230 RADIATION BIOLOGY 



picion that, in the cuinplex iiucleoproteiii association within the cell, the 

 resonance conditions may be significantly different from those of the iso- 

 lated nucleic acid and protein in solutions (see Chap. 5, this volume), and 

 hence any computations of absolute concentration or amounts from stand- 

 ards obtained on solutions must be considered provisional. For relative 

 concentrations this is less important, for it is a much less unlikely assump- 

 tion that A- is constant throughout the limited range under study. 



Deviations from Lambert's law that extinction is proportional to thick- 

 ness are uncommon sources of error if photometric analysis is properly 

 carried out, since there are no conditions within a thick sample which 

 differ from those within a thin one. It has been suggested that, within 

 a cytological preparation, error may arise because the color reaction 

 occurs only at the surface of the section, or because a colored product 

 piles up to form a sort of opacjue screen on the surface. This has not 

 yet been found. Conformity to Lambert's law is easily tested in cyto- 

 logical preparations, and it has been repeatedly demonstrated that light 

 loss is proportional to thickness of the absorbing layer (Pollister and Ris, 

 19-1:7; Pollister and Swift, 1950). A possible source of failure is dichroism 

 as a result of orientation of chromophores (Commoner, 1949; Commoner 

 and Lipkin, 1949). No case of such error has yet been detected in visible 

 and ultraviolet studies (Pollister and Swift, 1950), and there is evidence 

 that moderate nucleic acid orientation would have little effect on the 

 ultraviolet absorption measurements of cells carried out with unpolarized 

 light (Thorell and Ruch, 1951). It is perhaps a safe rule that dichroism 

 is unlikely to be a complicating factor except in objects which are con- 

 spicuously birefringent (e.g., skeletal muscle). Marked dichroism is a 

 potential tool for study of molecular orientation within the cell. For 

 example, Caspersson (1940b) has demonstrated that the ultraviolet 

 dichroism of grasshopper sperm heads is due to orientation of the pyrimi- 

 dine chromophores, and infrared dichroism has been employed to detect 

 orientation of protein polypeptide chains (Goldstein, 1950). 



When carrying out in vitro photometric analysis, the usual method of 

 isolating specific absorption from nonspecific is by subtracting the light 

 loss of a blank, which is either the solvent alo?ie, or a solution of the sample 

 substance in which a color test, which is the basis of the photometric 

 analysis, has not been developed. The wide usefulness of the photo- 

 metric approach in ([uantitative chemical analysis depends largely upon 

 these simple methods of extracting the essential datum from what is 

 actually in most cases an extremely complicated optical phenomenon 

 (see Chaps. 1 and 5, this volume). The cytological use of photometric 

 analysis likewise depends on, in one way or another, the relation of light 

 loss within the cell to specific absorption of a given chemical substance. 

 The elimination of the nonspecific component has been l)est achiexed in 

 methods of photometry of color reactions for nucleic acids and proteins, 



