Cellular tomography.

Traditionally, cellular specimens and tissues have been prepared for electron microscopy by chemical fixation or rapid freezing followed by freeze substitution and plastic embedding. Once embedded in a solid polymer, sections are obtained by room-temperature microtomy. More recently, two developments added a new twist to cellular electron microscopy: first, the rediscovery of tomographic 3-D reconstruction methods, which do not depend on any type of averaging procedures and hence are well suited for large irregular structures such as cellular organelles and large-macromolecular assemblies. Second, new advances made for the preparation of sectioned vitrified cellular specimens enable imaging them not only after plastic embedding but also directly in their vitrified state, recorded under strict cryo- and low-dose conditions. Today, tomography strongly benefits from an ever-evolving computer environment that now allows handling very large datasets with reasonable speed. Cellular tomography can be divided into four different classes: (A) tomography on thick (~300 nm) sections prepared from plastic embedded chemically fixed or freeze-substituted cells and tissues. (B) Large-scale tomographic 3-D data of entire cells obtained by serial sectioning of plastic embedded chemically fixed or freeze-substituted specimens obtained by computationally merging tomographic data from many consecutive sections within the grid plane and also in Z-direction. (C) Cryo-electron microscopy (cryo-EM) techniques now allow recording tomograms of intact vitrified small cells (e.g., bacteria) without substitution or sectioning. (D) Cryo-EM and cryo-tomography are increasingly applied to thin vitrified (<100 nm) obtained under strict cryo-conditions, including the during data recording in the microscope. Without extending to molecular tomography, which is described elsewhere in this book, this chapter leads the reader through cellular tomography by following the specifications outlined above.