INSTRUMENT CLASSIFICATION AND APPLICATIONS 



All excellent and exhaustive account of 

 this subject with an extensive bibliography 

 has been published by Davies (8). It is also 

 treated extensively by Hale (18). Both these 

 accounts discuss the errors which may be 

 encountered in this work. 



In biological applications it is usually 

 necessary to make use of the highest possible 

 powers, so only instruments which allow 

 this can be used. In particular, instruments 

 in which slit illumination is used are of little 

 value. 



The nature of the object may also have a 

 bearing on the type of microscope to be used. 

 If the object is in the form of small isolated 

 patches, such as a widely scattered distribu- 

 tion of cells or groups of cells, each small as 

 compared with the diameter of the field, a 

 shearing type of instrument can be used, such 

 as that described by Smith (6). If, on the 

 other hand, the object is of the same size as 

 the field or somewhat larger, the Dyson (1) 

 microscope may be more useful. If the object 

 is not too thick, interference contrast can 

 still be obtained even if the object occupies 

 the whole cross-section of the reference 

 beam. Under these conditions it is still pos- 

 sible to make meaningful measurements of 

 the path difference. The reason for this is 

 that the object in the reference beam is so 

 far out of focus that the effective background 

 path difference does not vary appreciably 

 over the limited area of the cell being meas- 

 ured. 



Applications to Opaque Specimens 



The literature on the use of interference 

 microscopy to opaciue specimens is much less 

 extensive than that covering biological 

 applications. However, it is being used to an 

 increasing extent for the measurement of 

 surface finish in engineering (12) and to the 

 control of the groove profile of diffraction 

 gratings. 



For opaque specimens the interference 

 method has considerable advantages over 

 the phase contrast technique. It is funda- 



mental in phase contrast that the surface 

 of the specimen acts as part of the optical 

 train which images the illuminating annulus 

 on to the phase ring, and it obviously will be 

 quite unsuited for this purpose if the surface 

 introduces large path differences, as for 

 example in the case of a machined sui'face. 

 Even if the surface is relatively smooth, a 

 spherical or cylindrical object will introduce 

 obvious difficulties. 



In addition, in view of the large phase 

 differences commonly observed in reflecting 

 specimens, the image seen by phase contrast 

 may bear very little resemblance in detail 

 to the actual surface topography. An ex- 

 ample of this (a diffraction grating) is shown 

 in Reference 9. 



Interference microscopes of Class I are 

 immune to both these difficulties. 



An investigation has been made by Tol- 

 mon and Wood (19) of an error which may 

 arise in any high-power interference micro- 

 scope, but Avhich is most often met with in 

 connection with opaque specimens. This 

 concerns the relationship between the fringe 

 spacing and the angle of slope of the surface; 

 they show that errors of the order of 10% 

 may arise if the angle of obliquity of the 

 reflected beam is not taken into account. 



A discussion of the micro-interferometry 

 of opaque objects with a description of 

 several instruments is given by Perry (20). 



REFERENCES 



1. Dyson, J., Proc. Roy. Soc, (London), A204, 



170-187, 1950. 



2. LiNNiK, W., see Kinder, W., Zeiss Nachr., 



August, 1937. 



3. Smith, F. H., British Patent No. 639,014, 



June '21, 1950. 



4. Dtson, J., Nature, 171, 743 (1953). 



5. Lebedev, A. A., Rev. Opt., 9, 385 (1930). 



6. Smith, F. H., Research, 8, 385-395 (1955). 



7. Davies, H. G., in "General Cytochemical 



Methods," Ed. J. F. Danielle, New York, 

 pp. 55-161, Academic Press, 1958. 



8. FRANgoN, M., "Le Microscope a Contraste de 



Phase et le Microscope Interf^rentiel." 

 Paris: Editions du Centre National de la 

 Recherche Scientifique, p. 113, 1954. 



419 



