The x-ray microanalysis of frozen-hydrated sections in scanning electron microscopy: an evaluation

The present status of the technique to measure concentrations of electrolyte elements and dry mass in or approximately 1 mum thick frozen-hydrated sections of soft biological tissues with electron probe X-ray microanalysis in a scanning electron microscope is critically reviewed. The technique is to quench-freeze fresh specimens to less than -180 degrees C, cut approximately 1 mum thick hydrated cryosections ( less than or equal to -70 degrees C), transfer on to a cold stage (less than -170 degrees C) of a suitable microanalytical arrangement, obtain scanning transmission images to identify the cell and tissue compartments, locate an electron probe (several mum2 to 100 nm) on the areas of interest and collect X-ray quanta. The X-ray quanta collected with suitable spectrometers (WDS and EDS) and processed with a computer using a comprehensive programme based on continuum normalization procedures ('Hall' programme). The cryosections are analysed first in a hydrated state and second after dehydration within the microanalyser column to obtain directly elemental concentrations in muM kg-1 wet wt and muM kg-1 dry wt of the compartments identified under the beam. The local water-fractions are estimated and the elemental concentrations converted into muM 1(-1) water. In the past 7 years the technique has been applied to obtain fully quantitative information on Na, K, Cl, P, S, Ca and H2O in more than ten types of tissue.