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SummaryThe inelastic mean free path of 120 keV electrons in vitrified ice layers has been determined in an energy-filtering TEM. From the ratio of the unfiltered and zero-loss-filtered image intensities recorded with a slow-scan CCD camera, the relative sample thickness t/L can be calculated. For calibration, the geometric ice thickness was measured by imaging a tilted view of a cylindrical hole which had been burnt into the ice layer. The total inelastic mean free path was found to be 161 nm, and the partial inelastic mean free path for an acceptance angle of 4·2 mrad was 232 nm. These results were built into a standard protocol for use in cryo-electron microscopy allowing on-line measurements of local ice-layer thicknesses by zero-loss-filtered/unfiltered imaging.

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The nuclear envelope (NE) physically separates the genetic machinery residing in the nucleus from protein synthesis taking place in the cytoplasm. Bidirectional transport of proteins, RNAs and RNP particles between these two compartments is mediated by the nuclear pore complexes (NPCs), large (-120 MDa) supramolecular assemblies embedded in the NE and being built of -100 different polypeptides, called nucleoporins. Extensive structural studies have revealed the 3D architecture of NPCs, and epitopes of several nucleoporins have been localized within their 3D structure.In an attempt to go beyond the current consensus model of the NPC (Fig. lc, inset), we have embarked on a more systematic structural analysis of the molecular architecture of native NPCs. This is achieved by field-emission (FETEM; Fig. la) or energy-filtering (EFTEM; Fig. lb) TEM of completely unfixed and unstained Xenopus oocyte NEs embedded in thick (i.e. -200 nm) amorphous ice so that the 3D organization of the cytoplasmic and nuclear periphery of the NPCs (i.e. the cytoplasmic fibrils and nuclear baskets) is fully preserved (Fig. lb).