those we are accustomed to of examining valves of diatoms under the microscope. If these photographs be held in front of a strong light, they at once convey different impressions to the mind, the hemispheres appearing depressions in the one, and raised beads in the other. Both are prints from the same negative, but in mounting are reversed; and therefore the apparent dissimilarity is due to a slight inequality of illumination, which the mind accepts as light and shade.
Very similar appearances to those described will result if a thin plate of glass were studded with minute, equal, and equi-distant plano-convex lenses, the foci of which would very nearly lie in the same plane. If the focal surface, or plane of vision, of the objective be made to coincide with this plane, a series of bright points will result, from the excess of light falling on each lens. If the plane of vision be next made to coincide with the surfaces of the lenses, these points would appear dark, in consequence of the rays being refracted towards points now out of focus. Lastly, if the plane of vision be made to coincide with the plane beneath the lenses that contain their several foci, so that each lens may be, as it were, combined with the object-glass, then a second series of bright points will result from the accumulation of the rays transmitted at those points. Moreover, as all rays capable of entering the objective are concerned in the formation of the second series of bright focal points, the first series being formed by the rays of a cone of light only, it is evident that the circle of least confusion must be much less, and therefore the bright points better defined in the first than in the last series.
There are no set of objects which have given rise to more discussion as to their precise character than the scales of the podura (Lepidocyrtus cervicollis), to the intimate structure of which Mr. Smith turned his attention, and succeeded, I am inclined to think, in his attempt to settle the structure of these very minute scales, and which heretofore have been described as “notes of exclamation.” By the aid of the same power as that employed in the examination of the pleurosigma formosum, the old conventional markings have disappeared, and well-defined “featherlets” have taken their place. By careful focussing up and down, a series of whitish pin-like bodies is to be seen, with an intervening secondary structure. A micro-photograph of a portion of a scale taken by Mr. Smith shows that these pin-like bodies are inserted in a fold of the basement membrane, which, in his opinion, furnish unmistakable evidence of the fact that these projecting bodies are real, and must no longer be looked upon as mere ghosts. Quite recently, a micro-photograph of a portion of a podura scale was placed in my hands, taken by Mr. J. W. Gifford with a Swift’s 1⁄12-inch apochromatic objective, of numerical aperture 1·40, and a deep eye-piece, having a combined magnifying power of 3,827 diameters. [Fig. 206] shows a portion of the photograph which, it will be admitted, supports Mr. Smith’s view of the structure of the podura scale.
Fig. 206.—Podura Scale, taken with a 1⁄12 Swift’s Immersion × 3,827.
Many other errors of interpretation are not unknown to the experienced operator with the microscope, arising, for the most part, from an influence exerted by peculiarities in the internal structure of certain objects; for example, that offered by the human hair, and which, when viewed by transmitted light, presents the appearance of a flattened-out band, with a darkish centre, due to the refractive influence of the rays of light transmitted through the hair. That it is a solid or tubular structure is proved by making a transverse section of the hair-shaft, when it is seen filled up by medullary matter, the centre being somewhat darker than the outer part. It is, in fact, a spiral outgrowth of the epithelial scales, overlapping each other, imparting a striated appearance to the surface. A cylindrical thread of glass in balsam appears as a flattened, band-like streak, of little brilliancy. Another instance of fallacy arising from diversity in the refractive power of the internal parts of an object is furnished by the mistakes formerly made with regard to the true character of the lacunæ and canaliculi of bone structure. These were long supposed to be solid corpuscles, with radiating opaque filaments proceeding from a dense centre; on the contrary, they are minute chambers, with diverging passages—excavations in the solid osseous structure. That such is the case is shown by the effects of Canada balsam, which infiltrates the osseous substance.
Air bubbles are a perplexing source of trouble. The better way of becoming accustomed to deceptive appearances of the kind is to compare the aspect of globules of oil in water with bubbles of air in water, or Canada balsam.
The molecular movements of finely divided particles, seen in nearly all cases when certain objects are first suspended in water, or other fluids, are a frequent cause of embarrassment to beginners. If a minute portion of indigo or carmine be rubbed up with a little water, and a drop placed on a glass slide under the microscope, it will at once exhibit a peculiar perpetual motion appearance. This movement was first observed in the granular particles seen among pollen grains of plants, known as fovilla, and which are set free when the pollen is crushed. Important vital endowments were formerly attributed to these particles, but Dr. Robert Brown showed that such granules were common enough both in organic and inorganic substances, and were in no way “indicative of life.”[42]
Professor Jevons succeeded in throwing light on these curious movements. He showed that they were not due to evaporation, as some observers contended, as they continue when all possibility of evaporation is cut off, when the fluid is surrounded by a layer of oil, and enclosed in an air-tight case: but as Professor Jevons pointed out, these movements are greatly affected by the admixture of various substances with water, being increased by a small quantity of gum, and checked by a drop of sulphuric acid, or a few grains of some saline substance, which increases the conducting power of water for electricity. The Brownian movement, now termed pedesis, much depends upon the size of the particles, their specific gravity, and the nature of the liquid in which they are immersed.
The correct conclusions to be drawn by the microscopist regarding the nature of an object will necessarily depend upon previous experience in microscopic observations, a knowledge of the class of bodies brought under observation, and the skill of the observer in the use of the instrument—that is, in securing the best focus possible with any objective brought into use. I am indebted to Messrs. Beck for the following series of illustrations, showing the effect of under and over correction of the objective.