29 : 5/ Microscopy 545 



objective to give the image which finally is magnified by the eyepiece. 

 If the diffraction plate introduces about a half-wavelength phase 

 difference, then the deviated and undeviated beams will subtract from 

 one another, giving a dark spot where they are equal. In this fashion, 

 an almost transparent object, which refracts the light even slightly, 

 will appear dark. This occurs because some of the light going through 

 it will be in the deviated beam and some in the undeviated beam, and 

 these two are combined to form the final image. 



Absorbing Film 

 Deviated Beams 



deviated Beams I 



- Dielectric for Phase Shift 

 Glass 



Undeviated Beams 



Figure 6. A diffraction plate. 



In the phase-contrast microscope, an object or area scattering or 

 refracting very little light will appear bright, as its image will be pro- 

 duced almost entirely by the undeviated beam. Moreover, an object 

 strongly refracting the light passing through it will appear bright because 

 its image will be produced by the deviated beam. By varying the 

 amount of absorption in the undeviated beam, one can increase the 

 contrast of various objects. By making the phase change slightly 

 wavelength dependent, one can make colorless, almost-transparent 

 objects appear colored. This process is called colored phase-contrast 

 microscopy. 



The extreme of absorbing the undeviated beam consists of removing it 

 altogether. In this case, one has the dark-field microscope as a special 

 example of the phase-contrast microscope. 



Phase-contrast microscopy is very useful for counting living sperm 

 cells, observing changes within living cells, and showing structures not 

 readily apparent with staining. The resolving power, using phase- 

 contrast microscopy, is, in general, higher than with dark-field micros- 

 copy but is lower than with bright-field light microscopy. In particular, 

 many small objects are surrounded with an extra ring (halo) which 

 cannot be removed by focusing or by altering the phases. 



5. Interference-Contrast Microscopy 



In the phase-contrast microscope, the final image is produced by the 

 interference of beams coming through the same or neighboring regions 



