X-ray microanalysis with the environmental scanning electron microscope: interpretation of data obtained under different atmospheric conditions.

X-ray microanalysis of non-biological and biological specimens was carried out in an environmental scanning electron microscope (ESEM) over a range of atmospheric conditions. Introduction of water vapour into the specimen chamber lead to direct X-ray contribution from oxygen atoms, an increase in extraneous background (causing reduced P/B ratios of other elements), X-ray absorption (also reducing P/B ratios) and broadening (skirting) of the electron beam. Similar results were obtained after introduction of an argon atmosphere. These effects were reduced under conditions of minimal chamber atmospheric pressure and maximal accelerating voltage. Because of beam skirting, quantitative X-ray microanalysis of biological specimens in a water vapour atmosphere was only valid where the sample was spread over a wide area (leading to mean elemental values for the whole preparation). Unless appropriate correction factors or changes in instrumentation can be implemented, quantitative analysis of wet specimens in ESEM cannot be applied to discrete specimens or to limited areas within a mixed sample.