Increased resolution of cytophotometric measurements by mean extinction and area parameters of Tradescantia nuclei.

Cells of the mature petal contain two types of nuclei that differ in the degree of chromatin compaction, and it was found that the degree of compaction was a source of error in cytophotometric measurements of Feulgen-stained DNA. The error, which was observed as a deviation from the Beer-Lambert law, resulted in underestimation of total nuclear Feulgen-DNA values. It was shown, however, that in spite of this deviation, segregation of the nuclei into discrete 2c and 4c DNA content classes was still possible because (1) the mean extinction of a nucleus is a function of nuclear area, and (2) nuclei with different amounts of DNA have dissimilar mathematical functions. The cytophotometric results were supported by autoradiography and biochemical measurements. In addition, it was demonstrated that the error was greater for nuclei with the more compacted chromatin and that a large portion of the error was associated with the nuclear periphery.