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.