ilicro:7 



now being visible. These visible intensity differences are related to 

 the ordinarily invisible structural details of the object wtiich do have 

 in themselves slight variations in thickness or refractive index, but 

 which are not sufficiently great to be visible in the bright-field 

 microscope. The phase shifting plate installed in the objective supplies 

 a one-quarterwave shift in phase of the undiffracted light rayr,; and when 

 these are combined with the diffracted light rays, the resultant inter- 

 ference effects of the two portions of the light which had come from the 

 object form the image of the object. 



Various kinds of phase contrast objectives are available and the pro- 

 spective user may wonder what the differences are and which lenses to 

 chose. Perhaps, some further explanation will help in this matter. 

 Differences in contrast of the specimen image can be obtained depending 

 on whether the undiffracted rays of light, as they pass through the 

 phase shifting plate, are accelerated or retarded one-quarter wave 

 length with respect to the diffracted rays of light. If the undiffracted 

 rays are accelerated, the resulting contrast appearance of the object is 

 called "positive" or "dark contrast." "Dark" contrast reveals the 

 object structures in a manner similar to that seen in a specimen stained 

 with hematoxylin when viewed with a regular bright-field microscope. 

 That is, denser structure appear darker. Thus, in "dark contrast" phase 

 the regions of the object having the greater optical path (thickness X 

 refractive index) appear darker than the surrounding background. 



If the phase shift of the undiffracted rays was to retard the rays one- 

 quarter wave length with respect to the diffracted rays, "negative" or 

 "bright contrast" results. Regions in the specimen having greater 

 optical path appear bright against a darker background. The image is 

 similar to that seen by darkfield illumination. The general preference 

 is to use "dark contrast" phase because of the similarity of image 

 appearance to that obtained with stained objects. 



The phase altering plate serves the additional function of balancing the 

 intensity of the undiffracted rays of light with respect to the diffracted 

 rays. If one or the other of the two rays is too intense, it "drovms" 

 out the other, obscuring the image contrast. Regulation of this light 

 balance by means of a suitable metalic film deposited on the phase 

 altering plate provides a way of controlling the contrast which can be 

 ranged all the way from no contrast to a psuedodarkfield contrast 

 appearance. The best results for most work lies somewhere in between 

 so that the various degrees of density of the subject are visible as 

 gradations of darkness. 



The ability of the phase microscope to make visible structures which are 

 very difficult to see by the bright-field microscope is remarkable and 

 have proved of great value in the study of biological materials, I am 

 sure you know of this. However, there is one inherent aspect to phase 

 contrast microscopy wtiich has limited its usefulness in nematology, at 

 least. This is the necessity of working with very thin subject materials. 

 Even the body diameter of a sinall nematode renders it too thick for 



