29 : 5/ Microscopy 



547 



This pattern will, in general, consist of sharp contour lines representing 

 equal differences of optical path length 3 between the object and its 

 surroundings. The interference-contrast microscope is most useful when 

 the maximum difference in the optical path lengths in the region of in- 

 terest is less than a quarter of a wavelength. Usually, if a rapid 

 change occurs in the optical path length as at the edge of a cell, the inter- 

 ference pattern will be very complicated. 



Col lima ted Beam 



I 



Metallic Coatings 



—Object Specimen 

 |j- *- To Objective 



■Coverg/ass 



Slide 



Figure 8. Multiple-beam interferometer adapted for biological 

 specimens. The final image is made up of beams of light 

 which have passed through the specimen many times. Small 

 differences in optical pathlength are made more significant by 

 multiple reflections. 



The interference-contrast microscope, shown in Figure 8, essentially 

 superimposes the specimen on one plate of an interferometer. If the 

 specimen is platelike in nature, distinct equal height contours will be 

 produced. The specimen height can then be measured to a small 

 fraction of a wavelength. In fact, with crystalline samples the limit of 

 resolution is of the order of 0. 1 nux. However, the width- and the length- 

 resolving powers are somewhat less than for the bright-field light micro- 

 scope. 



The greatest single advantage of interference-contrast microscopy is 

 that it enables the measuring of the heights of particles (or determining 

 the optical path lengths) with a precision that cannot be equalled by any 

 other method. In the usual biological applications, one is more 

 interested in determining the length and width; for these the inter- 

 ference-contrast microscopes of Figures 7 and 8 offer only slight advan- 

 tages over the phase-contrast microscope. Even the heights can be 



3 The optical path length is defined as the product of the path length times 

 the index of refraction. It enables one to find the length of the path in wave- 

 lengths. 



