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X-ray microanalysis of hydrated biological specimens.

Using a wide angle backscattered electron detector (BED), glutaraldehyde fixed or unfixed specimens of biological soft tissues such as hen oviduct, kidney, liver, duodenum of mouse as well as mitochondrial fraction from rat liver were observed under low vacuum (0.3 to 0.5 torr) at magnifications from x300 to 10,000. The backscattered electron images (BEI) of glutaraldehyde fixed hen oviduct were correlated to X-ray microanalysis. Intracellular secretion granules in such unhydrated, uncoated and unstained slices of hen oviduct preserved Ca even after glutaraldehyde fixation. Backscattered electron images of unfixed duodenum and kidney of mice were not satisfactory for observing intracellular structures, though X-ray microanalysis could detect P,S,Cl and K over cut surfaces of such hydrated unfixed tissue. Compared with frozen dehydrated specimen, the hydrated unfixed tissue tends to preserve more potassium and less chlorine. Cytochemical reaction product of succinate dehydrogenase (SDH-ase) activity in isolated mitochondria from rat liver was detectable in hydrated condition. Quick and easy assessment of localized elements in surgical specimens by combination of wet-SEM and X-ray microanalysis will be a new tool for clinical application of SEM. In spite of obvious limitations of this method, especially of its spatial resolution both in backscattered electron image and in bulk specimen X-ray microanalysis, the combination of wet-SEM and X-ray microanalysis provides information which has not been available in the past.