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. Bj&ouml;rkman. 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).

<ol>
	<li>Jensen, G. J., Briegel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+electron+cryotomography+is+opening+a+new+window+onto+prokaryotic+ultrastructure.">How electron cryotomography is opening a new window onto prokaryotic ultrastructure.</a> Curr. Opin. Struct. Biol. 17, 260-267 (2007).</li><li>Li, Z., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryotomography:+a+new+view+into+microbial+ultrastructure.">Electron cryotomography: a new view into microbial ultrastructure.</a> Curr. Opin. Microbiol. 12, 333-340 (2009).</li><li>Frank, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+biological+complexity.">Three-dimensional imaging of biological complexity.</a> J. Struct. Biol. 138, 85-91 (2002).</li><li>Iancu, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryotomography+sample+preparation+using+the+Vitrobot.">Electron cryotomography sample preparation using the Vitrobot.</a> Nat. Protocols. 1, 2813-2819 (2007).</li><li>Studer, D., Graber, W., Al-Amoudi, A., Eggli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+for+cryofixation+by+high-pressure+freezing.">A new approach for cryofixation by high-pressure freezing.</a> J. Microsc. 203, 285-294 (2001).</li><li>Ladinsky, M. S., Pierson, J., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitreous+cryosectioning+of+cells,+facilitated+by+a+micromanipulator.">Vitreous cryosectioning of cells, facilitated by a micromanipulator.</a> J. Microsc. 224, 129-134 (2006).</li><li>Pierson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+technique+of+vitreous+cryo-sectioning+for+cryo-electron+tomography:+Electrostatic+charging+for+section+attachment+and+implementation+of+an+anti-contamination+glove+box.">Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box.</a> J. Struct. Biol. 169, (2009).</li><li>Suloway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated,+sequential+tilt-series+acquisition+with+Leginon.">Fully automated, sequential tilt-series acquisition with Leginon.</a> J. Struct. Biol. 167, 11-18 (2009).</li><li>Fernando, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markov+random+field+based+automatic+image+alignment+for+electron+tomography.">Markov random field based automatic image alignment for electron tomography.</a> J. Struct. Biol. 161, 260-275 (2008).</li><li>Kremer, J. R., Mastronarde, D. N., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> J. Struct. Biol. 116, 71-76 (1996).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-axis+tomography:+an+approach+with+alignment+methods+that+preserve+resolution.">Dual-axis tomography: an approach with alignment methods that preserve resolution.</a> J. Struct. Biol. 120, 343-352 (1997).</li></ol>

No conflicts of interest declared.

We show in this video article how to do ECT of bacterial cells. Limited by space and time, we only show the general protocol. More details can be found in papers, online or other information provided by the manufactures of the equipment and software developers, and from ECT research groups. Depending on the different bacterial species studied, the equipment used and the structural interest in the cells, the protocol and the experimental parameters need to be tested and optimized.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Poly-L-Lysine</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P8920</td>
<td>0.1% solution</td>
</tr>
<tr>
<td>MKIII Vitrobot</td>
<td>&nbsp;</td>
<td>FEI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gold colloid solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1527-25ml</td>
<td>10 nm</td>
</tr>
<tr>
<td>Bovine serum Albumin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P2489</td>
<td>10%</td>
</tr>
<tr>
<td>Nr 1 Filter paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1001 055</td>
<td>&nbsp;</td>
<tr>
<td>Dextran</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>31389</td>
<td>15-25%, MW 39000</td>
</tr>
<tr>
<td>Brass-domed planchet</td>
<td>&nbsp;</td>
<td>Swiss Precision Company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bal-Tec HPm 010 high pressure freezer</td>
<td>&nbsp;</td>
<td>Leica Microsystems Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cryo-ultra microtome UC6/FC6</td>
<td>&nbsp;</td>
<td>Leica Microsystems Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Diamond cryo trim 45</td>
<td>&nbsp;</td>
<td>Diatome US Inc</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Static line II ioniser</td>
<td>&nbsp;</td>
<td>Diatome US Inc</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CX100 Dry shipper</td>
<td>&nbsp;</td>
<td>Taylor-Wharton-Cryogenics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MSH loading station</td>
<td>&nbsp;</td>
<td>Gatan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ti E/B inverted microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>Custom</td>
</tr>
<tr>
<td>Cryo LM stage</td>
<td>&nbsp;</td>
<td>FEI</td>
<td>&nbsp;</td>
<td>Custom</td>
</tr>
<tr>
<td>TF30-polara</td>
<td>&nbsp;</td>
<td>FEI</td>
<td>&nbsp;</td>
<td>With UltraCam from Gatan</td>
</tr>
<tr>
<td>Amira</td>
<td>&nbsp;</td>
<td>Visage Imaging</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electron cryotomography of bacterial cells

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).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), 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.

Watch this Scientific Journal Video about Electron Cryotomography of Bacterial Cells at JoVE.com

Electron Cryotomography of Bacterial Cells

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

electron-cryotomography-of-bacterial-cells

Research

JoVE Journal

Biology

25.2K 조회수.  California Institute of Technology - Caltech. 가까운 원시 상태에서 세균 세포의 ultrastructure 연구를 전자 cryotomography (요법)를 사용하는 방법을 우리는 "macromolecular"(~ 4 NM) 해상도, 여기를 보여줍니다.