TIIIOKY AND TECIIMQl ES 



of area .4. Hence the reference beam for area 

 B goes through an area C, whicli is a distance 

 d to the right of B. This area C should be 

 kept clear when making measurements in 

 area B. 



Because of the separation of the object 

 and reference beams, any non-parallelism 

 such as a wedge in the slide, coverglass, or 

 moiniting medium becomes part of each 

 reading. However, since the optical paths 

 are determined by subtracting a reading in 

 the surround from a reading in the object, 

 the contribution of a uniform wedge drops 

 out. In order to be safe the reading in the 

 surround should be close in distance and in 

 time to the reading on the object. To detect 

 such a wedge the slide may be turned, pref- 

 erably by means of a circular stage, and any 

 change in luminance of the background 

 noted. 



In the systems employing polarized light 

 a birefringent specimen should be aligned 

 with its optic axis parallel to the vibration 

 direction of the polarized light (parallel to 

 the direction of shear). For manj^ biological 

 specimens the birefringence is too small to 

 cause appreciable error. For highly bire- 

 fringent specimens, methods have been de- 

 scribed by Faust (34). 



It has been shown (22) that with the AO- 

 Baker shearing system there is a condenser 

 aperture which produces the best contrast. 

 The user can determine this condition of 

 best contrast either by qualitative observa- 

 tions or by the use of a photomultiplier. 



With the above precautions precisions of 

 from one tenth to one three-hundredth of a 

 wavelength can be expected, depending on 

 the system used, the size and uniformit}^ of 

 the specimen, and the care of the observer. 



Special -Purpose Modifications 



As mentioned earlier, the total dry mass of 

 cells or nuclei can be determined by inte- 

 grating the measured dry mass per unit area 

 over the area of the specimen. In cases where 

 the measurements must be done c^uickly or 



Fig. 12. The fiekl of view of an AO-Baker 

 shearing micro.scope. The object has a sharp image 

 at B, a blurred image at A. Area C should be kept 

 free of material which would cast a blurred image 

 on area B. 



the specimen is very irregular, it may be 

 possible to employ an automatic integration 

 technique (35, 36, 37). In the .system of 

 Mitchison, Passano, and Smith (36) the in- 

 tegration is done opticallj'^, but care must be 

 taken that the maximum optical path differ- 

 ence is less than about X/8. 



The index of liquids available only in small 

 quantities can be measured by a technicjue 

 developed by Smith and Iverson (38). The 

 liquid is allowed to fill a small Avedge, and 

 the interference bands seen in the image are 

 measured. 



The optical absorption of specimens can 

 be determined by employing a modification 

 of the AO-Baker system (39). If the object 

 beam passes through an absorbing specimen 

 but the reference beam does not, the emerg- 

 ing beams will be unequal in amplitude as 

 well as ill phase. Polarimetric technicjues, 

 including a sensitive half-shade device and/ 

 or photoelectric comparison, yield both the 

 amplitude ratio and phase difference. Meas- 

 urements at various wavelengths in the 

 visible spectrum ar(^ possible. 



REFERENCES 



1. DiTCHBURN, R. W., "Light," p. 360, Blackie 



and Son, Ltd., London, 1952. 



2. Merton, T., Proc. Roy. Soc. (London), A189, 



309 (1947). 



3. Mellors, R. C, Kupfer, A., and Hollen- 



433 



