General considerations of X-ray microanalysis of frozen hydrated tissue sections

X-ray microanalysis of frozen hydrated sections can be applied for analysis of diffusible or highly mobile elements which might be displaced or lost when conventional preparatory techniques are used. Once frozen the specimen must be handled in such a way that it becomes vacuum compatible for subsequent analysis. Analysis cannot be accomplished without compartment recognition. Initial freezing can be considered successful if ice crystal damage does not exceed the dimension of the compartment to be analyzed. As compartment size decreases ice crystal artifacts become a significant problem. The application of special freezing methods including use of non-penetrating cryoprotectants is an important advance in freezing methodology for X-ray analysis. Cryosectioning is a fracturing process at low temperatures which can affect morphology and may affect analysis. An understanding of this process can aid in optimizing cryosectioning for use in preparing frozen hydrated and frozen dried section for X-ray microanalysis. Special equipment is necessary for both freeze dried as well as frozen hydrated specimen; while special transfer devices are necessary for frozen hydrated specimen transfer. Because of the design of cold stages special consideration for reducing extraneous background radiation is an intrinsic part of their application to biological X-ray analysis.