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

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 approximately 13 eV, it can be deduced that bubbles of molecular hydrogen are evolved during irradiation at doses of > 10(4) nm-2, and that these bubbles contain gas at pressures in excess of one thousand atmospheres.