260 PROGRESS IN MICROSCOPY 



that connecting the object and image intensities is no longer feasible, 

 thus preventing microspectrophotometric measurements altogether. 

 Besides all the foregoing causes of error, diffraction laws set a limit 

 to the size of the smallest objects observable in microspectrophoto- 

 metry. When the dimensions of the object approximate the wave- 

 length, a non-negligible portion of the object-diffracted light is lost 

 and cannot enter even the most powerful objectives. Therefore, an 

 absorbance error is made. On the other hand, distribution of the 

 diffracted light varies as the wave-length. In relation to the incident 

 light, the spectral distribution of the light diffracted and focused on 

 the image is altered. But such modification is not due to absorption 

 of the object, thus giving rise to another error. Furthermore, when 

 reflecting objectives are involved, the occluding effects of the small 

 mirror on the diffraction disk are to be considered. The reinforced 

 diffraction rings further reduces the dimensions of an object below 

 which microspectrophotometric measurements can no longer be made. 

 It seems that the smallest dimensions of investigated objects should 

 not fall below 4 to 5 /. approx. At a 2500 A wave-length, details ap- 

 proximating one micron can be measured whereas, in the infra-red, 

 close to / = 20 11 measurements should be restricted to objects whose 

 dimensions approximate 80 i-i. 



6. PHOTOMETRIC EYEPIECES 



In some cases, simple visual instruments may prove to be serviceable. 

 They usually consist of an eye-lens enabling comparison of the lumi- 

 nance of an imaged area with that of a comparison area of variable 

 luminance. In Leitz's microscope photometer (Berek) the same source 

 illuminates the specimen and the comparison area (Fig. 10.16). There 

 is no need, therefore, to stabilize the light source. The light-beam 

 originated by the source of light (not shown in Fig. 10.16), is split 

 in two by the semi-reflecting cube A/,. One of these portions travels 

 along the path (I) and serves to iUuminate the microscope normally. 

 The other portion travels along the path (2) outside the microscope 

 and serves as comparison beam. The two beams meet again in the 

 cube Mi. One half of the cemented face of Mi is opaque and reflective; 

 it is illuminated by the comparison beam (2). The other half lets 

 through the microscope-originated light. The microscope eyepiece Oo is 

 so designed as to allow focusing simultaneously the investigated image 

 and the line bounding the comparison area. 



