Quantitation in Histochemistry 



21 



to grave errors in quantitation, especially in the case of sub- 

 stances with a high optical density, i.e., of dark shades. An 

 extreme example will be given to shown this point. It will 

 be taken for granted that the sections are of a very uniform 

 thickness, a condition not easy to fufil. 



Let us assume that the field is occupied by a homogeneous 

 colored layer the light transmission of which it 5 per cent. If 

 the same amount of colored material is distributed in dis- 

 continuous spots occupying only half the field, transmission 

 of the entire area will go up to 50 + 2.5 = 52.5 per cent. If 



T=5% 



T=52.5% 



T=90.5% 



Fig. 1 



it is distributed over only one-tenth of the field, transmission 

 will rise to 90 + 0.5 = 90.5 per cent ( see Fig. 1 ) . In this, way 

 the same amount of colored matter distributed over the same 

 area in different ways may read 5 per cent, 52.5 per cent, or 

 90.5 per cent, which is a spread of eighteen fold in terms of 

 transmission. 



The error decreases rapidly with increasing transparency 

 of the color. If the transmission of the uniform layer is not 

 5 but 50 per cent, the corresponding readings will be 50, 75, 

 and 95 per cent; and if the uniform transmission is 80 per 

 cent, they will be 80, 90, and 98 per cent. However, since the 

 absolute amount of colored matter is a function of the loga- 

 rithm of transmission, the actual error in quantitation would 

 be 30:1, 13.5:1, and 12:1, respectively. 



