PHASE-CONTRAST MICROSCOPY 



65 



wards. Zernike's initial work was published before the last war but 

 effective development of phase-contrast microscopy was not under 

 way before 1946. Complete investigation of phase contrast requires 

 some mathematical data which are not indispensable, however, if the 

 study is restricted to basic phenomena. P is a transparent object 

 observed in a compound microscope (Fig. 2.1). The filament of the 



Direct light 



diffracted light 

 Fig. 2.1. Diagram of the ray paths through a microscope. 



source So is focused on to the iris diaphragm A of the condenser 

 through the lens L. The diaphragm D^^ is in the focal plane of the 

 condenser's optical system C. Under such conditions, were the source 

 but a pin-point, a beam of parallel rays would pass through the 

 specimen P. In fact, the source is always spread over a certain area: 

 each of its points originate a pencil of parallel rays so that an infinity 

 of such pencils pass through the specimen at various angles. Against 

 the lens L is the iris diaphragm Do whose distance from the condenser 

 is such that C actually images Dq on to the specimen. The specimen 

 P is observed by the microscope proper, consisting of the objective O^ 

 and the eyepiece Oo. The afore-mentioned Kohler illuminating system 

 is used. 



Let us consider, for instance, a small transparent bacterium A, 

 assuming the lamp Sq to be a source of monochromatic light. The 

 bacterium is invisible, not on account of its size, but merely because 

 its contrast in relation to the remainder of the field is too low. Owing 

 to the incident light, the bacterium diffracts a small amount of light 

 in a cone that widens out as the bacterium is smaller. In Fig. 2.1 

 the hatched area shows the portion of diffracted fight that passes into 

 the microscope. 



Let Di be substantially diaphragmed down, thus using only but 

 a small portion of the filament imaged at S[ . In this plane, the beam 

 of direct light covers but a very narrow area whereas the beam of 

 diffracted light overspreads amply. This point is essential and will 



