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An electron microscopy technique, where the sample is rapidly cooled to a cryogenic temperature before imaging using a transmission electron microscope. Viruses, assemblies of biological macromolecules, proteins and cell organelles are typical samples. The sample is in a water solution, which is transformed into a vitreous state, not into crystalline water, upon rapid cooling. Formation of ice crystals damages the sample and prevents the microscopist from obtaining good-quality images. Therefore the speed of cooling is critical. Initial cooling is often done in a bath of cold organic liquid, e.g. ethane or propane, but after vitrification the sample is handled in liquid nitrogen, which is not explosive like the organic coolants. The intense electron beam of the microscope will cause radiation damage to the sample, and the miscroscopist must take great care not to fry the sample to death before collecting the desired images.

The chief advantage of this preparation technique is that the sample is preserved in a near-native state with its 3-dimensional structure intact. Cryoelectron microscopy is often used together with automated image analysis to reconstruct a 3-dimensional structure of e.g. a virus particle or a protein complex from tens or hundreds of 2-dimensional images of individual particles. In a non-crystalline sample, the orientation of particles is random and therefore the elecron microscope records 2-dimensional projections of a population of particles in random orientations. Due to the nature of the transmission electron microscope, information about internal structure of the particle is contained in the projection. If the sample is homogeneous, i.e. all particles can be assumed to be copies of the same thing, the 2d images can be aligned and a 3d reconstruction made. The computation is carried out in Fourier space. The alignment of single particles is much easier if the particles have internal symmetry, which is why viruses often make good samples. Structures of asymmetric macromolecular complexes such as the ribosome have also been succesfully reconstructed.

2-dimensional crystals can also be used as samples. In this case the particles in the sample are already aligned in the one-particle thick crystal lattice, which is imaged from different angles. 2d crystals have been used to determine structures of integral membrane proteins. Averaging of images of many particles is not possible when the sample is not homogeneous. For instance, each cell organelle such as a mitochondrion is unique, and a 3-dimensional reconstruction can only be carried out by tomography, where images of the same particle are recorded from different angles.

The resolution obtained by cryoelectron microscopy and image reconstruction is typically around 20 Ångströms and can be below 10 Å in a favourable case. The resolution depends on the density of the electron beam, the number of images averaged in the reconstruction and sample quality. X-ray crystallography is the most common method used to determine structures of biological macromolecules and it can achieve much better resolutions. However, crystallography requires the sample to be crystallized, which can be time-consuming and may prove impossible. Growing of protein or virus crystals is regarded more of an art than a science. Electron cryomicroscopy, on the other hand, does not require a crystalline sample.

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