538 Microscopy /29 : 2 



types of microscopes, are emphasized. This is by no means an exhaustive 

 survey of the varieties of microscopes used in current biological research. 

 New and different variations are being introduced and used. However, 

 the basic theory outlined in this chapter remains unaltered, as do the 

 goals of obtaining more and more contrasting images of almost identical 

 objects and at the same time decreasing the lower limit of resolution. 



2. The Bright-Field Light Microscope 



The essentials of the bright-field light microscope (ordinary light microscope) 

 are shown in Figure 1. Light from a source strikes the mirror and 

 passes through the condenser and diaphragm. The condenser can be 

 adjusted so that the light is focused on the specimen or so that the light 

 is a parallel beam at the specimen. The diaphragm can be regulated 

 to control the light reaching the specimen. The objective forms a 

 real image of the specimen about 20 cm above in the microscope tube. 

 This real image is finally magnified by the eyepiece which forms a 

 virtual image 25 cm from the eye. The magnification of the objective 

 is the ratio of the imag# distance divided by the object distance. The 

 image distance is fixed by the geometry of the microscope, but the 

 object distance can be varied. The stronger the lens, the smaller this 

 distance and hence, the greater the magnification. 



The useful magnification is limited by the resolving power; that is, 

 there is a certain minimum distance of separation below which the images 

 of two points cannot be separated, no matter how great the magnifica- 

 tion may be. This limit of resolution is determined both by the wave- 

 length of light used and by the geometry of the objective. The dis- 

 cussion which follows is an outline of the mathematical proof of the 

 equation for the limit of resolution. 



To develop the expression for the limit of resolution of a microscope, 

 consider first the simpler problem of a grating placed in the path of a 

 parallel light beam, as shown in Figure 2. If the diffracted light then 

 passes through a converging lens, all parallel rays incident on the lens 

 will be focused onto a line in the focal plane of the lens. At some lines 

 in the focal plane, the various light rays originating from different lines 

 on the grating will cancel, whereas at others they will reinforce. These 

 bright lines are called the diffraction orders. From Figure 2, one can see 

 that bright lines will occur at angles 0, satisfying the relationship 



nX = bsind (1) 



where n is an integer, A the wavelength, and b the space between lines 



