GENERAL MKKOSCOPY 



necessary for the microscopist to be pre- the numorifal aperture of the system by 



occupied with optics; nevertheless, there arc 1000. This rule is based upon the relationship 



certainrules which must be observed in order between the resolving limits of the eye and 



to realize the best instrumental performance, the microscope system. The resolving limit 



of the human eye is 0.2-0.3 mm at the 

 Resolving Limit, Useful Magnification, normal viewing distance of 25 cm. Therefore 

 and Contrast ^q render visible the structure resolved by a 

 Detailed accounts of the factors governing lens of 1.4 numerical aperture, a minimum 

 resolution, and, in turn, useful magnification magnification of 1000 X is required. If a 

 have been given in many publications (1, 2). photomicrograph is going to be viewed at 

 The resolving limit of a microscope may be distances greater than 25 cm, then a mag- 

 defined as the ability to register two objects nification exceeding that indicated by this 

 whose centers are separated by the least rule is warranted. 



distance, d, at which the objects may be Until now^ we have been dealing with 



detected as distinctly separate. The mini- lateral resolution, ignoring vertical resolu- 



mum object diameter is usually not less than tion, or as it is usually termed, "depth of 



twice the least detectable distance of separa- field." As lateral resolution increases, depth 



tion, the latter varying directly with the of field decreases. Therefore, to perform an 



wavelength of light, X, and inversely with observation of structure in depth, usually 



the refractive index, n, of the medium in some compromise is necessary. Obviously, 



which the object is immersed and with sin it is not always of greatest advantage in a 



a, where a is one-half the angular aperture study to exploit upper resolving limits of a 



of the objective lens. Thus, system. 



The rendition of contrast also is of prime 



d = - — : — . consideration. Although a subject may con- 



2m sin a . , , , i . i ■ 



tarn structure on a resolvable scale, this 



The resolution of a microscope is controlled structure may not be visible microscopically. 



simultaneously by the numerical aperture This lack of visibility is frequently due to 



(N.A. = n sin a) of the objective and con- the inherent optical properties of the speci- 



denser lenses. In fact, the numerical aper- men, namely, that the structure possesses 



ture of a system may be expressed as the the same light-transmitting or reflecting 



average of these two numerical apertures: qualities as the surrounding or background 



,, . , XT A medium. Contrast can be improved, both by 



TVT . / . \ N.A.obi + N.A.cond . , , , 



N,A. (system) = . optical methods and by specimen prepara- 



tion. 



For highest resolution, oil immersion lenses A microscope "sees" structure because of 



are available today with numerical apertures the optical characteristics inherent in a given 



up to 1.4, and a corresponding resolving limit specimen preparation which interact with 



of the order of 0.2 micron. Since no lens, the incident illumination and modify it. 



objective, or condenser can exceed a nu- Consequently, contrast may be improved by 



merical aperture of 1.0 in air, it is essential better optical monitoring of these altera- 



to use high index immersion oil on the con- tions or by enhancing the degree of light 



denser as well as on the objective to realize alteration by the sample through suitable 



the ultimate in resolution and magnification preparative methods. The most commonly 



from a light microscope. encountered optical factor controlling con- 



i The useful magnification of a microscope trast is the numerical aperture of a system, 



may be roughly calculated by multiplying As with depth of field, there is a loss of con- 



364 



