Reliability of intracellular water and ion distributions as measured by X-ray microanalysis--a review

X-ray microanalysis can be an important tool to reveal the spatial relationships between polyelectrolytes, ions, and water as they occur within cells and tissues in vivo. To reach this goal, at least two of these three closely interrelated variables should be measured independently. Moreover, the absence of systematic errors should be proven. The present review discusses the probability of artificial ion and water shifts between intracellular compartments due to the growth of dendritic ice crystals much larger than the cross-sectioned remnants commonly seen in frozen-dried sections. Considering the possible mechanism of ice crystal growth it is concluded that ions and water are not translocated over large distances. Moreover, problems associated with the preparation of a sample for water content estimations are discussed here. The importance of an appropriate pre-freezing treatment is highlighted, as is the importance of fast freezing. The risk of artificial water shifts between compartments with different freezing properties is discussed and the absence of clefts between compartments or haloes around them as seen in frozen-dried sections is taken as an appropriate criterion. Constancy of section thickness and retention of full hydration of cryosections are necessary prerequisites for many of the techniques and conditions to fulfill these requirements are given.