Scanning Microscopy


In this paper we give a brief outline of our bulk specimen technique developed to measure intracellular water concentration in frozen-hydrated biological specimens by means of energy dispersive X-ray microanalysis. Fractured surface of the deep-frozen tissue samples is analyzed in an electron microscope (a specimen area of 15x11.5 𝝁m is scanned) using 20 kV accelerating voltage and 1-5 pA effective beam current (measured in the specimen). Strong electric charging, which is the main problem associated with the low temperature X-ray microanalysis of frozen-hydrated specimens, is reduced by choosing optimum temperature range for the measurements (170-185 K) and by etching a thin surface layer on specimen surface. The main advantage of the method over other X-ray microanalytical techniques using sections and bulk specimens for water and dry-mass content determinations in cells (which are shortly reviewed) is the simple specimen preparation, the easy sample handling and the good stability of specimen during measurements. The main disadvantage is the poor spatial resolution as compared to the analysis of sections.

Measurements with our method provided meaningful results of the change in intracellular water contents in various postmitotic cells of rats dependent on age. The observed decline of the intracellular water contents results in increased ionic strength and slower diffusion in old cells than in young ones. These effects may be implicated in senescent deterioration of cell metabolism.

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