CHAPTER IV 



THE TECHNICS OF 

 PHASE MICROSCOPY 



The phase microscope utilizes absorption and optical path differences 

 for the control of the contrast visible in the image. Increase or decrease 

 of contrast, or reversal of bright and dark elements with increased or 

 decreased contrast, facilitates the work of the microscopist. Phase 

 microscopy is most valuable for specimens too transparent for effective 

 examination with other types of microscopy. When the specimen detail 

 consists of small differences in optical path due to variations in refractive 

 index and thickness, regions of absorption, or both, the phase micro- 

 scope should be used. Such specimens may be unstained living cells 

 and tissues, suspensions and emulsions, glass, plastics, minerals, poorly 

 stained or slightly pigmented objects, and similar materials. The phase 

 microscope is used also in the orientation and preparation of specimens 

 to be examined with the electron microscope. 



When the image is brighter or lighter than its surround (Fig. IV. IC) 

 it is described by the term bright contrast; dark contrast (Fig. IV. ID) 

 designates a specimen darker than its surround. When the optical path 

 of the specimen is greater than that of its surround the A+ diffraction 

 plate will give bright contrast and the A— and B— diffraction plates 

 dark contrast. When the surround has the greater optical path the 

 specimen will show in dark contrast with the A+ and in bright con- 

 trast with the A— diffraction plates. The contrast can be determined 

 by focusing in the same direction slowly through the specimen. When 

 the contrast is bright above the specimen, dark when in exact focus, 

 and then bright on continued focusing, dark contrast is being observed. 

 Likewise when the focus is dark as the specimen is approached, bright 

 at exact focus, and then dark, the contrast is called bright. 



Bright contrast is preferred, in general, for counting, for the study of 

 motion, for very small specimens, for stereophotomicrography, and with 

 optical staining. Dark contrast is preferred for measurement, for 

 specimens to be compared with stained material from other methods, 

 and for enhanced contrast with absorbing specimens. When appropriate 

 the B— dark contrast is useful for measurement. The Eberhard 



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