Immersion freezing of cell monolayers for cryo-electron tomography.

Cryo-transmission electron microscopy (cryo-TEM) is not limited to the visualization of suspended macromolecules in their native hydrated state. It can also be used to elucidate the molecular architecture of peripheral parts of adherent cells: After cultivation of the cells directly on EM grids, they are physically fixed by immersion freezing to yield cells embedded in thin, crystal-free layers of frozen water. Subsequently, specimens can be visualized using cryo-electron tomography (cryo-ET). This protocol outlines the production of protein-coated gold particles as suitable fiducial markers for electron tomography, and how to cultivate cells on grids with a carbon support film. It also describes how to freeze cells to obtain optimal and reproducible results using the Leica EM GP immersion freezer, and how to assess the results.