The preparation, examination and analysis of frozen hydrated tissue sections by scanning transmission electron microscopy and X‐ray microanalysis

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Abstract
SUMMARY: A method is reported for preparing, examining and analysing frozen hydrated tissue sections using transmission electron microscopy and X‐ray microanalysis. Use of this method permits localization and measurement of water soluble or diffusible elements within the hydrated cell matrix. Since any change in total fresh weight of the specimen will affect the concentration of all components, great care has been taken to demonstrate that the mass neither increases nor decreases and to ensure that the tissue remains frozen‐hydrated. Criteria for assessing whether or not the tissue remains frozen‐hydrated are reported.After quench freezing, 1–2 μm thick sections of mouse liver were cut at 193°K and picked up on a specially designed annular specimen holder covered with an aluminium coated nylon film. Using a transfer device which prevents contamination of the tissue sections while maintaining them at a low temperature (below 143°K), the sections are transferred either to the vacuum evaporator cold stage or the scanning microscope cold stage. The tissue sections may be coated with an aluminium layer to improve electrical and thermal conductivity. The specimens are examined in the scanning transmission imaging mode and analysed using an energy dispersive X‐ray analyser. Concentration of intra‐nuclear and intracytoplasmic K, P, S and Cl are reported for mouse hepatocytes as ratios of the characteristic radiation to the continuum radiation used as a measure of mass. Ratios for all four elements were higher in the nucleus than the cytoplasm. Examples are given of this method as applied to plant and insect tissue.