ultramicroscopy Cryo-electron energy loss spectroscopy: observations on vitrified hydrated specimens and radiation damage

Abstract Valence electron energy loss spectroscopy (EELS) has been used to characterize the composition of frozen-hydrated specimens in the electron microscope. Fine structure in the energy range up to 30 eV provides a means of distinguishing between vitreous and crystalline ice. Some features of the ice spectrum can be understood in terms of transitions between molecular orbitals in the water molecule and by the existence of excitons in the solid. Spectra from hydrated biological specimens can be analyzed to obtain quantitative estimates of the water content by fitting contributions from the ice and organic components, EELS also provides information about the radiation chemistry that occurs when hydrated specimens are exposed to the electron beam. From the observation of the hydrogen K-edge at ~ 13 eV, it can be deduced that bubbles of molecular hydrogen are evolved during irradiation at doses of > 10 4 nm -z, and that these bubbles contain gas at pressures in excess of one thousand atmospheres

ultramicroscopy 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 solutions 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