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Electron Tomography

To obtain a 3D model for objects that are too thick or vary too much in size and shape for single particle electron microscopy (≥ 1 micrometer thick), e.g., small cells, cellular organelles and tissues, macromolecules and their complexes. Viruses in a cellular environment. Sub-tomogram averaging may be possible on samples which demonstrate multiple copies of particular sub-features.

Samples are fixed at room temperature or vitrified by plunge freezing or by high-pressure freezing. The sample is generally embedded in a resin and cut into thin slices if necessary. Frozen samples may be cut directly using cryo-sectioning. Imaging is performed at various tilt angles in the microscope using the lowest possible electron dose. Imaging under low dose conditions minimizes physical damage of the sample during data acquisition, but leads to extremely low signal-to-noise conditions. Sub-tomogram averaging of common features recovers the signal by an intensive computational procedure, yielding structural resolutions down to ~1 nm.

Tomography generates three-dimensional models of cellular samples by computational integration of different views, allowing the visualization of the proteins in a close-to-native environment. Resolution tends to be lower than for other methods such as single particle EM.

The sample can only be tilted by ± ~ 60° in the microscope. Thus, some 3D information (the so called missing wedge) is missing.

Sample requirements
Samples need to be sufficiently small and concentrated for plunge freezing or high pressure freezing ~ <1 um. Tissue specimens need to be fresh and may need to be dissected and trimmed to around 1mm cubed for resin embedding.