Application of scanning electron microscopy to X-ray analysis of frozen-hydrated sections. II. Analysis of standard solutions and artificial electrolyte gradients

Abstract

New specimen handling and analytic techniques for the application of x-ray microanalysis to studies of cell and organ biology have been recently described (Saubermann et al., 1981, J. Cell Biol. 88:257-267). Based on these techniques, absolute quantitative standardization has been etablished through x-ray analysis of frozen-hydrated and then dried sections of independently measured standard solutions of elements. These experiments demonstrate that the specific techniques employed have a probable error of <10%. Artificial electrolyte gradients established in gelatin were subjected to analysis to determine whether there was elemental displacement under non-membrane-limited conditions at the temperatures employed for sectioning (-30° to -40°C). No significant difference was observed between such gradients in serial sections cut at -30° and -80°C. Similarly, no additional ice-crystal-damage artifact was found in sections cut at -30°C when compared with sections cut at -80°C. Thus, in terms of ice-crystal size, gradient maintenance, and compartmental differentiation, cryosectioning at -30° to -40°C was not associated with redistribution imcompatible with 1- to 2μm spatial resolution, nd absolute measurements of elemental concentration were practical within regions of this size.