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Zero‐loss energy‐filtered imaging of frozen‐hydrated proteins: model calculations and implications for future developments

Energy‐filtered transmission electron microscopes operating in zero‐loss mode are used increasingly to study biological material in frozen‐hydrated conditions. The contrast enhancement and improved structural resolution obtainable by this method have been studied using Monte‐Carlo model calculations for the scattering processes occurring in such samples. Three models representing typical situations have been analysed, each normalized to minimal beam damage. It is shown that for proteins in thin layers of ice an optimal signal‐to‐noise ratio is achieved in the 80–120‐keV electron energy range. For proteins which have to be embedded in thicker ice layers, a considerably higher acceleration voltage is required. In particular, electron energies above 200 keV would be desirable for electron diffraction work on microcrystals.