Cryo-electron microscopy (cryoEM) is a method of imaging protein complexes in their native or near-native state without chemical fixation or dehydration, thus preserving structure. To achieve this, very thin layers of aqueous sample are suspended over holes in a graphitized carbon support film and vitrified (ie, frozen without forming crystals) by plunging rapidly into liquid ethane. Frozen grids are transferred into the cryo-electron microscope at liquid nitrogen temperature to maintain the vitrified state and are imaged with low doses of electron radiation to avoid damage. Images represent projected density, and three-dimensional representations of the original structure can be obtained by back-projection or equivalent methods.
Single-particle methods (somewhat mis-named) involve collecting thousands of micrographs with thousands to millions of images of individual particles that are assumed to be in a uniform state, or at most in several distinct states. Particle images that resemble each other, save for translation and in-plane rotation, are collected into groups and these groups are combined in a three-dimensional sense to resolve a preliminary structure. The resolution and quality of the structure is improved by refinement of the orientations assigned to each particle, and microscope and camera distortions are also accounted for. This method has yielded the highest resolution structures to date, including apoferritin (a test sample) to 1.2 Ångstroms. Particular successes include icosahedral virus particles, which are difficult for other methods due to their size, membrane proteins that are difficult to crystallize, and amyloid fibrils.
Cryo-electron tomography (cryoET) is used to collect a tilt series of images of the same object, typically a non-uniform sample such as a bacterium or organelle, and is otherwise similar to the single particle methods in using back-projection to calculate three-dimensional models from the tilt series. Resolution is limited by the large electron dose used in collecting data, but developments such as sub-tomogram averaging continue to advance the reach of cryo-electron tomography. When samples may be too thick for electrons to penetrate, thin sections of interest, called lamellae, may be milled from the frozen sample using a Focused Ion Beam mill (FIB-mill), which is a scanning electron microscope equipped with a second beam of ions that perform the milling.