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Evaluation of a preparative method for x ray micro analysis of soft tissue

, : Evaluation of a preparative method for x ray micro analysis of soft tissue. Cytobios 28(109): 17-34

The cryopreparative methods designed for the radioautography of diffusible substances were adapted to produce freeze-fried sections of soft tissues for electron probe microanalysis. The evaluation of these methods for use with X-ray microanalysis with particular reference to: the preservation of cellular morphology, the introduction of structural artefact by ice crystal formation, the preservation of natural elemental distributions under experimental conditions where known quantities of diffusible elements are present, the effects of ice crystal formation upon possible artefactual elemental redistributions, the effect of section thickness on elemental quantitation and the effect of tissue excision on element translocation reported. Freeze-dried sections were prepared from mouse pancreas and from 20% (wt/vol) solutions of bovine serum albumin (BSA) and gelatin containing known amounts of inorganic salts and were analyzed in a scanning electron microscope fitted with energy dispersive X-ray detecting equipment. With the preparative methods used, the morphology of pancreatic acinar cells was well preserved. Acinar cell boundaries, nuclear boundaries, chromatin, nucleoli, ergastoplasm and zymogen granules were readily discernible. Ice crystals were present within sections of BSA, but 70% had a cut surface area < 1 .mu.m2 and over 90% had a cut surface area of < 2 .mu.m2. Characteristic peak-to-continuum ratios of elements in the BSA sections remained constant over an order of magnitude change in magnification. Elemental redistributions were not detected until the magnification was such that the analyzed area fell totally within the confines of a single ice crystal. No differences in elemental peak-to-continuum values were obtained between 2 and 4 .mu.m sections of the gelatin-salt solution. Tissue excision did not cause element translocations when compared to tissues frozen in situ. Apparently this method is valid for preparing tissues for microanalysis under these conditions (analysis of nuclear, cytoplasmic and secretory compartments) and is limiting only when analyses are conducted at very high magnifications.

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