Quantitative X-Ray Microanalysis of Ultra-Thin Resin-Embedded Biological Samples

For X-ray microanalysis, unlike materials samples, most biological specimens present a complex of characteristics which prescribe the preparative procedures, determine the microanalytical configurations and limit the achievable statistical accuracy. The five principal limitations are the high water content, the ionised state and high mobility of the majority of the inorganic elements of interest, the low, sometimes trace concentrations of these elements, the ubiquity and diversity of the organic molecular and structural components of the cellular and extracellular matrix, often rendering quantification of their component elements redundant, and the sensitivity of biological material to radiation damage and beam-induced mass loss, particularly in hydrated samples. For mobile elements cryofixation is the only acceptable initial processing step. Spatial resolution requirements and beam sensitivity restrictions usually indicate microanalysis of thin section (<= 1 mu m) by energy dispersive detectors. Amongst briefly reviewed options for production of thin sections, the merits of low temperature freeze-drying, vacuum resin embedding and dry sectioning are presented, particularly for the location of targets, which are difficult to locate deep within tissues. Evidence is presented that plastic embedding need not cause elemental translocation and that intracellular gradients and physiological changes in diffusible elements are preserved by this preparative route. Prospects for improved accuracy in quantification are considered.