PROTOPLASM 9 



lowest and highest positions of the objective no real image can be formed, by refraction, within 

 the tube of the microscope. As the iris is opened, the number of separate images diminishes. 

 The same facts are even better shown by the use of Abbe's diffraction plate, as supplied by 

 Zeiss. One of the figures on this plate consists of a series of rhombic clear areas, obtained by 

 removing the silver coating by scratching a set of crossing lines and then preparing a 

 photographic negative. The real structure is shown in the photograph of Fig. 8. With narrow 

 iris, as in the previous case, a number of different images can be obtained, four of which I have 

 photographed in Fig. 9. More details will be found in an article by J. W. Stephenson (1877, 

 p. 87). The facts given here are sufficient to show that, by diffraction, structures can be seen 

 which are quite unlike those actually present. It will be noted that the condition favouring 

 their production is that of a narrow cone of illumination, due to a small aperture of the 

 substage iris diaphragm. Some interesting photographs of diffraction images will be found 

 in Edser's "Light" (p. 433). In these cases the images are more or less similar to the 

 real objects. 



The presence of different structures in a cell, even supposing that they are 

 colourless, can be detected if they have refractive indices differing from that 

 of the surrounding substance. Light rays will be. deflected and give rise to 

 darker and lighter spaces. 



Colourless glass beads in air, observed under transmitted light by a low power lens, show 

 dark and light rings ; if immersed in oil of the same refractive index as themselves, they 

 become invisible. Ordinary immersion oil is very nearly correct for this purpose. 



Now most of the various structures in living cells possess very nearly the 

 same refractive index, a fact which renders this mode of microscopic vision 

 of limited use. Moreover, even when images are seen, they have only an indirect 

 relation to the forms of the objects themselves, as is evident from the appearance 

 of beads in air by transmitted light. 



Suppose, however, that in the above experiment we take coloured beads. 

 It will be found that, when immersed in oil, a beautifully clear and distinct image 

 is obtained, whereas in air it is obscured by refraction. This shows what is to 

 be aimed at in microscopic observation. Put shortly, we desire coloured objects, 

 mounted in 'a medium of the same refractive index as themselves, and, to avoid 

 diffraction, illuminated by a wide angled cone of light. This latter is obtained 

 in " critical illumination" by which an image of the source of light is produced, 

 in or very close to the plane of the object, by a substage condenser with iris 

 opened as widely as the numerical aperture of the objective will permit. For 

 details the textbooks must be consulted (for example, Spitta's " Microscopy," 

 pp. 209-226). It is sufficient here to emphasise the fact that if, in a particular 

 case, the light of "critical" illumination is too brilliant, it must not be reduced 

 by narrowing the iris, nor by putting the condenser out of focus, but by the 

 interposition of a screen of the necessary degree of opacity. 



The mode of vision by absorption of certain components of light by coloured 

 objects is therefore, par excellence, the method to be aimed at. Unfortunately, 

 it is of but limited application to living cells, where so many of the constituents 

 are colourless. There are, however, two cases where it can be used for such 

 objects, and it is, of course, the aim of all histological staining processes. The 

 two cases referred to are, firstly, photography by ultra-violet light, and secondly, 

 intra-vital staining. 



ULTRA-VIOLET PHOTOGRAPHY 



Certain structures in the cell, although transparent to all visible wave lengths 

 of light, and therefore colourless, are more or less opaque to ultra-violet light. So 

 that if our eyes were sensitive to this light the objects in question would appear 

 coloured. Now the photographic plate is sensitive to ultra-violet light, and 

 Kohler (1904, pp. 129-165 and 273-304) has shown the possibility of photo- 

 graphing cells by this means. Fig. 10 gives photographs illustrating the fact. 

 It will be noted that, although transparent and colourless to ordinary light, the 

 nucleus is particularly opaque to light of the wave length of the ultra-violet. 

 Unfortunately, the method has not as yet been made much use of, owing to the 

 necessarily elaborate nature of the apparatus required. 



Related to the method described above is that in which the fluorescence produced by ultra- 



