SLIGHTLY ABSORBING SPECIMENS 183 



microscopy and shows more detail within the less dense parts of the 

 specimen. Regions showing little detail, as the eosine-stained part of 

 a spinal cord, may reveal added information, such as inclusion bodies, 

 when examined with the phase system. Combined staining and mount- 

 ing methods should also be helpful (Zirkle, 1940). 



Since the eye is more sensitive to small bright regions on a dark 

 background than to the reverse, it may be helpful to reverse a dark 

 image to bright contrast with an A+ phase objective for detailed study. 

 Some ray tracks in photographic emulsions show better in this manner 

 (Section 9 of Chapter V). 



In general, the phase microscope is most useful for studying absorbing 

 specimens when their image is too weak for clear vision with brightfield 

 methods. The IB— 0.25X type of diffraction plate usually provides 

 the best improved contrast, although the 2.5B— 0.25X plate may give 

 greater visibility with some specimens. The A+ plate is useful when 

 reversal of the contrast is desired or when the specimen has a lesser 

 optical path than its surround. With a few absorbing materials the 

 A— type of diffraction plate gives better contrast than the others. 

 Typical examples of the application of phase microscopy to stained 

 materials are given in Table V.2, although the European work may need 

 revaluation as they have the opportunity of using other than A — 

 diffraction plates. 



Old, faded preparations may have enough stain left for good visibility 

 with the phase microscope long after becoming invisible with the bright- 

 field microscope. Thus some museum collections may have their value 

 restored. Likewise light staining combined with phase contrast may 

 reveal more detail than either method would alone. Dr. Jack Schultz 

 reports that chromosomes slightly stained with light green have marked 

 densiphase contrast. Lindegren (1947) obtained better visibihty of 

 yeast structure with a B— phase objective with his acetoformol- 

 toluidin blue chromosome stain. Barer (1947, 1948a, h) and Haselmann 

 (1948a, b) report investigations under way with stained material. 

 Interpretation of densiphase contrast is more difficult, because optical 

 path differences may occur within the pigmented regions, as well as 

 selective absorption of radiation of certain wavelengths. When bleach- 

 ing of the absorbing material is possible without otherwise damaging the 

 specimen, direct comparisons may be made. Measurement with phase 

 before staining may yield pertinent information on the finer structure of 

 specimens. Modern controlled staining methods should be combined 

 with phase microscopy (Petrunkevitch, 1937; Stowell and Alber, 

 1943). Spectrophotometric methods offer further promise. 



