A CRITigi'E OF CYTOCIIEMICAL METHODS 225 



object is both larger and more lightly colored than another, the cytolosist 

 is almost completely helpless to answer the obvious (juestion of whether 

 the decrease of color is entirely due to dilution in the larger mass. The 

 relation of volume to light absorption is easily computed from an actual 

 figure, a measurement of extinction, but such a (juantitative datum is 

 absolutely necessary. No amount of experience can train a cyto- 

 logist's eye to operate as a microscopic photometric device. These 

 measurements must be made with objective photometers, with which a 

 transmission is measured from which concentration may be estimated and 

 amount computed (where the form and homogeneity of the cytological 

 object are favorable). 



4-2. PHOTOMETRIC TECHNIQUE 



The simplest photometer is a photographic plate, which can be used to 

 determine relative intensities, from comparison with a density-intensity 

 calibration curve. This curve may be independently measured and used 

 for a whole series of plates (Caspersson, 1936; Pierce and Nachtrieb, 1941) 

 or may be measured from a series of intensities through a rotating sector 

 (Cole and Brackett, 1940) or a calibrated wedge (Uber, 1939) which is 

 photographed on the same plate with the cells. The photographic 

 method seems at first glance easy and obvious, a simple modification of 

 the technique of photomicrography. For accurate results, however, it is 

 far more complicated. Plate exposure and development must be rigidly 

 standardized, and the negative density must be measured with a fairly 

 elaborate photoelectric apparatus. The latter must, in fact, be nearly 

 as sensitive as a photometer for direct measurement of microscopic slides 

 — hence, in most cytological w^ork, the latter is an easier technique. 

 Photographic photometry is indispensable for some problems, for 

 example, where ultraviolet absorption measurements are to be made upon 

 living cells (Thorell, 1947; Malmgren and Heden, 1947; Mellors et al, 

 1950). 



For direct absorption measurements of fixed preparations at a single 

 wave length, the relatively simple device indicated in Fig. 6-7 is adequate 

 (Pollister and Moses, 1949). Photomultiplier tubes are sensitive enough 

 to allow measurement of areas less than 1 m', tor all methods for nucleic 

 acid and protein. Other devices employing photomultiplier tubes have 

 been described by Lison and Pasteels (1951) and by Pollister (1952c). 

 The data obtained are pairs of measurements, a first (h) through part of 

 the cell, a second (h) through an empty part of the slide, outside the sec- 

 tion. From these, transmission (7') can be computed (as Ix/h) and 

 extinction either computed (as logio /o /x) or obtained from a conversion 

 table (Erode, 1943). 



If absorption curves are to be measured, the apparatus described must 

 be supplemented by means of dispersing the spectrum either before or 



