Zero-loss energy filtering as improved imaging mode in cryoelectronmicroscopy of frozen-hydrated specimens

One of the remaining problems in attaining higher structural resolution with cryoelectronmicroscopy of frozen-hydrated specimens is the low contrast of micrographs taken close to the electron optical focus. By measuring electron energy loss spectra (EELS) of ice layers we show that a large fraction of incident electrons undergoes an inelastic electron-plasmon scattering process. Thus these electrons do not carry structural information of the protein but increase the background of the electron image and therefore reduce the contrast of the negative. Here we report the improvement in contrast gained by filtering out inelastically scattered electrons using an energy-filtered transmission electron microscope (EFTEM). This gain in contrast permits a dramatic decrease in defocusing values, resulting in improved structural resolution. In addition, the increased signal to noise ratio allows the recording of micrographs at a reduced electron dose. This should result in less damage to vitrified and unstained proteins and other beam-sensitive specimens.