Methods Mol Biol 2007 Jan 01;369:407-29. doi: 10.1007/978-1-59745-294-6_20.

Three-dimensional cryotransmission electron microscopy of cells and organelles.

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Cryoelectron microscopy of frozen-hydrated specimens is currently the only available technique for determining the "native" three-dimensional ultrastructure of individual examples of organelles and cells. Two techniques are available, stereo pair imaging and electron tomography, the latter providing full three-dimensional information about the specimen. A resolution of 4 to 10 nm can currently be obtained with cryotomography. We describe specimen preparation by means of plunge-freezing, which is straightforward and rapid compared with conventional EM techniques. We detail the considerations and preparation needed for successful cryotomography. Frozen-hydrated specimens are very radiation-sensitive and have low contrast because they lack heavy metal stains. The total electron dose that can be applied without damage to the specimen at a given resolution must be estimated, and this dose is fractionated among the images in the tilt series. The desired resolution determines the number and magnification of the images in the tilt series, as well as the objective lens defocus used for phase contrast imaging. The combination of the desired resolution and the maximum number of images into which a given dose can be fractionated sets an upper limit on specimen thickness. Because of these constraints, careful choice of imaging conditions, use of a sensitive CCD camera system, and microscope automation, are important requirements for conducting cryoelectron tomography.

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