290 H. K. SCHACHMAN AND R. C. WILLIAMS 



all the electrons entering the specimen leave it with their velocities un- 

 changed in magnitude and changed only in direction — the effects of elastic 

 scattering. An electron lens that would image all electrons leaving the 

 specimen, scattered or not, would deliver an image devoid of information, 

 since it would have no contrast from point to point. Actually, an electron 

 lens operates at a numerical aperture of only about 0.01 (as shown above), 

 the aperture limitation being usually effected by the insertion of a metal 

 disk with a very small central hole in the back focal plane of the lens. This is 

 the so-called "objective aperture." Its effect is to block all electrons scattered 

 through an angle greater than about 1°. Thus, a relatively thick specimen 

 that scatters electrons widely will be imaged less brightly than one that 

 scatters electrons throughout only a narrow cone. Contrast phenomena are 

 the direct result of scattering effects, the more higlily scattering objects 

 appearing relatively dark in the image. 



In the observation of particulate materials, such as suspensions of virus 

 particles, the limitations imposed upon the effective resolving power of the 

 electron microscope are largely those brought about by deficiencies in 

 contrast. Even when a very small objective aperture is used it is still a fact 

 that a virus like tobacco mosaic, with a diameter of 150A, appears with very 

 low contrast indeed, as ordinarily photographed. Although it is difficult to 

 set even an approximate figure, it might be estimated that with materials 

 of the chemical composition of viruses the effect of low contrast in electron 

 images changes the useful resolution of the electron microscope from its 

 theoretical value of about 6 A to a figure more like 50 A. 



Fortunately, the contrast exhibited by smaU objects can be artificially 

 enhanced. One way is to employ shadowing (Williams and Wyckoff, 1946), 

 whereby a thin film of a heavy metal is cast obliquely upon the specimen 

 surface. The result is that the condensed metal is unevenly distributed, 

 owing to topographical variations, affording contrast through the great 

 electron-scattering power of even a thin film of a heavy metal (Fig 5). By 

 application of this technique the effective resolving power of the electron 

 microscope approaches 15 A, where it is apparently limited by the structure of 

 the shadowing film. Another method of enhancing contrast is to impregnate 

 the specimen materials with a stain of high electron-scattering power— a 

 volume stain, as distmct from the surface-staining effects of shadowing. Osmic 

 acid (Porter and Kallman, 1953) and phosphotungstic acid (Hall et al., 1945) 

 have been the compounds of choice in such staming procedures, but it 

 appears that neither is particularly effective when applied to virus particles 

 imder reasonably normal pH conditions (HaU, 1955). 



Not only is the contrast in the images of small particles dependent upon 

 their electron-scattering power per unit thickness, but it is influenced from 

 one micrograph to another by the exactness of focus. This influence is 



