Lien vers Pubmed [PMID] – 20461053
Lien DOI – 10.3791/1943
J Vis Exp 2010 May; (39):
While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy. Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with “macromolecular” resolution (approximately 4nm).(1, 2) In ECT, cells are imaged in a vitreous, “frozen-hydrated” state in a cryo transmission electron microscope (cryoTEM) at low temperature (< -180 degrees C). For slender cells (up to approximately 500 nm in thickness(3)), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.(1, 2) In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.