MICROSCOPY 



The ability to resolve neighboring objects as being distinct and sepa- 

 rate depends on the degree of contrast between the objects and the 

 resolving power of the viewing device. In the case of the unaided human 

 eye, the limit of resolution is considered to be only about 0.1 mm (100,«) 

 under optimal viewing conditions. This means that if two adjacent ob- 

 jects are separated by a distance less than 0.1 mm they will appear as 

 one object to the eye. The limitation imposed by cell size on direct 

 observation with the eye requires that cells and their component parts 

 be studied with the aid of an instrument such as the microscope, which 

 operates not only to magnify but also to give increased resolution 

 (Figure 11-8). 



The component of the optical system of any microscope which is 

 responsible for the initial magnification of the specimen is the objective 

 lens (Figure 11-8). This lens also determines the resolving power of 

 the microscope, which is a function of ( 1 ) the light-gathering power or 

 numerical aperture (N.A.) of the objective lens, and (2) the wave- 

 length of radiation used to illuminate the specimen. The resolving 

 power, in terms of the size of object which may be seen, is determined 

 from the Abbe formula as follows: 



P- Q-61X 



where / is the wavelength of the illumination and N.A. is the numerical 

 aperture of the objective lens. The value, 0.61, is a constant represent- 

 ing the minimum difference in contrast that is detectable. Consideration 

 of the above formula shows that with objectives of equivalent numerical 

 aperture, the resolving power of a microscope is inversely proportional 

 to the wavelength of light used. That is, the resolving power increases 

 with decrease in wavelength of the illumination. For a microscope using 

 an oil-immersion objective of N.A. = 1.2, the Abbe formula gives a 

 limit of resolution of about 0.27 fi for visible light of mean wavelength 

 5400 A, and about 0.13^ for ultraviolet radiation of wavelength 2600 A. 

 On a similar basis, the same microscope using infrared radiation of 

 8000 A wavelength would have a resolving power of only about 0.4,(/. 



Microscopy includes many different kinds of instruments and tech- 

 niques. However, all microscopes, regardless of their design, are /nag- 

 nifying devices which produce an enlarged image of the specimen under 

 study. This means that any information derived by use of the microscope 

 must come ultimately from examination of the enlarged image it forms. 

 Image formation is determined largely by the particular wavelength of 



SURVEY OF CYTOLOGICAL TECHNIQUES / 223 



