Quantitation in biological X-ray microanalysis, with particular reference to histochemistry

Thin specimens consisting of various light and heavy elements in gelatine have been subjected to X-ray microanalysis to determine the relationship between the number of X-ray counts for a specific element expressed as a percentage of the continuum (the percentage counts) and the concentration of that element. For light elements, the relationship between the percentage counts and concentration is strictly linear. For heavier elements, the relationship is not linear, because of the increase of the continuum counts with (formula: see text). If a correction is made for the effect of (formula:see text), heavy elements also show a linear relationship between percentage counts and concentration. Within the limits of atomic number (Z = 56) and concentration (approximately 10%) studied here, it is shown that when X-ray microanalysis is carried out on bulk specimens consisting of various elements in gelatine, the relationship between X-ray counts and concentration for a particular element is linear. The problems in quantitation of the results of X-ray microanalysis caused by exogenous continuum and mass loss induced by irradiation are discussed. It is pointed out that when X-ray microanalysis is used to study histochemical and other staining procedures, allowance must also be made for the reduction in concentration of other elements in the specimen as a result of the addition of the stain to the specimen.