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Electron Cryotomography of Bacterial Cells

Chen, Songye and McDowall, Alasdair and Dobro, Megan J. and Briegel, Ariane and Ladinsky, Mark and Shi, Jian and Tocheva, Elitza I. and Beeby, Morgan and Pilhofer, Martin and Ding, H. Jane and Li, Zhuo and Gan, Lu and Morris, Dylan M. and Jensen, Grant J. (2010) Electron Cryotomography of Bacterial Cells. Journal of Visualized Experiments (39). Art. No. 1943. ISSN 1940-087X. PMCID PMC3149996. https://resolver.caltech.edu/CaltechAUTHORS:20110804-140527126

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Abstract

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 (~4nm). In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (< -180°C). For slender cells (up to ~500 nm in thickness), 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. 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.3791/1943DOIArticle
http://www.jove.com/details.php?id=1943PublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149996/PubMed CentralArticle
ORCID:
AuthorORCID
Chen, Songye0000-0001-5407-5049
Dobro, Megan J.0000-0002-6464-3932
Briegel, Ariane0000-0003-3733-3725
Ladinsky, Mark0000-0002-1036-3513
Shi, Jian0000-0003-3810-5302
Tocheva, Elitza I.0000-0002-4869-8319
Beeby, Morgan0000-0001-6413-9835
Gan, Lu0000-0002-8685-4896
Jensen, Grant J.0000-0003-1556-4864
Additional Information:© 2010 Journal of Visualized Experiments. This work was supported in part by National Institutes of Health Grants R01 AI067548, R01 GM081520, R01 GM086200, R01 AI049194, and P01 GM066521 to GJJ as well as the Howard Hughes Medical Institute, the Beckman Institute at Caltech, and gifts to Caltech from the Gordon and Betty Moore Foundation and Agouron Institute. MSL is supported by NIH grant 2R37-A1041239-06A1 to Pamela S. Björkman. Leica Microsystems Inc. kindly provided video content of cryosection collection. The representative results of Treponema primitia were collected and processed by Gavin E. Murphy, and published in Molecular Microbiology with the title "Novel ultrastructures of Treponema primitia and their implications for motility". (Murphy, G. et al., Mol. Microbiol. 67, 1184-1195, 2008).
Funders:
Funding AgencyGrant Number
NIHR01 AI067548
NIHR01 GM081520
NIHR01 GM086200
NIHR01 AI049194
NIHP01 GM066521
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Gordon and Betty Moore FoundationUNSPECIFIED
Agouron InstituteUNSPECIFIED
NIH2R37-A1041239-06A1
Issue or Number:39
PubMed Central ID:PMC3149996
Record Number:CaltechAUTHORS:20110804-140527126
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20110804-140527126
Official Citation:Chen S., McDowall A., Dobro M.J., Briegel A., Ladinsky M., Shi J., Tocheva E.I., Beeby M., Pilhofer M., Ding H.J., Li Z., Gan L., Morris D.M., Jensen G.J. (2010). Electron Cryotomography of Bacterial Cells. JoVE. 39. http://www.jove.com/details.php?id=1943, doi: 10.3791/1943
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:24694
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:04 Aug 2011 21:32
Last Modified:04 Oct 2019 23:00

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