Electron Microscopy of Cells

نویسنده

  • J. Richard McIntosh
چکیده

It is widely acknowledged that EM of macromolecules is entering a new phase of productivity (for review see Nogales and Grigorieff, 2001). It is less well known that comparable innovations are afoot in cellular EM. Studies of biological “ultra-structure” were of key importance in the early years of cell biology, but more recently their importance has waned significantly. This change has been largely because much of what could be seen by classical techniques for EM has long since been described; it has also derived from the cost of EM in both time and money and the innovations that have recently revolutionized light microscopy, making it ever more powerful for the study of living cells. However, recent improvements in both methods and instrumentation for EM are now allowing the structure of organelles and cellular subsystems to be characterized with unprecedented detail and reliability. Thanks to tomography, the three-dimensional (3-D) 1 structure of cells can now be visualized with 5–8-nm resolution (Frank, 1995; Baumeister et al., 1999). Thanks to high pressure freezing, cellular specimens of considerable size can now be well frozen, even without the addition of chemical cryoprotectants (Shimoni and Muller, 1998). With this and other methods for rapid freezing, samples become solidified within milliseconds (Gilkey and Staehelin, 1986), whereupon they are embedded in glass-like ice (Sartori et al., 1993). The cellular milieu is still aqueous, but rapid freezing has immobilized all the cell’s constituents before significant rearrangement is possible. Under these conditions, biological structure is trapped in an essentially native state, and ice crystals, which would deform the physiological organization, have had little time to grow. More rapidly frozen specimens retain impressive preservation of intracellular detail (Heuser and Reese, 1981). Frozen-hydrated cells can be examined directly in the EM by using a low temperature specimen holder, or they can subsequently be fixed by “freeze-substitution,” during which cellular water is replaced at 80 C to 90 C by an organic solvent that contains chemical fixatives to stabilize the biological structure before it is embedded in a matrix suitable for microtomy. Freeze-substituted samples appear similar to material prepared for EM by conventional methods, but they have a greater likelihood of displaying structures in their native state (for review see Steinbrecht and Muller, 1987; McDonald and Morphew, 1993). This review will describe recent progress in the 3-D imaging of both frozen-hydrated cells and those that have been preserved by freeze-substitution. We will then evaluate the impact of the resulting data on our understanding of cellular mechanisms.

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عنوان ژورنال:
  • The Journal of Cell Biology

دوره 153  شماره 

صفحات  -

تاریخ انتشار 2001