LIGHT (OPTICAL) MICROSCOPY 



the microscope. The shape or nature of such 

 particles cannot, of course, be determined, 

 but they can be detected, and some estimate 

 made of their size by inference from their 

 brightness, and the speed of their Brownian 

 movement, if the particles are suspended in a 

 hquid. 



Since the limitation on the smallest par- 

 ticle which can be made visible is imposed 

 by the level of energy of the illuminating 

 beam, the ultramicroscope uses an extremely 

 bright light source, such as a carbon arc, 

 and concentrates the light energy by means 

 of a medium-power microscope objective 

 into a horizontal beam at right angles to the 



IMAGE PLANE 



REAR FOCAL 

 PLANE OF OBJECTIVE 



CONDENSER - 



UNDIFFRACTED 

 ORDER 



LIGHT SOURCE 



Fig. 7. The phase contrast microscope. The 

 illumination is restricted to a hollow cone by the 

 Annular Diaphragm, which is imaged into the 

 Phase Shifting Element by the lenses. Interfer- 

 ence, producing a visible image, occurs between 

 the diffracted light passing around the Phase 

 Shifting Element, and the direct light passing 

 through this element. 



microscope optical axis. The object slide is 

 of special construction to admit this con- 

 centrated beam from the side. 



Phase Contrast. Phase contrast is an- 

 other form of illumination useful on trans- 

 parent objects, and in particular on live prep- 

 arations. The normal microscopic object is 

 seen because it has regions of different opac- 

 ity. In brightfield illumination a completely 

 transparent specimen is very difficult to see 

 in any detail, as all parts are equally dense. 

 Darkfield illumination shows up border ef- 

 fects in such specimens, due to edge scatter- 

 ing, refraction, and diffraction. Phase con- 

 trast, is of more value with transparent 

 media, due to its ability to reveal internal 

 detail. This method has found extensive use 

 in the study of transparent living prepara- 

 tions. 



Phase contrast is really more than just a 

 change in the form of illumination. It is bas- 

 ically a method of separating the diffracted 

 and undiffracted parts of the light, treating 

 them differently, and then re-combining 

 them under conditions such that they pro- 

 duce controlled visible interference effects. 

 The scheme produces a visible image of an 

 otherwise almost invisible object. 



The arrangement necessary for phase con- 

 trast is shown in Fig. 7. A clear annulus in 

 the focal plane of the condenser is imaged 

 at infinity by the condenser, and then re- 

 imaged by the objective in its upper focal 

 plane. The undiffracted light all passes 

 through this image, and, by means of an 

 annular phase pattern located in this focal 

 plane, is both reduced in intensity and given 

 a quarterwave phase shift with reference to 

 the diffracted light. The end effect of these 

 two changes in the undiffracted light, when 

 combined with the diffracted light passing 

 around the phase annulus, is to simulate the 

 phase and intensity distribution which would 

 be present in the objective focal plane if the 

 specimen had density variations rather than 

 refractive index variations. As a conse- 

 quence, the image formed by the interfer- 



452 



