Freeze-substitution as a preparation technique for biological X-ray microanalysis.

Freeze substitution can be carried out under anhydrous conditions in an acrolein-ether solvent. Embedding is done in epoxy or methacrylate resins. During this process at least some diffusible elements are virtually totally retained. Retention of elements in organ lumina and body fluid space occurs in situ. There is some evidence, as yet not unequivocal, that translocation of elements does not occur to any great extent, if at all in animal tissues. Resin embedded specimens are readily sectioned on a dry knife and good quality images are obtainable in a STEM. Sections of this type lend themselves to analysis of mass per unit volume since section thickness can be easily determined and standard are readily avilable. A method of measuring section thickness from beam current attenuation is described. Charging, drift, mass loss, contamination and beam current can all be constantly monitored by a simple method of collecting transmitted electron current. It is also possible to use transmitted electron current to determine mass thickness and to use this value as a measure of total mass in quantitative calculations. This avoids the necessity of determining the extraneous continuum contribution to background which must be done when employing peak to background ratios.