PHASE-CONTRAST MICROSCOPY 



75 



object which is an outline of index and thickness changes in the cell. 

 Figure 2.9(b) shows the structure of the phase-contrasted image. The 

 whole cell evinces but slight contrast in relation to the remainder of 

 the field, the discontinuities originated by the cell edges appear in m 

 and m' . The imaged inner details conform to their structure. 



Making a phase-plate opaque brings about a central dark ground. 

 Theoretically, contrast is maximal since the dephasing details are 



(b) 



m 



m 



Fig. 2.9. Image structure of an extended object. 



brightly outlined against a dark ground. However, this makes image 

 interpretation more tricky. It should be borne in mind, that, in phase- 

 contrast microscopy, intensities are proportional to the object-origi- 

 nated phase (equations 2.4 and 2.5) whereas those on dark ground 

 are proportional to the squared phase. If the object dephases but 

 slightly, viz. if cp is small, its square is still smaller. Therefore a dark 

 ground requires intense sources that emphasize the minutest surface 

 defects such as dust and so forth. Under such conditions the details 

 displacing the phase inconsiderably are always clouded by the diffracted 

 hght, originated by improperly focused defects. 



5. PERCEPTION LIMIT AND RESOLVING POWER. EFFECTS OF STRAY LIGHT 



Let us consider an object not discernible in an ordinary microscope 

 because it is transparent and not owing to its minute size. Therefore 

 it exhibits no contrast with the remainder of the field. When observed 

 in a phase-contrast microscope with an absorbing phase-plate, the 

 imaged contrast is educed from expression 2.9. This expression implies 

 that the microscope is perfectly devoid of stray hght and the values 

 so derived are therefore critical. 



Let us consider a small transparent disk, giving rise to phase 

 difference cp, and the diameter of which is, e.g. 4 to 5 times that of 

 the diffraction disk. The image obtained in phase-contrast microscopy 



