Ronald R. Warner

Water content from analysis of freeze-dried thin sections.

The analytical electron microscope and related electron beam instruments are uniquely able to measure the water content of biological tissue at a subcellular level. An analysis of the Rick procedure for quantifying this water content (Dörge et al., 1978; Rick et al., 1979) reveals a hidden assumption; namely, that the sample and standard densities, in the hydrated state, must be equal. In general this will not be valid. Equations are derived that do not invoke this hidden assumption. These equations show that errors in using the Rick technique for measuring water content should be small, a few per cent, unless the sample contains a very heterogeneous distribution of mass or the water content of the standard is a poor representation of the sample. For freeze‐dried samples with extensive mass heterogeneity, such as skin, the equations for calculating water content presented in this paper should be used.

Inaccuracies with the Hall technique due to continuum variation in the analytical microscope

We have identified a source of extraneous (non‐sample) continuum in analytical microscopes with high‐angle EDS detectors. The extraneous continuum is generated by specimen‐scattered electrons but is not associated with a characteristic peak. This peakless continuum varies in magnitude with position across a grid and can cause large errors in quantitation with the Hall technique. The use of a modified specimen holder and an objective lens with a widened exit bore in the lower pole piece reduces the peakless continuum to acceptable levels in our instrument. Peripheral standards attached to the specimen can further reduce the impact of this extraneous continuum. Analytical microscopes with horizontal EDS detectors can also have serious problems with continuum variation across a grid, although the effect is likely due to X‐ray absorption by the specimen holder rather than extraneous continuum.

Gallium: a new supporting medium for specimen preparation

Gallium offers a useful alternative to conventional plastic and paraffin embedding media. Samples can be inserted into or removed from gallium cleanly and intact in minutes. Gallium can be polished, has good sectioning properties, is electrically conductive, tolerates high beam currents, is usually not a detectable sample component, and probably would not be a solvent for sample constituents. Samples can be immobilized in gallium within a few degrees of room temperature. Gallium has low toxicity, can be obtained in ultrapure form, is not expensive, and is reusable. Gallium has been used to prepare thin and thick sections and to expose bulk sample cross sections for STEM, SEM, microprobe and light microscope analysis. Gallium does not infiltrate; consequently it is not likely to be useful for soft biological samples.