An x-ray microanalytical method for measuring in vivo element and water concentrations, relating to osmoregulation, in cells and tissues of the posterior eye.

Osmoregulation is critical for cell and tissue survival yet there are relatively few methods available to determine osmotic gradients from water and elemental concentration either in single cells or across multiple cellular layers of tissue. X-ray microanalysis of frozen-hydrated preparations in a scanning electron microscope is one such powerful, sensitive, non-destructive technique. Here we use x-ray microanalysis to quantitatively analyse intracellular element concentrations and oxygen concentrations, as a proxy for water concentrations, in selected individual cells of the posterior eye. Using frozen-hydrated preparations of the retinal complex of chicken eyes, it is shown that structural preservation is sufficient to identify cell layers and individual cells. The quantitative analysis of selected areas in the photoreceptor layer, inner nuclear layer and ganglion cell layer, where specific cell types were known to be present, provided measurements of intracellular element concentrations comparable with the analysis of individual cells. It is also shown that in the cells of the retinal pigment epithelium and outer photoreceptor segments elemental analyses were reasonably consistent at the cellular level in different depth levels of the same sample. Comparison of oxygen concentrations, as a proxy for water concentration, at two accelerating voltages (15 and 5kV) indicated that at 15kV oxygen concentration was largely derived from intracellular water. Water concentrations could be calculated and concentrations of diffusible elements (Na, K) could be defined in mmol l-1 . From the latter it is possible to calculate osmotic concentrations of individual cells and osmotic gradients across the tissue. This article is protected by copyright. All rights reserved.