Current concepts of x-ray microanalysis of frozen hydrated tissue

X-ray microprobe analysis is a technique that has had many applications to material science, metallurgy, and mineralogy. Over the past decade, many biological applications have been considered. Common problems with such applications have been specimen preparation, analytic considerations, and interpretation. Frequently, biological problems involve analysis of elements that are diffusible. This increases the difficulties in applying X-ray analysis to biological questions since the preparative techniques might disturb the location and concentrations of elements before analysis can be done. Furthermore, the final preparations are rarely ideal from a purely analytic perspective. This report deals with a specialized preparative technique that aims at providing a specimen that retains elements in their original concentration within aqueous material. Thus, broadly speaking, this technique is similar to soluble-compound-localization methods, except that in our technique the water is left in place during analysis. One major benefit of using a frozen hydrated X-ray microanalytic technique is that it is possible to measure the water fraction of a selected analytic compartment. Additionally, one can measure elemental concentrations in aqueous material found in lumens, which may enable the investigator to relate luminal content with adjacent cell composition. X-ray microprobe analytic techniques for frozen hydrated tissue can be conveniently divided into four steps: freezing, cryosectioning (or cryofracturing), transfer and tissue handling, and analysis (analytic algorithm). Each step has the potential to affect analysis. While a complete discussion of each step is beyond this brief review, the author will attempt to emphasize salient points regarding each step