THE PHYSICAL PROPERTIES OF INFECTIVE PARTICLES 291 



especially unfortunate whenever attempts are made to assess the effective- 

 ness of staining procedures on very small particles. If the image of a specimen 

 object is formed further from the lens than its proper position it is said to be 

 "underfocused." An underfocused image of a circular object is characterized 

 by having a bright interference halo around its periphery, lending to the 

 image an enlianced contrast. Resolving power is decreased by miderfocusing, 

 but in the interests of contrast it is common practice to miderfocus slightly. 

 An overfocused image has both a loss of resolving power and of contrast. 



b. Techniques of Electron Microscopij. With many biophysical methods the 

 theoretical complexities and uncertainties underlying the interpretation of 

 the experimental data outweigh in importance a consideration of the tech- 

 niques used to secure the data. In some cases the techniques are well- 

 established and have not changed fundamentally for years. In electron 

 microscopy there is no great body of theory that has to be considered in 

 interpretation of micrographs, and in those cases where interpretation is 

 obscure the most relevant factor is usually a consideration of the technique 

 employed. Methods assume a relatively large importance in electron micro- 

 scopy, also, because it is a new field in which the full usefulness of the instru- 

 ment has yet to be explored. 



i. Virus Suspensions, Morphology. The electron microscopy of suspensions 

 of virus particles, either intact or disintegrated, can be conveniently divided 

 into two types of investigation. One of these is morphological in character, 

 where the shape, size, and structural arrangement of virus particles or their 

 subunits are being examined. The other is essentially quantitative in nature, 

 where the purpose is to count the numbers of various kinds of particles in a 

 known volume of suspension. The development of techniques for the former 

 type of investigation has tended toward enhancement of the effective 

 resolving power of the microscope and toward the preservation of structure 

 of the virus particles. In the latter type of work considerations of morphology 

 are secondary. Rather, experimental advances have been directed toward 

 securing specimen fields that are qualitatively and quantitatively represen- 

 tative of the entire suspension being investigated. 



As has been indicated above it is necessary to enhance the contrast 

 exhibited in the images of small virus particles before anything useful can 

 be told about their structural details, either interior or exterior. Unfortun- 

 ately, electron stains for virus particles are so poorly developed that little 

 can be said about enhancement of interior detail. The exterior form and 

 surface detail, however, can be greatly accentuated by the application of 

 shadowing methods (Fig. 6). The principal objectives in the development of 

 these methods are to secure a substrate surface that is devoid of apparent 

 irregularities, and to obtain shadowing films that show no evidence of granu- 

 larity. While conditions have existed for some time by which detail of the 



