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.
