542 Microscopy /29 : 3 



a compound lens, namely the objective, limits the resolving power. 

 However, this introduces no major changes in the foregoing theory. 

 Equation 5 can still be used to find the limit of resolution of a micro- 

 scope. Using blue light A, about 0.45 micra, and a numerical aperture 

 of 1.25, 1 one can find that the limit of resolution b is 



, 0-45 nQ . 



b = k rrrt = 0.2 micra 



2 x 1.25 



If the light, other than parallel, is used to illuminate the specimen, the 

 constant 2 in the denominator of the right-hand side of Equation 5 will 

 not be correct. However, the error is so slight that it may be ignored. 



The bright-field light microscope can also be used to determine the 

 amounts of various pigments present in regions whose linear dimensions 

 are larger than the limit of resolution. To accomplish this, a micro- 

 scope is combined with a spectrophotometer to produce a microspectro- 

 photometer. In this variation, the light source is passed through a 

 monochromator ; the eyepiece is replaced by a lens which forms a real 

 image at the surface of a light-sensing device such as a photomultiplier 

 tube. By measuring the relative optical density of neighboring portions 

 of a cell at various wavelengths, it is possible to locate many pigments 

 such as cytochromes within a cell. In all microspectrophotometers, 

 resolution is sacrificed to make quantitative absorption measurements 

 possible. 



The bright-field light microscope is a standard tool. In order that it 

 be useful, the specimen must possess regions in which different amounts 

 of light are absorbed and must have linear dimensions larger than 

 0.2 micra. The former restriction makes it necessary to fix and stain 

 most specimens. The microscope in itself cannot help to distinguish 

 artifacts due to fixing and staining from the inherent properties of the 

 specimen. Further, the limit of resolution prohibits the observation of 

 virus particles and makes it difficult to resolve the details of the structure 

 of bacteria and mitochondria. 



3. The Dark-Field Microscope 



This section and the three following deal with methods of increasing the 

 contrast of the images seen in a microscope without resort to staining 

 and fixing techniques. By far, the oldest of these methods is dark-field 

 microscopy, also called ultramicroscopy . In this method, the specimen is 

 illuminated by a beam of light which has such a shape that no direct 



1 For air, the numerical aperture, NA, is always less than one. The value 1.25 

 is typical of a high quality of immersion lens. 



