What is cryo-EM?

Cryo-electron microscopy (cryo-EM), also called electron cryo-microscopy, is a method for obtaining images and 3-D reconstructions of macromolecules. In cryo-EM the specimen-typically an unstained protein preparation embedded in vitreous ice-is held at cryogenic temperatures while images are formed in the transmission electron microscope.

Cryo-EM is actually a family of methods of specimen preparation for 3-D electron microscopy. The classical preparation methods for cell biology, involving fixation, sectioning, plastic embedding, and staining, do not provide the resolution for visualizing nanometer-sized structures such as single macromolecules and macromolecular complexes. For these smaller structures, replicas by heavy-metal shadowing or casts by negative staining provide a view of surface topography. Negative staining is a particularly simple and rapid way to obtain a surface view of macromolecular complexes, but the resolution of negative-stain images is reputed to be limited by the grain size to ~20 Å, and is subject to drying and flattening artifacts.

Why not image a protein molecule directly, without stain, in the electron microscope? We can, but the protein is rapidly blown to bits while any image is acquired. This is because the high-energy electrons break the chemical bonds in the protein, forming volatile fragments that quickly vanish into the microscope's vacuum system. The problem can be solved by immobilizing the protein at a very low temperature, embedded in ice. This way many chemical bonds can be broken while the atomic nuclei remain approximately in place, and much larger electron doses (~10-20 electrons per square angstrom of specimen area) can be applied before destroying the specimen. If the aqueous film containing the protein specimen is frozen very rapidly and maintained below -160 °C, the water remains glassy and no ice crystals form.

Cryo-EM methods were proposed more than 50 years ago and developed in the 1970s and 1980s. A modern cryo-EM specimen consists of an aqueous film that spans holes in a thin carbon film (FIGURE 1). The film is blotted to ~1,000-Å thickness, and the grid is rapidly plunged into liquid ethane. Cooled by liquid nitrogen, the ethane is maintained well below its boiling point and therefore serves as an excellent cryogen. Then the cryo-samples are visualized at -170 °C, and 3D structures can be determined.

Cryo-EM has been successfully employed to study structures of macromolecules. Here are some amazing examples.