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, nondestructive 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 5 kV) indicated that at 15 kV 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. From the latter it is possible to calculate osmotic concentrations of individual cells and osmotic gradients across the tissue.Lay Description Understanding many cellular processes, in both healthy and diseased states, depends on knowing how the water content of cells and their surrounding fluids is controlled. The transport of water is generally down its concentration gradient or against the osmotic concentration gradient defined by solutes such as sodium, potassium and chloride dissolved in the water. We have refined a microanalytical method, that detects the x-rays emitted from specific elements when they are bombarded by electrons in a scanning electron microscope, to apply it to the analysis of the retina of the eye. The method facilitates the measurement of the elemental composition, water and osmotic concentration gradients of cells and tissues in the eye, that may be involved in the development of myopia, or short sightedness, a condition that afflicts many people including some 80 - 90% of children in Asia.