In cryo-electron tomography (cryoET), cells in aqueous media are applied to EM grids and plunge-frozen into a mixture of liquid ethane and propane, a cryogen so efficient that it cools the sample ~104 K/s, so rapidly that water molecules do not have time to rearrange into a crystalline lattice of ice.
The result is a thin layer of vitreous ice, an amorphous solid that preserves cellular structure. Samples are subsequently kept at ~80 K to prevent ice crystallization (hence the “cryo”) and imaged in a transmission electron microscope as they are tilted one or two degrees at a time. This results in a series of projection images that can be digitally reconstructed into a 3-D tomogram of the cell (much like a CT scan).
CryoET avoids many artifacts of traditional EM methods (including fixation), providing unparalleled structural information about native cells. Resolution of relatively thin biological samples is ~4-6 nm. For structures present in multiple copies and/or in multiple tomograms, sub-tomogram averaging can increase the resolution to better than 1 nm.
CryoET therefore neatly bridges the gap between structures of individual proteins (for instance, from X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy single particle reconstruction) and cellular macrostructure (from light microscopy). In combination with these methods and others (such as biochemistry and bioinformatics), cryoET may one day allow us to determine the function and location of each protein in the cell.