1902 Encyclopedia > Microscope > Immersion System

Microscope
(Part 4)




(A) INTRODUCTION (cont.)

Immersion System.—It was long since pointed out by Professor Amici that the introduction of a drop of water between the front surface of the objective and either the object itself or its covering-glass would diminish the loss of light resulting from the passage of the rays from the object or its covering-glass into air, and from air into the front glass of the objective. It was obvious to him, moreover, that when the rays enter the object-glass from water, instead of from air, both its refractive and its dispersive action will be so greatly changed as to need an important constructive modification to meet the new condition. This modification seems never to have been successfully effected by Amici himself ; but his idea was taken up by the two eminent Paris opticians, MM. Hartnack and Nachet, who showed that the application of what is now known as the "immersion system" to objectives of short focus and large angular aperture is attended, not merely with the advantages expected by Professor Amici, but with others on which he did not reckon. As the loss of light by the reflexion of a portion of the incident rays increases with the obliquity of their incidence, and as the proportional loss is far smaller when the oblique rays pass into glass from water than when they enter it from air the advantage of increasing the angular aperture is more fully experienced with "immersion" than with "dry" objectives,—just as Professor Amici anticipated. But, further, the immersion system allows of a greater working distance between the objective and the object than can be attained with a dry or air objective having the same angular aperture; and this increase affords not only a greater freedom of manipulation, but also a greater range of "penetration " or "focal depth." Further, the observer is rendered so much less dependent upon the exactness of his covercorrection that it is found that water-immersion objectives of high power and considerable angular aperture, extremely well adapted for the ordinary purposes of scientific investigation, can be constructed without it,—a small departure from the standard thickness of covering-glass to which such objectives are adjusted by the maker having scarcely any effect upon the distinctness of the image. It is now the practice of several makers to supply two fronts to objectives of 1/10 or 1/12 inch focus, one of them fitting the objective for use "dry" (that is, in air), whilst the substitution of the other converts it into a water-immersion objective. And in the objectives constructed on Mr Wenham’s systbm na change in the front glass is needed, all that is necessary fcr making them work as immersion-lenses being a yet closer approximation of the f ront lens to the second combination, which can be made by the screw-collar.

Within the last few years, however, the immersion system has undergone a still further and most important development, by the adoption of a method originally suggested by Mr Wenhain (though never carried out by him), and independently suggested by Mr Stephenson to Professor Abbe of Jena, under whose direction it was first worked out by Zeiss (the very able optician of Jena), who has been followed by Powell and Lealand of London, as well as by several other constructors of achromatic objectives both in England and elsewhere, with complete success. This method consists in the replacement of the water previously interposed between the covering-glass and the front glass of the objective by a liquid having the same refractive and dispersive powers as crown-glass, so that the rays issuing at any angle from the upper plane surface of the covering-glass shall enter the plane front of the objective, without any deflexion from their straight course, and without any sensible loss by reflexion,—even the most oblique rays that proceed from the object keeping their direction unchanged 'until they meet the back or convex surface of the front lens of the objective. It is obvious that all the advantages derivable from the system of waterimmersion will be still more thoroughly attained by this system of "homogeneous" immersion, provided that a fluid can be obtained which meets its requirements. After a long course of experiments, Professor Abbe found that oil of cedar wood so nearly corresponds with crown-glass, alike in refractive and in dispersive power, as to serve the purpose extremely well, except when it is desired to take special advantage of the most divergent or marginal rays, oil of fennel being then preferable. There are, however, strong objections to the use of these essential oils in the ordinary work of research; and it seems not unlikely that a solution of some one or more saline substances will be found more suitable. In addition to the benefit conferred by the water-immersion system, and more completely attained with the homogeneous, it may be specially pointed out that, as no correction for the thickness of the covering-glass is here required, the microscopist can feel assured that he has such a view of his object as only the most perfect correction of an air-objective can afford. This is a matter of no small importance, for while, in looking at a known object, the practised microscopist can so adjust his air-objective to the thickness of its covering-glass as to bring out its best performance, he cannot be sure, in regard to an unknown object, what appearances it ought to present, and may be led by imperfect cover-correction to an erroneous conception of its structure.

It has been recently argued that, as the slightest variation in the refractive index of either the immersion fluid or the covering-glass, a change of eye-pieces, or the least alteration in the length of the body-in a word, any circumstances differing in the slightest degree from those under which the objective was corrected—must affect the performance of homogeneous-immersion objectives of the highest class, they should still. be made adjustable. The truth of this contention can, no doubt, be proved, not only theoretically, but practically,—the introduction of the adjustment enabling; an experienced manipulator to attain the highest degree of perfection in the exhibition of many mounted objects, which cannot be so well shown with objectives in fixed settings. But it may well be questioned whether it is likely to do the same service in the hands of an ordinary working histologist, and whether the scientific investigator will not find it preferable, when using these objectives, to accept what their maker has fixed as their point of best performance. The principal source of error in his employment of them lies in the thickness of the optical section of the object; for the rays proceeding from its deeper plane, having to pass through a medium intervening between that plane and the cover-glass, whose refractive and dispersive indices differ from those of the glass and immersion-fluid, cannot be brought to so accurate a focus as those proceeding from the plane immediately beneath the cover-glass. The remedy for this, however, seems to lie rather in making the preparation as thin as possible than in the introduction of what is likely, in any but the most skilful and experienced hands, to prove a new source of error. Every one who has examined muscular fibre, for example, under a dry objective of very high power and large aperture, well knows that so great an alteration is produced in its aspect by the sligh test change in either the focal adjustment or the cover- correction that it is impossible to say with certainty what are the appearances which give the most correct optical expression of its structure. This being a matter of judgment on the part of each observer, it seems obvious that the nearest approach to a correct view will be probably given by the focal adjustment of the best homogeneous immersion-objectives, in fixed settings, to the plane of the preparation immediately beneath the cover-glass (see Jour. Roy. Micros. Soc., 1882, pp. 407,854,906).

In every particular in which the- water-immersion system is superior to the dry, it is itself surpassed by the oil or other homogeneous system, the anticipations of those by whom it was suggested being thus fully realized. But the advantages already spoken of as derivable from the use of the "immersion system" are altogether surpassed by that which the theoretical studies of Professor Abbe have led him to assign to it, and of which he has practically demonstrated its possession. For he has shown (as will be explained below) that the interposition of either water or oil so greatly increases the real "aperture" of the objective that immersion-objective’s may be constructed having a far greater virtual aperture than even. the theoretical maximum (180º) of the angular aperture of an air-objective.





The same eminent physicist, working on the basis supplied by the mathematical investigations of Professor Helmholtz and himself on the undulatory theory of light, has further established an entirely new doctrine in regard to the production of highly magnified representations of closely approximated markings. All that has hitherto been said of the formation of im ages by the compound microscope relates to such as are produced, in accordance with the laws of refraction, by the alteration in direction which the light-rays undergo in their passage through the lenses interposed between the object and the eye. These, dioptric images, when formed by lenses free from spherical and chromatic aberration, are geometrically correct pictures, truly representing the appearances which the objects themselves would present were they enlarged to the same scale and viewed under similar illumination. And we seem justified, therefore, in drawing from such microscopic images the same conclusions in regard to the objects they picture as we should draw from the direct vision of actual objects having the same dimensions. The principal source of error in such interpretations arises out of the "nterference" to which the rays of light are subjected along the edges of the minute objects through which they pass, or along any such lines or margins in their inner part as are sufficiently opaque to throw a definite shadow. For every such shadow must be bordered, more or less obviously, by interference -or diffraction-spectra; and thus the images of strongly-lined objects with very transparent intermediate spaces may be so troubled or confused by these "diffraction-spectra" as to render it very doubtful what interpretation is to be put upon their appearances.

A good example of this kind is afforded by the scales of the gnat or mosquito, which are composed of a very delicate double membrane, strengthened by longitudinal ribs on both sides, those of the opposite sides uniting at the broad end of the scale, where they generally terminate as bristle-sbaped appendages beyond the intermediate membrane. These are crossed by fine markings, which are probably ridge-like corrugations of the membrane, common to both sides of the scale. Between each pair of longitudinal ridges there may be seen, under certain adjustments of focus and illumination, three uniform parallel rows of beads, which have been supposed to represent a true structure in the membrane. By Dr Woodward (colonel in the United States army), however, it has been shown that this beaded appearance is merely the result of the "interferences" produced by the longitudinal and transverse lines of the scale. For the longitudinal diffraction-lines are clearly seen, alike in the microscopic iinage, and in photographs (fig. 13), to extend in to em ty space beyond the contour of the scales, almost as far as the ends of the bristles in which the parallel ribs terminate; and they vary in number with the varying obliquity of illumination, so that in the same scale two, three, four, or even five rows of beads can be seen, and photographed at pleasure, in every intercostal space. [Footnote 265-1]

Every microscopist who has worked much with high powers is well aware of the difficulty of distinguishing between real and speetral markings—a difficulty which can only be overcome by training and experience. It seems, however, to have been now tully ascertained by pro-fessor Abbe that it is only through such diffraction-spectra that the microscope can make us acquainted with the minutest structural features of objects, since, according to the calculations of Professor Helmholtz and himself (based on the constants of the undulatory theory), no amount of magnifying power can separate dioptrically two lines, apertures, or markings of any kind, not more than 1/2500 of an inch apart. The visual differentiation or "resolution" of lines or other markings whose distance lies within that limit is entirely the result of "interference,"—-the objective receiving and transmitting, not only dioptric rays, but the inflected rays whose course has been altered in their passage through the object by the peculiar disposi-tion of its particles, and combining these rays into a series of diffraction-spectra, the number and relative position of which bear a relation to the structural arrangement on which their production depends. If the objective be per-fectly corrected, and all the diffraction-spectra lie within its field, these will be recombined by the eye-piece so as to form a secondary or "diffraction" image, lying in the same plane with the dioptric image, and coinciding with it, while filling up its outlines by supplying intermediate details. But where the markings (of whatever nature) are so closely approximated as to produce a wide dispersion of the interference-spectra, only a part of them may fall within the range of the objective; and the recombination of these by the eye-piece may produce a diffraction-image differing more or less completely (perhaps even totally) from the real structure; while, if they should lie entirely outside the field of the objective, no secondary or diffraction image will be produced. And thus, while the general form of such an object as a diatom-valve may be correctly given in a dioptricx image, its surface may appear quite unmarked under an objective of small aperture, however great its magnifying power, though covered with regularly disposed markings when seen through an objective of wider aperture with perhaps only half the magnifying power.

It is obvious, however, that, while the dioptric image represents the actual object, the diffraction-image thus formed by the reunion of a portion of the interference pencils is only an optical expression of the result of their partial recombination, which may represent something entirely different from the real structure. For it has been proved experimentally, by placing finely-ruled gratings in the position of objects, and by limiting the, apertures of objectives by diaphragms with variously disposed perfora-tions, that the same arrangement of lines shall be presented to the eye by differently lined surfaces, and different arrangements by similarly lined surfaces, according to the numbers and relative positions of the reunited spectra. Hence it is clear that there must be an essential difference in character and trustworthiness between the images dioptrically formed of the general outlines and larger details of microscopic objects and those representations of i their finer details which aregiven by the recombination of their diffraction-spectra, [Footnote 266-1] and that the confidence to be placed in the latter class of representations will be greater in proportion to the completeness of the recombination of the separated interference-spectra, which, again, will be proportional (accurate correction of the aberrations being assumed) to the aperture of the objective. [Footnote 266-2]

The combined advance of scientific theory and of practical skill in the application of it have now brought up the compound achromatic microscope to an optical perfection that renders it capable of actually doing almost everything of which, in the present state of optical theory, it can be regarded as capable. The resolution of Nobert’s nineteenth band, having 112,595 lines to an inch, which was long regarded as the crux of microscopists, is now found so easy as to leave little room for doubt that, if a new test were obtainable having the minimum visibile of 118,000 lines to the inch, an oil-immersion objective would be found to resolve it. But the experience of the past makes it evident that, as no limit can be set to the advance of optical theory, results yet more remarkable may be still expected to arise, every such advance being turned to account by the practical skill which experience has now enabled the best constructors of achromatic objectives to attain. [Footnote 266-3]

The progressive improvements thus effected in the construction of microscopic objectives have been accompanied by other improvements, alike in the optical and in the mechanical arrangements by which the best performance of these objectives can be secured; and it will be desirable now to describe in succession the most approved forms of the eye-piece, the objective, and the illuminating apparatus respectively, and then those of the instrument as a whole, pointing out the special adaptiveness of each to the requirements of different classes of scientific investigators.





Footnotes

265-1 Monthly Micros. Jour., vol. xv. (1876), p. 253.

266-1 Thus it is still a moot point whether the microscopic appear-ances seen in the siliceous valves of diatoms (figs. 8-11) are the optical representations of elevations, depressions, or perforations, or of internal molecular arrangements not involving any inequality of surface,

266-2 This doctrine was first fully developed by Professor Abbe in the Archiv für Microsk. Anatomie, vol. ix. (1874), and is more fully expounded in his subsequent contributions to Jour. Boy. Micros. Soc. See also the papers of Mr Stephenson and Mr Crisp in that journal, and in the preceding Monthly Microscopical Journal.

266-3 Any good workman can now make by the dozen such small-angled 1/4 inch objectives as Mr A. Ross produced with much pains and labour fifty years ago. It was not until 1844 that, with the honourable emulation of surpassing what Professor Amici had then accomplished, he produced a 1/12 inch of 135º, which, by taking advantage of some very heavy flint-glass he had, he afterwards increased to 170º.


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