A quantitative microprobe analysis of elements in cortical and nuclear cells of the calf lens

The outer cortical cells in the calf lens remain transparent under conditions that produce opacity in central nuclear cells. The nuclear cells opacify by a mechanism of cellular restructuring that is associated with a cytoplasmic phase separation while cortical cells do not opacify by this mechanism. In this study the differences in elemental composition of nuclear and cortical cells were analyzed using X-ray emission spectroscopy (XES) of tissue that was prepared for scanning electron microscopy. It was necessary to develop special methods of fixation and dehydration to prevent significant distortion of lens tissue and minimize solubilization and redistribution of elements during the histological processing of the tissue. We calibrated the microprobe for the quantitative analysis using gelatin standards which contained known concentrations of sulfur, potassium, phosphorus, chlorine, and cesium. The standard curves were used to determine proportionality constants, which related the intensity of X-ray emission to the molar concentration of each element, and to determine the minimum detectable levels of each element. An important finding is that the intensity of the X-ray emission is dependent on sample density only at low protein concentration. At the high protein concentrations that exist in lens, the intensity is not affected by sample density.(ABSTRACT TRUNCATED AT 250 WORDS)