Scanning Electron Microscopy


The element distribution in biological cells and tissues can be revealed by electron probe microanalysis from ultrathin cryosections. In particular, the distribution of physiologically important and often mobile elements such as Na, Mg, P, S, Cl, K, and Ca can be studied in cryosections on an ultrastructural level. The cryopreparation technique required for this purpose consists of 1. cryofixation, 2. cryosectioning, 3. cryotransfer including freeze-drying and carbon coating if necessary, 4. energy dispersive X-ray microanalysis in a cold stage equipped scanning transmission electron microscope. The lateral analytical resolution of this method is less than 50 nm in freeze-dried ultrathin (about 100 nm thick) cryosections. The detection limit is about 12 mMol/kg dry weight for elements with an atomic number higher than 12. For sodium this value is about 48, for magnesium about 36 mMol/kg dry weight. Good cryofixation without or at least with very small ice crystals with a diameter of 50 nm or less is found to be necessary not only for recognition of ultrastructural details but also for reliable evaluation of X-ray spectra. Carbon coating of frozen-hydrated sections reduces the mass loss observed in uncoated frozen specimens.

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