MICROSPECTROSCOPY AND MICROSPECTRO PHOTOMETRY 259 



conduces to under-eslimale absorbance of the investigated object 

 because the background brings about a drop in contrast of the object 

 in relation to the field surrounding it. Such background is best mi- 

 nimized by setting a second monochromator next to the first (double 

 monochromator). The role of the stray light in the microscope is 

 prominent, too. The undesirable haze it generates on the image may 

 distort measurements. To minimize stray light to the utmost, the 

 illuminated area of the specimen should be as small as possible so 

 that the other areas, not involved in the measurement, do not add 

 their own quota of stray light. This is why an inverted microscope 

 is particularly suitable. 



Owing to the condenser aperture, beams at various angles may 

 pass through the object. Then, absorption is not the same as when 

 parallel beams are involved. Blout, and later Walker, have investi- 

 gated the measurement corrections required. If the N.A. of the 

 condenser is 1, measuring an object (living cell) of index 1-37, conduces 

 to a 12 per cent over-measurement (absorbance measured close to 

 unity). If the object has an index of 1 53 (fixed cell), the error does not 

 exceed 4 per cent and, when the N.A. of the objective is 0-6, the two 

 errors drop to 5 per cent and 1 per cent, respectively. Another cause 

 of error may stem from the lack of uniformity of the object itself. 

 Let us now consider an object of constant thickness and another one 

 whose thickness varies. The projected areas of both objects on the 

 observation plane are identical. Assuming that the images of both 

 objects can enter the cell through the aperture T (Figs. 10.5 and 10.6). 

 it is found that measurements diverge and an error regarding the quan- 

 tity of substance is made. To minimize such an error (caused by the 

 non-uniformity of the object) the diameter of the aperture should be 

 reduced to a bare minimum so that the small portion of the image 

 passing through T^ can be considered as uniform. Due consideration 

 must be given to aberrations of the objective. For instance, objectives 

 comprising two spherical mirrors, of N. A. exceeding 050 (spherical 

 aberration corrected but field reduced by the coma), can be used in 

 microspectroscopy but the objects investigated must, of course, be 

 kept within the small field where coma is admissible (10 [x for an 

 aperture of 0-65 at 2500 A). Outside this field the coma causes 

 a considerable drop in the maximal central intensity thus reinforcing the 

 diffraction rings. Since the lossof maximal-centre energy is proportion- 

 al to the square of the distance to the centre of the field (D. S. Grey), 

 coma effects increase rapidly beyond the admissible field, to the extent 



