CHARACTERIZATION OF FROZEN-HYDRATED SPECIMENS BY EELS

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A method has been developed for measuring water content in subcellular compartments of frozen hydrated tissue that depends on variations in the valence excitation energy loss spectrum between water and the organic constituents of a cell. Single-scattering distributions are first obtained from ice and protein by applying the Fourier-logarithmic deconvolution technique. The resulting reference spectra are then normalized by applying the Bethe sum rule to the differential oscillator strengths in order to derive the differential cross sections. A direct estimate of the water content can be obtained by applying a least squares fitting algorithm to spectra from hydrated specimens. Tests have been performed on thin cryosections of rapidly frozen bovine serum albumin standards, containing 70%, 80%, and 90% protein, by recording parallel-EELS spectra in the field-emission scanning transmission electron microscope (STEM). It was found that an accuracy of approximately 1% could be achieved in the determination of water content. The method has been applied to cryosectioned erythrocytes and cerebellar cortex.