378 



NATURE 



[November i8, 1920 



Microscopy with 



By J. E, 



""T^HE microscope is now so widely used in all 

 A branches of science and in industry that it 

 is not surprising- to find an increasing demand for 

 greater optical efficiency. It must be admitted 

 that in comparatively few cases is the instru- 

 ment used under such conditions as to secure 

 the best possible result ; but this is due to lack 

 of appreciation of the principles involved, and 

 will be remedied only by a wide educational 

 effort. Even when the greatest optical efficiency 

 is secured, the limitations are soon felt. The 

 chief need is for increased resolution, that factor 

 on which the delineation of minute structure 

 depends. Advances of great value have been 

 made in methods of rendering visible minute 

 objects, but it must be clearly realised that, while 

 this greater visibility can be secured, no informa- 

 tion as to the form or structure of objects which 

 are below the resolution limits is to be obtained 

 by this means. Increased magnification is by 

 some workers still regarded as desirable, but un- 

 less this is accompanied by proportionally in- 

 creased resolution, the results are worse than 

 useless, and can lead only to serious errors of 

 interpretation. 



Two factors mainly govern resolution — namely, 

 the numerical aperture of the objective, and the 

 mean wave-length of the illuminant. No increase 

 of numerical aperture has been obtained since the 

 classic researches of Abbe, resulting in the pro- 

 duction of apochromatic objectives ; and in the 

 present state of knowledge there appears little 

 likelihood of any substantial advance in this direc- 

 tion. By using light of short wave-length, a 

 promising field of research is at once opened up. 

 An increase of resolution is obtained even with 

 visible light if the violet or blue end of the spec- 

 trum is utilised, but the increase is much more 

 definite if ultra-violet light is used, although the 

 image is no longer a visual one. 



The computation of microscope objectives for 

 use with ultra-violet light presents considerable 

 difficulties, as only two substances sufficiently 

 transparent to these radiations are available — 

 quartz and fluorite. So long ago as i860 Spencer 

 in .America used ffuorite for this purpose, and at 

 a much later date Boys in this country suggested 

 the possibility of using fused quartz. In 1904 

 Kohler, of Jena, succeeded in computing objec- 

 tives entirely of fused quartz, some earlier ones 

 which were fluorite-quarfz combinations being 

 thereby superseded. Ultra-violet light, therefore, 

 became available for microscopic work, but the 

 practical difficulties in the use of the apparatus 

 are so considerable, calling for almost more 

 knowledge of physical than of microscopical 

 methods, that it has been used by few. 



The' results obtained, particularly in biological 

 work, are in many cases of great interest, as, in 

 addition to the advantages already indicated, there 

 NO. 2664, VOL. 106] 



Ultra-violet Light. 



Barnaku. 



is the further important point that organisms are 

 dealt with and photographs obtained of them in 

 the living state. The classic researches of Hartley 

 showed that organic substances which are p>er- 

 fectly transparent to ordinary light have very 

 definite absorption regions or bands in the ultra- 

 violet, and that their absorption is, in many in- 

 stances, so characteristic that it constitutes an 

 accurate method of identification. To a consider- 

 able extent, this fact is of value when using ultra- 

 violet in microscopy. Objects that show little or 

 no structure by transmitted light are seen to be 

 highly organised when examined by ultra-violet 

 radiations, and the structure .seen is in part de- 

 pendent on the wave-length of the light used. 

 Objects for examination by this method must be 

 dealt with in the living state, or at least in such 

 a manner that no change takes place in their con- 

 stitution. It follows that none of the ordinary 

 methods of mounting such things as micro- 

 organisms, in which staining, hardening, fixing, 

 drying, or heating is resorted to, can be em- 

 ployed. The method is, in fact, its own staining 

 process, differentiation of structure depending on 

 the difference of absorption in ultra-violet, and 

 not on complex staining processes, which are in 

 some cases causing appearances not a.s.sociated 

 with the organism itself. Further, apart from the 

 alteration that may take place in the tissues them- 

 selves as the result of such processes, their em- 

 ployment in the method under consideration would 

 render them opaque to the radiations used, and, 

 therefore, useless for the purpose. The organisms 

 or tissues are simply mounted in any suitable fluid, 

 such as water, normal saline. Ringer's solution, 

 etc., which is transparent to ultra-violet light and 

 the photograph is taken at once. The result is an 

 image that, whether it shows more or less than a 

 stained preparation, is a representation of the 

 object in the living state, and with greater resolu- 

 tion than can be obtained in the microscope by 

 any other process. 



Such a method is obviously one to be tried 10 

 its utmost whatever practical difficulties may be 

 involved, and there is little doubt that in time it 

 will be recognised as what it really is — the only 

 great advance in microscopic technique for a 

 generation. The apparatus as used by the present 

 writer is in its essentials the same as that devised 

 by Dr. Kohler (Fig. i), although in many points 

 of detail improvements have been devised. The 

 quartz objectives are three in number, their equi- 

 valent focal lengths being 6 mm., 25 mm., and 

 1-7 mm., their effective numerical aperture being 

 respectively 250, 17, and 07. It will at once be 

 appreciated that in cases where the full aperture 

 can be utilised the two higher powers are of much 

 greater N.A. when used with light of 275 /x/i 

 wave-length than any objective available for use 

 with ordinary light. These two are glycerine 



