This method can help answer key questions in the structure biology field, such as the preparation of sensitive proteins, where a limited amount of protein is available. The main advantage of this technique is that compared to standard sample preparation methods, only minute amounts of sample are required, and that no harsh paper blotting step is involved. The implications of this technique extends towards the single cell analysis by visual proteomics, or the diagnosis of protein-related disease.
We first had the idea for this method when we had the technology available to solve high resolution structures with only about 100, 000 individual particles. To begin, prepare the sample as described in the accompanying text protocol. Next, assemble the ethane cup, cryo box holder and spider.
And fill the cryogen container to the brim with liquid nitrogen. After a few minutes, the cup will be cool and free of liquid nitrogen. Then open the ethane gas bottle, and slowly let the gas stream into the ethane cup.
Let the cup fill with liquid ethane until the level is two to three millimeters below the top. This will take a few minutes and requires about five milliliters of liquid ethane. Remove the spider, place the polystyrol lid on top, and place the cryogen container on the mounting in the cryo writer.
Grasp the glow discharged EM grid with the cryo writer tweezers, knot the tweezers on the electromagnet, and screw the micromanipulator to align the grid flat on the stage, carbon film side up. Back in the software, double-click on grid_save. Then adjust the microcapillary position, so that the tip is approximately 10 micrometers above the grid surface.
Make sure that the microcapillary can move freely across the grid, without touching it anywhere. And if necessary, withdraw the microcapillary a few micrometers. Then bring it to position back to the center of the grid, and save the new position as grid.
Move microcapillary back to home position. Next, flush the microcapillary with a few tens of nanoliters of system liquid, and use lint-free tissue to remove any drops from the microcapillary. With everything now prepared, start the macro script.
The macro first moves into position, and dispenses five nanoliters of system liquid to remove any air bubbles at the tip, and then moves to samples position. Next, it aspirates 65 nanoliters of sample, and then infuses five nanoliters back into the sample tube. This accounts for system backlash, and it allows for synchronized writing.
After it moves to the grid position, it initiates a writing pattern, which will cause the microcapillary to move across the grid and simultaneously dispense 45 nanoliters of sample. Then it moves the microcapillary back to the center of the grid, lowers it another 10 micrometers, and withdraws excess sample liquid. Finally, the macro withdraws the microcapillary and turns off the electromagnet, which initiates the plunge freeze mechanism.
Once released from the magnet, carefully release the magnetic adaptor from the plunger and quickly transfer the grid from the ethane cup into the cryogen container, containing the cryo box, placing the grid into a free slot. To begin, prepare cells as described in the accompanying text protocol, and mount the indium tin oxide slide on the microscope insert. Use two screws to fix the slide on the aluminum insert, and to ensure electrical contact between the indium tin oxide coating of the slide and the electrically grounded aluminum frame.
Then remove the cell culture medium from the well, and wash the cells twice with 300 microliters of electroporation buffer. Following the last wash, keep the cells in the electroporation buffer. Next, place the aluminum insert that is holding the indium tin oxide slide in the live cell incubator stage, on the set-up.
Using the microscope, locate the cell culture and choose an area with no cells. Approach the microcapillary tip on the slide surface and gently touch it. Then withdraw the tip in 100 micrometers and save the position as cells.
Now quickly leave the cell culture and flush the microcapillary tip with a few tens of nanoliters of system liquid, then put it into the cell culture again. Dispense a few nanoliters during immersion to the PDMS well to ensure that no air bubbles are trapped at the tip. Double-click at the cell's position and gently approach the surface of the indium tin oxide slide and upon contact, retract the tip 10 micrometers.
At this point, select a nearby cell for lysis. and place the tip of the microcapillary above the targeted cell. Then start the macro script for single-cell lysis.
Once started, the script will ask for a position that the microcapillary should be moved to after a cell has been successfully lysed. Enter the desired position, for example grid, for the EM grid. The macro will then proceed without user intervention, and will begin by moving the microscope's stage in cell culture 100 micrometers to the left, where it takes a snapshot of the targeted cell.
Then 15 nanoliters of double-distilled water dispenses from the microcapillary tube, displacing and diluting the high salt buffer, applying osmotic pressure to the cell. Next, the stage moves back to position the tip above the targeted cell again. There, a predefined voltage burst is applied, and after 500 milliseconds, the pump system starts to aspirate three nanoliters of sample at a flow rate of two microliters per minute.
Finally, the stage moves to the left again, which allows the cell to be inspected. A window appears asking for user input. If the cell was successful, enter yes to take a snapshot of the lysed cell.
Afterwards, the microcapillary moves to the endo location, immersed in the reservoir liquid. Leave the microcapillary immersed for eight to 12 minutes, depending on the nozzle geometry. Next, dispense five nanoliters of the conditioned cell lysate onto the grid.
Withdraw the microcapillary and let the conditioned sample slowly dry on a dew point stage. Finally, remove the grid from the stage and store it at room temperature in a grid box. Shown here are typical cryo images of frozen samples using the CryoWriter set up described.
In the grid overview, the periphery of the vitreous ice can be clearly seen. Upon closer examination of a grid slot with the holey carbon film containing vitreous ice, white holes can be found, indicating that not all holes were filled with sample buffer. In one of the sample containing carbon holes, the tail of a bacteriophage can be clearly seen with detail.
Using the CryoWriter set up to perform single-cell visual proteomics, one can see things such as individual proteins, for example filamentous actin and membrane patches with attached proteins. The image you can see in panel 6b is negatively stained with a 2%negative stain, based on an organotungsten compound. Panel c shows a single-cell lysate prepared for cryo EM.After watching this video, you should have a good understanding of how to prepare negative stain or cryo EM grids, from nanoliter total volumes of protein samples or single-cell extracts.
Don't forget that working with liquid ethane and nitrogen can be extremely hazardous, and precautions, such as wearing safety glasses and a lab coat, should always be taken by performing this procedure.
