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X-ray microanalysis of frozen-hydrated specimens.

The preparation of frozen-hydrated bulk specimens and sections for X-ray microanalysis starts with cryofixation, which is done either by rapid immersion into liquid propane, propane jet fixation or metal mirror fixation. Bulk specimens appropriate for the analysis in a scanning electron microscope (SEM) are obtained by cryofracturing the samples, coating, usually by a thin carbon layer, and cold transfer into the cold stage of the microscope. The X-ray microanalysis of bulk specimens is affected by an internal space charge which makes quantification difficult. Frozen-hydrated dry cut sections, varying in thickness between 60 and 2000 nm, are prepared by means of cryoultramicrotomy. After cold transfer into the cold stage of a scanning electron microscope or a scanning transmission electron microscope (STEM) the sections are analyzed in the frozen-hydrated and freeze-dried state. The reliability of the results with regard to structural recognizability and X-ray spectra depends considerably on the state of hydration. Particularly ultrathin sections in STEM show very low contrast and a great mass loss in the frozen-hydrated state in comparison with the freeze-dried state. In spite of the available concepts for quantification of X-ray data to obtain physiologically important wet weight concentrations of diffusible elements, the radiation damage at present turns out to be the most serious problem for X-ray microanalysis of frozen-hydrated sections.