Water distributions of hydrated biological specimens by valence electron energy loss spectroscopy

A technique has been developed for measuring the water distribution in thin frozen hydrated biological specimens by means of electron energy loss spectroscopy (EELS). The method depends on the quantification of 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 a multiple-least-squares fitting procedure to reference spectra for water and the organic constituents. The direct EELS approach has an important advantage over other indirect methods that are based on X-ray generation or elastic scattering measurements since these are applied to freeze-dried specimens where differential shrinkage between compartments may produce errors. Precision and accuracy of the EELS method have been tested on cryosectioned solution of bovine serum albumin; data have also been obtained from cryosections of rapidly frozen erythrocytes. Results suggest that a precision of better than +/- 5% (s.d.) is attainable from a single measurement and the accuracy may be as high as +/- 2% if repeated measurements are made. The lateral spatial resolution of the water determinations is limited by radiation damage to approximately 100 nm which is of the same order as the specimen thickness.