CHAPTER IT 



Phase Contrast Microscopy 



1. TRANSPARENT OBJECTS 



A TRANSPARENT object is an object that can only be distinguished 

 in the medium encompassing it by the variations of its refractive index 

 or its thickness. No variation in hght-intensity will detect its presence, 

 the field of view remaining uniform. Such objects, frequently called 

 "dephasing", obviously escape normal observation although their role 

 in microscopy is a large one. 



Transparent objects are many in biology and microscopists have 

 to stain them in order to render them visible. This method is par- 

 ticularly detrimental when living objects are observed and this fact 

 gives much prominence to phase- and interference-contrast methods 

 as they enable one to study living tissues under conditions just as 

 good as when studying tissues that died after staining. 



Transparent objects are met with in many other branches of science 

 besides biology. For instance, inequalities on metallic surfaces having 

 a uniform reflection act as transparent objects and can be observed 

 by either phase- or inteiference-contrast methods. Structural uneven- 

 ness in crystals, index variations in an inhomogeneous liquid, de- 

 formations of a liquid surface and so forth are transparent objects. 

 Even when not unduly small, transparent objects are not perceivable 

 in ordinary microscopes because they lack in contrast when compared 

 to the remainder of the field. Both contrast and interference micro- 

 scopes render such objects visible. 



2. PRINCIPLE OF THE PHASE-CONTRAST METHOD 



The phase-contrast method was invented by the Dutch physicist 

 Zernike who was awarded the Nobel prize in 1953. 



Originally used by its inventor for inspecting telescope mirrors, 

 the phase contrast technique was applied to microscopy shortly after- 



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