A quantitative cryo-scanning X-ray microanalysis protocol for the examination of the eye

Analysis of elements present in fluids contained in small, poorly accessible sections of biological tissue is challenging. The choroid of the eye, which is a vascular tissue approximately 100 microm thick, surrounds the retina for the purposes of nutrient supply and metabolite removal, and which in the chick shows dramatic volumetric change in response to visual experiences. Because fluid homeostasis is critical to good vision, a complete understanding of the ionic changes driving large shifts in ocular fluids is required. However, the structure of the choroid and retina make extraction of pure fluids for analysis extremely difficult. Elemental x-ray analysis on a transverse chorioretinal specimen was performed after rapid freezing of a whole chick eye in liquid nitrogen, and mechanically fracturing the frozen globe. Using a Polaron Cryotrans System on a Cambridge S-360 scanning electron microscope and a Kevex Quantum detector, spectra were obtained for blood vessels, lymphatic vessels and vitreous that were readily visible at 265x. Analysis was performed on a frozen control solution of the elements found in the vessels. The elements and their concentrations found in blood vessels by x-ray analysis compared well with those from whole blood as established by conventional means. The analysis for lymph yielded results compatible with expectations; no other published data for small lymphatics enable a direct comparison. In conclusion, x-ray analysis can be used to acquire information that is otherwise unobtainable from tissue in situ. The same bulk-frozen elemental microanalysis protocol would have application to other organs and tissues when access to the site would destroy the integrity of the tissue under investigation.