The application of EDXS to the biological sciences

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Abstract
The distribution of chemical elements in soft tissues may be faithfully preserved by very rapid freezing. Most often the material is then cryosectioned and the sections frozen‐dried prior to analysis, but direct analysis in the hydrated state is an established alternative. For bulk specimens, the shape of the analysed volume is uncertain. But whichever current model is accepted, analytical spatial resolution must generally be limited to the order of 1 μm. Such specimens can be suitable for the specific analysis of cytoplasm, cell nuclei and large extracellular spaces but not for study on a finer scale. Analytical spatial resolution in the range 200–500 nm is obtainable with sections cut ∼ 1 μm thick. In the frozen‐hydrated state, small extracellular spaces can be analysed but multiple scattering obscures intracellular detail in the STEM image. The irradiation required for an EDXS analysis, approximately 50 nanoCoulomb (50 nanoAmpere seconds), need not produce intolerable radiation damage when spread over an area 200 nm or more in diameter. Finer structure, for example mitochondria and regions of rough or smooth endoplasmic reticulum, can be identified and analysed in frozen‐dried cryosections cut ∼ 100 nm thick. Recently such features have been visualized in 100 nm frozen‐hydrated sections where the water is vitreous. This opens the prospect of analysing material where elemental distributions have been preserved on a very fine scale, since one might avoid even the ionic shifts from aqueous solution to supramolecular structures which must occur on freeze‐drying. But radiation damage may be prohibitive when an irradiation of 50 nanoCoulomb is concentrated into a hydrated area less than 200 nm in diameter.

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