Micro: 5 



or crystaline properties. 



Since V/orld War II, important developments in light microscopy have come 

 about. The theoretical liiriits to the amount of useful magnification and 

 the degree of resolution of microscope lenses have long been knovm and 

 refinements to attain the theoretical limits are being incorporated into 

 the new microscopes. Surface coatings of lenses, new optical designs 

 with new kinds of glass, all are helping the microscopist. In addition, 

 there hag been a trend in the development of the light microscope to 

 Utilize the interference phenomenon of light. This trend has been for 

 the purpose of making the invisible, or near invisible, structures of 

 specimens visible. Such structures are not necessarily difficult to see 

 because of smallness of size, but rather because of their transparency. 



This particular kind of problem was lirst overcome with the development 

 of the phase contrast microscope . Commercially available models became 

 available shortly after World War II. Within the past two years, another 

 new type of microscope became available and has as a main characteristic 

 the ability of making invisible transparent structures visible to the eye. 

 This microscope is called the interference contrast microscope . The 

 principle utilized in both types of microscopes is the phenomenon of 

 interference of light waves. Variations in light intensity come about 

 as two beams of light are combined and their waves reinforce or interfere 

 with each other, depending upon their phase relationships. Variations 

 in light intensity in the specimen render it visible. Despite the in^jli- 

 cation in having two different names, phase and interference contrast 

 microscopes both are light interference type of instruments. They do 

 have important differences and applications as will now be discussed. 



Phase Contrast Microscopy 



The optical path in the phase microscope is diagrammed in Figure 1. A 

 single beam of light coming from the illuminating source is focused as 

 a hollow cone of light onto the specimen by the substage condenser. In 

 accordance with the structure and refractive properties of the specimen, 

 the light beam becomes refracted into various individual light rays 

 going in various directions. The phase objective is designed in such a 

 way that the light rays it intercepts from the object structure are 

 divided into two beams of light. These beams are made up of (l) the 

 light rays which enter the microscope objective directly after having 

 passed through the object without being deviated or diffracted by the 

 structure, and (2) the light rays which enter the objective lens after 

 having been diffracted from the original cone of light by the object 

 structure. In order to utilize the interference phenomenon it is 

 necessary to put one of the light beams out of phase with the other. 

 This is done by a phase altering plate which is placed in the objective. 

 The two light beams, consisting of the diffracted and undiffracted rays, 

 are then recombined into focus at the image plane where they are viewed 

 and further magnified by a regular ocular or eyepiece. However, because 

 one of the beams is out of phase with the other, when the two are recom- 

 bined interference occ\irs. This results in light intensity differences 



