A CRITIQUE OF CYTOCHEMICAL METHODS 209 



loss on the vertical axis, and wave length or frefjuency on the horizontal 

 axis. The shape of the absorption curve is of qualitative value in identi- 

 fying the specific absorbing atomic configuration, the chromophore. 

 When a sample contains two or more nonreacting absorbing substances, 

 the compound absorption curve results from addition of the individual 

 components. If the curves of the chromophores are considerably differ- 

 ent in absorption coefficient at some wave lengths, the compound curve 

 can be analyzed into the individual curves of its components by solving 

 simultaneous equations (Stearns, 1950). 



As Tables 6-1 and 2 indicate, in order to be detectable in the cell by 

 absorption (i.e., by contrast), substances must reach a concentration 

 many thousand times that which is sufficient for analysis in an absorption 

 cuvette. This imposes a fundamental limitation on the whole method of 

 interpreting the chemical composition of cellular components from visual 

 microscopical appearance or, indeed, from the most careful microscopical 

 absorption measurements with an objective photometer. Very small 

 quantities can be detected, as little as 10~''* g in a single small granule, 

 which may be perhaps no more than a millionth of the entire cell volume. 

 Therefore, in one sense, these are very "sensitive" techniques in the 

 vocabulary of the microchemist (see Benedetti-Pichler and Rachele, 

 1940). But, as a means of demonstrating a complete picture of distribu- 

 tion of a substance within the cell, these microscopic methods are deplor- 

 ably inadequate. Obviously, if the concentration within a small granule 

 just reaches the threshold for detection (which experience shows to be 

 roughly equivalent to an extinction of 0.03 or about 7 per cent absorption; 

 see Tables 6-1 and 2, column 4), then outside this spot of high local con- 

 centration there can be a relatively enormous amount of substance which 

 is below the detectable absorption or contrast. It is a simple matter to 

 compute in any cell, from data like those of Tables 6-1 and 2 and the 

 volume relations within the cell, the maximum possible amount of sub- 

 stance that could escape visualization or measurement. Conclusions 

 about localization and distribution of intracellular substances from micro- 

 scopical data must always take into account this interrelation between 

 absorption and intracellular geometry. It must be emphasized that 

 microscopic methods alone can prove neither the exclusive localization of 

 a substance within a small intracellular structure nor the complete absence 

 of a substance from any part of the cell.- Such conclusions can come 

 only from a combination of methods of chemical analysis of cell isolates 

 and cytology, as pointed out by Pollister, Himes, and Ornstein (1951). 



^ One escape from tliis limitation on microscopic detection and estimation lies in 

 developing stains which are fluorescent and tests based on fluorescence. Since 

 fluorescence is seen or measure4 as total intensity against a dark field, i.e., zero inten- 

 sity (instead of by subtraction from a field of high intensity), the dye or color reaction 

 can be detected readily in concentrations as low as one one-thousandth of the minimum 



