410 JAMES HILLIER 



to the considerable reduction in resolving power that accompanies 

 the introduction of an aperture. In the electron microscope the 

 wavelength is so small that the numerical aperture can be as small 

 as 0.003 before there is any impairment of resolving power due to 

 diffraction effects. The observed loss in resolving power is the result 

 of a technical difficulty of manufacturing a sufficiently small aperture 

 and having its surface uniformly conducting electrically. There is, 

 in this regard, the additional problem of contamination of the aper- 

 ture due to electron bombardment and the subsequent charging of 

 the resulting semi-insulating coating. 



2. Limitations Due to Preparative Techniques 



In the use of the electron microscope in the field of biology, by 

 far the most important limitation of the method is the set of condi- 

 tions imposed on the preparation of the specimen. Of these, the most 

 important is the fact that the specimen must be dried. Next in 

 importance is the fact that the specimen must be prepared so that it 

 is less than a quarter of a micron in thickness. Finally, the specimen 

 must be able to withstand high vacuum and often high temperatures 

 due to the electron bombardment. 



Desiccation Artifacts. Any microscopist interested in biological 

 materials is familiar with the disastrous distortion that many speci- 

 mens undergo in the final stages of drying. At the same time he is 

 aware that under some conditions it is possible to dry biological ma- 

 terials without any apparent change as far as the light microscope is 

 concerned. Generally speaking, the electron microscopist has not 

 been quite as aware of the serious nature of this problem because the 

 objects that tended to show the maximum distortion on drying were 

 in general objects that were too large for the electron microscope in 

 any case. At the same time, studies on the size and shape of viruses 

 under natural conditions have agreed remarkably well with those 

 obtained with the electron microscope in the dried condition, so that 

 the electron microscopist is reasonably certain that for objects of the 

 size of viruses drying appears to have little, if any, effect. Inter- 

 mediate between these extremes lie the bacteria, which to the casual 

 observer appear reasonably normal in the electron micrographs. 

 Critical bacteriologists, on the other hand, have realized that most 

 of the published electron micrographs of bacteria have shown evidence 

 of considerable shrinkage of the protoplasm away from the cell wall. 



