182 THE TECHNICS OF PHASE MICROSCOPY 



glass are available. A cover glass can be anchored with a bit of Scotch 

 tape when the oil immersion objective is to be used. Since the air 

 between the specimen and the cover glass reduces the numerical aperture 

 to about 0.9 there is little gain with an immersion objective at such 

 decreased resolution. 



These tiny specimens exhibit Brownian motion which interferes with 

 clear observation. When the choice of surrounding medium is not 

 important, a more viscous mounting medium may be helpful. When 

 measurements are required, it is usually necessary to take a photo- 

 micrograph at a speed that will give a sharp picture and then measure 

 the photomicrograph (see Section 3.6). 



When the refractive indices of a two-phase system are known, it is 

 possible to identify the inner and outer phases. Particles of several 

 sizes are examined with an A+ and A— phase objective. The phase 

 of higher index will be bright with the A+ and dark with the A — 

 diffraction plates (see Section 9 of Chapter \T). To avoid a possible 

 ambiguous example it is well to pass judgment only after regions of 

 several different sizes have been observed and all found to exhibit the 

 same contrast behavior. 



Degree of homogenization, uniformity of particle size, and presence 

 of undissolved crystalloids can be determined l)y phase microscopy. 

 With proper cells and electrodes electrophoretic charge and migration 

 may be determined for otherwise transparent specimens. Some caution 

 should be exercised when inhomogeneous systems are examined, to avoid 

 incorrect interpretation; e.g., it may not be possible to distinguish a 

 spherical bacterium from a similar globule of fat in milk, when their 

 path differences are the same. Shape differences are diagnostic, and 

 slight differences in path may be observed with diffraction plates of 

 different transmissions. Specific examples and recommendations will 

 be cited in the following chapters. 



3.4. Slightly absorbing specimens 



The generalization of the theory for phase microscopy included both 

 absorbing and retarding specimens and indicated that phase microscopy 

 is useful with pigmented materials as well as with colorless ones (Bennett 

 et al., 19-16; Barer, 1949). When the contrast from highly absorbing 

 specimens is good, adding densiphase contrast may not make a noticeable 

 difference, but, when the absorption contrast is low, the added contrast 

 may be enough to make the specimen visible. Even with contrasty 

 preparations (metal-impregnated nervous tissue) the contrast added with 

 phase brings out details that would be overlooked with brightfield 



