Name: Electron Cryotomography of Bacterial Cells (Video) | JoVE Journal
Uploaded: 2010-05-06T13:20:00.000+00:00
Duration: 14 min 23 s
Description: Learn electron cryotomography to visualize bacterial ultrastructure at near-native states using plunge freezing and high-pressure freezing techniques. This protocol enables 3D reconstruction of cellular architecture for cell biology research.

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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/pubmed?term=((How+electron+cryotomography+is+opening+a+new+window+onto+prokaryotic+ultrastructure%5BTitle%5D)+AND+%22Curr.+Opin.+Struct.+Biol%22%5BJournal%5D)">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/pubmed?term=((Electron+cryotomography%3A+a+new+view+into+microbial+ultrastructure%5BTitle%5D)+AND+%22Curr.+Opin.+Microbiol%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+imaging+of+biological+complexity%5BTitle%5D)+AND+%22J.+Struct.+Biol%22%5BJournal%5D)">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/pubmed?term=((Electron+cryotomography+sample+preparation+using+the+Vitrobot%5BTitle%5D)+AND+%22Nat.+Protocols%22%5BJournal%5D)">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/pubmed?term=((A+new+approach+for+cryofixation+by+high-pressure+freezing%5BTitle%5D)+AND+%22J.+Microsc%22%5BJournal%5D)">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/pubmed?term=((Vitreous+cryosectioning+of+cells%2C+facilitated+by+a+micromanipulator%5BTitle%5D)+AND+%22J.+Microsc%22%5BJournal%5D)">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/pubmed?term=((Improving+the+technique+of+vitreous+cryo-sectioning+for+cryo-electron+tomography%3A+Electrostatic+charging+for+section+attachment+and+implementation+of+an+anti-contamination+glove+box%5BTitle%5D)+AND+%22J.+Struct.+Biol%22%5BJournal%5D)">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/pubmed?term=((Fully+automated%2C+sequential+tilt-series+acquisition+with+Leginon%5BTitle%5D)+AND+%22J.+Struct.+Biol%22%5BJournal%5D)">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/pubmed?term=((Markov+random+field+based+automatic+image+alignment+for+electron+tomography%5BTitle%5D)+AND+%22J.+Struct.+Biol%22%5BJournal%5D)">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/pubmed?term=((Computer+visualization+of+three-dimensional+image+data+using+IMOD%5BTitle%5D)+AND+%22J.+Struct.+Biol%22%5BJournal%5D)">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/pubmed?term=((Dual-axis+tomography%3A+an+approach+with+alignment+methods+that+preserve+resolution%5BTitle%5D)+AND+%22J.+Struct.+Biol%22%5BJournal%5D)">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.

<table><tbody><tr><td>Poly-L-Lysine</td><td>Sigma-Aldrich</td><td>P8920</td><td>0.1% solution</td></tr><tr><td>MKIII Vitrobot</td><td>FEI</td><td></td><td></td></tr><tr><td>Gold colloid solution</td><td>Sigma-Aldrich</td><td>G1527-25ml</td><td>10 nm</td></tr><tr><td>Bovine serum Albumin</td><td>Invitrogen</td><td>P2489</td><td>10%</td></tr><tr><td>Nr 1 Filter paper</td><td>Whatman, GE Healthcare</td><td>1001 055</td><td></td></tr><tr><td>Dextran</td><td>Sigma-Aldrich</td><td>31389</td><td>15-25%, MW 39000</td></tr><tr><td>Brass-domed planchet</td><td>Swiss Precision Company</td><td></td><td></td></tr><tr><td>Bal-Tec HPm 010 high pressure freezer</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Cryo-ultra microtome UC6/FC6</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Diamond cryo trim 45</td><td>Diatome</td><td></td><td></td></tr><tr><td>Static line II ioniser</td><td>Diatome</td><td></td><td></td></tr><tr><td>CX100 Dry shipper</td><td>Taylor-Wharton</td><td></td><td></td></tr><tr><td>MSH loading station</td><td>Gatan</td><td></td><td></td></tr><tr><td>Ti E/B inverted microscope</td><td>Nikon Instruments</td><td></td><td>Custom</td></tr><tr><td>Cryo LM stage</td><td>FEI</td><td></td><td>Custom</td></tr><tr><td>TF30-polara</td><td>FEI</td><td></td><td>With UltraCam from Gatan</td></tr><tr><td>Amira</td><td>Visage Imaging</td><td></td><td></td></tr></tbody></table>

electron cryotomography of bacterial cells

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.

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 ...

In this video, 3D images of bacterial cells in near native states at molecular resolution are generated by electron cryo tomography. This is accomplished by first plunge freezing intact cells, or alternatively high pressure freezing and cryosectioning the cell pellets. A series of M projection images are acquired and used to generate a 3D reconstruction, or Tom Graham, which is segmented and further computationally analyzed.Hi, I'm Grant Jensen from the Division of Biology at Caltech, and this is Sonya Chen who's organized our presentation. Today we're gonna show you how to do electron cryo tomography of bacterial cells. Typically, a single individual would do the entire procedure, but because our protocols have been developed through shared experience, we will include everybody who's presently working in the lab.In addition to Sonya and me on the video, you'll see Megan Dobro, Ariana, Bri, Miguel, Alistair McDowell, mark Rodinsky, and Jen. She other lab members produced the computer screen captures and helped prepare the accompanying written protocol including Morgan Bebe, Martin Piller, Jane Ding, Joe Lee, Lou GaN, Aliza Tova, and Dylan Morris. Ariana will start us off by showing you how we plunge freeze bacterial cells.For thin bacterial cells, a suspension of intact cells is plunge frozen across an EM grid. Here the process is demonstrated using a commercial automatic plunge freezer begin by examining the bacterial cultures to ensure that they're free of contaminants and at an appropriate confluence or concentration glow discharge the carbon coated EM grid for two to four minutes. Next, mix 100 microliters of colloidal gold solution with 25 microliters of a 5%BSA solution.Centrifuge the mixture at 18, 000 G for 15 minutes. After the centrifugation, remove the supernatant and resuspend the gold pellet. In 20 microliters of cell solution, place the EM grid in the Vitra bot at 100%humidity.Then apply four microliters of the cell mixture to the glow discharged EM grid. The grid is automatically blotted from both sides with filter paper to remove excess liquid, then plunged rapidly into a liquid mixture of ethane and propane, which is cooled by liquid nitrogen. Carefully remove the grid from the ethane propane mixture and quickly transfer it into a grid storage box kept under liquid nitrogen For thicker cells, high pressure freezing prevents ice crystallization.Subsequent cryosectioning render samples thin enough for em. Imaging begin by centrifuging 15 milliliters of cells at 1000 RPM for three minutes. Following centrifugation, remove the supernatant mix 0.5 milliliters of the remaining culture pellet with 0.5 milliliters of 20%dextrin cryoprotectant.Then centrifuge again at 13, 000 RPM for 15 seconds. After the cells have been pelleted, remove the supernatant pipette 0.1 milliliter of the super pellet paste into a heat sealed micro pipette tip, which already contains 0.2 milliliters of 20%dextrin cryoprotectant centrifuge. The mixture in the micro pipette tip at 13, 000 RBM for 30 seconds.After the spin, remove the supernatant. A five to 10 microliter tight pellet is seen at the sealed tip. Transfer the paste to one half dome of a Teflon coated brass domed planchet already mounted in the high pressure freezer holder arm, then seal with its flat partner brass hat.Then promptly insert the arm assembly into the primed high pressure freezer. At 2100 bar pressure, the combined brass planche unit containing the cells is rapidly cooled by a jet of high pressure liquid nitrogen. After the cells have been cooled, rapidly removed the arm assembly and quickly plunge it into a bath of liquid nitrogen to prevent the planchette from warming.Next, transfer the planchette to a pre-cool cryo microtome vice-like chuck mounted securely in the cryo microtome chamber to expose the well frozen dome of cells for sectioning. The two brass planches are carefully separated under liquid nitrogen. A perfectly frozen dome of cells will be glassy in appearance and free of cracks and ice nucleation fissures.Using the cryo microtome shape the outer dome region to less than 0.2 square millimeters using the diamond trimming tool with a rapid sectioning speed retain and about 200 micrometer depth with antistatic spray directed at the low angle knife and copper support grids. In close proximity, carefully advance the diamond knife to the polished block face using a cutting speed of about 0.4 millimeters per second and a narrow cutting window. Begin sectioning 50 to 350 nanometer thick slices.Maneuver a fine eyelash applicator to snag the early sections, sliding off the low angle diamonds knife edge and to support and guide the ribbons of sections across the copper grid support. Once the grid is loaded, use a cooled polished silver probe to firmly press the ribbons. Store the grids in the grid boxes in a liquid nitrogen cooled dry shipper until the microscope is ready.The bacterial cells on the grid can be imaged using a cryo light microscope. Here, a custom Nikon inverted microscope, TIB with modified FEI cryo stage will be used. Load the grids into cartridges on the cryo station and clip them down using the copper clip rings, then place the cartridge in a tube in liquid nitrogen for transferring.Load up to two cartridges into the pre-cool cryo stage and move the grid into the viewing window. Then lower the condenser lens and focus the objective lens onto the grid. Find the cells and take images.For correlated LM and EM study. The grid is scanned around the area of the cells for locating the cells later in em. After imaging the grid, put the cartridge back into the tube and keep the tube in the liquid nitrogen until ready to be imaged in EM to obtain a 3D TOM of the bacterial cell.A series of projection images are taken while the sample is incrementally tilted along one or two Es in the cryo TEM here, the procedure is demonstrated using legon with a cryo T-E-M-F-E-I-T-F 30 polar load up to six cartridges into a multi selection holder on the cryo station. Then connect it to the TF 30 Polar Select one cartridge and insert it into the EM column. Start legend on client software on the EM computer.Input the image conditions for the legend on session. Select targets on images starting from lowest magnification and send them to the next step with higher magnification. Then queue up all targets for tilt series collection and submit all of them to the final tomography step.Acquire a series of images. The sample will be incrementally tilted along one or two axes in the cryo TEM. After the experiment is done, download the completed tilt series onto a local workstation for reconstruction.Tomo Grahams of the bacterial cells are calculated using specialized software. The program Raptor is used to reconstruct Toms automatically and e tomo can be used to do it semi automatically. Inspect the tilt series using Im MOD's 3D mod viewer and discard any that because of tracking or other errors are unlikely to produce.A good tommo raptor is run distributed over the Linux machines in the lab using the basic settings. It takes 20 minutes to two hours depending on the size of the file to generate the tomograph. When Raptor fails or an investigator wishes to ensure the reconstruction is optimal, EMO can be used to align the images carefully by hand.Follow the steps of each tab in the e tomo GUI to reconstruct the Tom Graham from the Tilt series. We use a web-based in-house database to organize store search and distribute the tomographic data. The main browsing page presents a thumbnail image and a featured YouTube like movie.For each tomo Graham. After the Tommo, Grahams are obtained, individual features can be segmented to help visualize their shapes. In 3D segmentation is the process of assigning each vle of the 3D image to a particular region or material.Here a mirror software is used to generate a surface model in which each region is shown with a different color and viewed in 3D movies and animations of the tomo. Grahams can be made to summarize projects and to illustrate findings in 3D graphics.Representation. Software such as Amira or Kymera can be used to render the individual still frames of the movie and commercial.Movie editing software such as Adobe Premier can be used to edit the movie commercial software. Maya is used to generate the animation. It can take the surfaces from Amira construct key frames and render into a movie.Here a representative tilt series of whole cell data is shown for the Spiro Treponema Promesa, followed by a slice by slice view of the reconstructed Tom Agram. Segmentation of the T Promesa cell distinguishes its outer membrane, inner membrane and surface structures, including surface bowls and surface hook arcade. The bacteria is flagella and ular motor, peri plasmic cone, and pillai.This mechanical model shows how the spirochete treponema Promesa swims and inner cylinder or core is enclosed by an outer sheath. The flagella move like paint rollers between the inner cylinder and outer sheath. As the flagella turn, the inner cylinder counter rotates within the outer sheath.In a non viscous environment, the cell rotates uselessly, but in viscous environments like those in which T Promesa is found in nature, external fibers or other objects resist rotation of the outer sheath, accelerating rotation of the inner cylinder, and providing materials that the cell can push against as it drills itself forward. Shown here are actual T promeus cells as observed through a light microscope. We've just shown you how to do electron cryo tomography of bacterial cells.Of course, we realize this type of work requires major investments. The cryo-electron microscopes and the fluorescence microscopes are sophisticated and expensive, and they also must be properly cited. For instance, the foundation of this room is a mechanically isolated slab of concrete, several feet thick, resting on specially compacted ground.The air conditioning system is also custom designed with large ducts and gentle curves to maintain a constant temperature without any drafts or turbulence. The curtains that you see are placed to reduce acoustic noise. And finally, we verify this room is free of interfering electromagnetic fields.While the operations we've shown you on the microscope are now largely automated, problems still frequently arise and require expert diagnosis. Nevertheless, we hope watching this movie has helped you appreciate the procedures and the instrumentation involved in this type of work. Thank you for your attention.

electron-cryotomography-of-bacterial-cells

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Source: Chetrit, D., et al. Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System. J. Vis. Exp. (2020)The video describes how to prepare Legionella pneumophila samples for cryo-electron tomography by combining bacterial culture, gold nanoparticle marking, and vitrification techniques. The process preserves native cellular structures and enables high-resolution imaging of membrane-associated secretion...

Preparation of Legionella pneumophila for Cryogenic Electron Tomography

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Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

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Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the Saccharomyces cerevisiae.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

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Cryo-STEM tomography provides a means to visualize organelles of intact cells without embedding, sectioning, or other invasive preparations. The 3D resolution obtained is currently in the range of a few nanometers, with a field of view of several micrometers and an accessible thickness in the order of 1 µm.

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This protocol standardizes cryo-electron tomography (cryo‑ET) for thick tissues by integrating high-pressure freezing (HPF) with the waffle method, cryogenic focus ion beam (cryo‑FIB) lift‑out, and dose‑symmetric, parallel acquisition, yielding uniformly thin lamellae and vitrified specimens for reproducible in situ analysis in the mouse hippocampus.

Cryo-Electron Tomography Workflow for Thick Tissues Demonstrated in Mouse Hippocampus

Learn electron cryotomography to visualize bacterial ultrastructure at near-native states using plunge freezing and high-pressure freezing techniques. This protocol enables 3D reconstruction of cellular architecture for cell biology research.

Electron Cryotomography of Bacterial Cells

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