CHARACTERIZATION OF FROZEN-HYDRATED SPECIMENS BY EELS

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The technique originated by us last year for measuring water distributions in thin, frozen-hydrated biological specimens by means of electron energy loss spectroscopy (EELS) has been further developed. This approach depends on quantifying subtle changes in the valence electron excitation spectrum as a function of composition. It involves determining the single-scattering intensities, calculating oscillator strengths, and applying multiple least squares fitting to reference spectra for water and organic constituents. The technique has now been extended to spectrum-imaging, so that two-dimensional water distributions can be obtained from hydrated cryosections. Automated processing of the spectrum-image data has been implemented, and ways of reducing the computation time are being investigated. Applications of the technique (e.g., analyzing the water content of rapidly-frozen hepatocytes and cryopreserved embryos) are in progress.