Scanning transmission electron microscopy (STEM) has been combined with parallel electron energy-loss spectroscopy (EELS) to develop new methods and experiments that complement conventional electron microscopy-based structural biology. To assess the potential of valence EELS for imaging the distributions of biological compounds in cells, high-resolution (0.5 eV) spectra have been recorded from specimens of protein, carbohydrate, nucleic acid, and lipids, as well as from vitrified water. These spectra, which were obtained at low electron dose, had charateristic fine structure in the energy range 4-12 eV, corresponding to absorption maxima in the vacuum ultraviolet spectrum. Knowledge of these spectra are important for quantifying water distributions in frozen-hydrated cryosections of tissue in the STEM. Further measurements have also been performed to determine the effects of radiation damage on the valence EELS spectra. Radiolysis of frozen-hydrated specimens subsequent to damage in the electron beam has been found to result in the formation of hydrogen bubbles. Improved methods have also been developed for quantifying EELS core edge spectra recorded in the difference mode from specimens containing very low concentrations of important elements such as calcium and phosphorus.
