Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

A common challenge in cryo-electron microscopy sample preparation is particle localization at the air-water interface, causing the particles to adopt preferred orientations and denature. Instead, it's better to have the particles rest on the grid surface, which finned film supports like graphene promote. Another challenge is the requirement for relatively high sample concentrations, which could also be addressed by graphene.

Graphene-coated grids provide numerous advantages for cryo-EM structure determination, but have historically been difficult to fabricate in-house reproducibly, as well as cost prohibitive to purchase commercially. This protocol allows for the robust deposition of monolayer graphene, thereby lowering the barrier to users employing these grids in their research. Graphene is unique because of its material properties.

It is a monolayer of carbon and therefore exhibits minimal background noise. Other cryo-EM grid supports, such as a thin layer of amorphous carbon or graphene oxide flakes, cannot be applied as a single monolayer and contribute more background signal. Now that we have established a robust protocol for the application of graphene to cryo-EM grids, we would like to pursue the functionalization of graphene-coated grids with affinity handles for on-grid capture directly from cell lysates.

This could alleviate another bottleneck in cryo-EM specimen preparation. Begin by preparing a graphene etching solution in a 50 milliliter beaker. To do so, add 4.6 grams of ammonium persulfate or APS to 20 milliliters of molecular grade water in the beaker, cover it with aluminum foil and allow the APS to dissolve completely.

To prepare the CVD graphene section for MMA coating, carefully cut a square section of the graphene, transfer the section to a coverslip in a clean Petri dish and cover the dish for transporting it to the spin coater. Next, set the spin coater to a 60-second high speed spin at 2, 500 rotations per minute. Carefully transfer the CVD graphene section to the appropriately sized chuck.

Press the take or absorb button to engage the vacuum pump and affix the graphene to the chuck. Then apply MMA to the center of the CVD graphene square. Close the lid, pull the vacuum, and immediately press the start or stop button.

After disengaging the vacuum pump once the spinning has stopped, using tweezers, carefully retrieve the MMA-coated CVD graphene. Invert the graphene such that the coated side is facing down, and place it back on the glass coverslip. Next, using flat tip tweezers, transfer the CVD graphene on the coverslip into the glow discharger and glow discharge for 30 seconds at 25 milliampere.

Return the graphene on the coverslip to the Petri dish. Finally, cover it for transporting it to the copper etching area. Begin by cutting the methylmethacrylate or MMA-coated graphene squares into grid sized pieces.

To do so, use two sets of reverse action forceps to support the CVD graphene square, while ensuring the MMA side faces up while attaching it to the forceps. To cut the graphene into small squares, use the two sets of reverse action forceps to hold the graphene right side up, anchoring it in position, as well as to hold the edge of the small square after cutting it away from the rest of the big square. Tilt the beaker containing ammonium persulfate or APS to place the squares in it at a shallow angle without sinking.

Then pick a square and make it contact the surface of the APS solution before releasing it. After covering the beaker with aluminum foil, incubate it overnight at 25 degrees Celsius. Next, to remove the MMA graphene films from the APS solution, first plunge a glass coverslip vertically into the solution and then move it laterally such that it touches a floating square.

Then carefully remove the coverslip while ensuring that the film adheres completely to the side of the coverslip upon removal. Plunge the cover slip vertically into molecular grade water in a 50 milliliter beaker to dislodge the attached film into the water. In a similar manner, transfer all the films to the beaker.

Let the films remain in the beaker for 20 minutes before they are ready to be adhered to cryo-EM grids. To attach the methylmethacrylate or MMA-coated CVD graphene to the grids, use the previously prepared grid-sized coated graphene squares kept in a beaker containing molecular grade water. Using negative action tweezers, gently dip a grid vertically into the water with the carbon side facing a floating MMA graphene square.

Once in contact with the square, carefully remove the grid from the water while ensuring the square fully adheres to the carbon side of the grid. Place the grids on a clean coverslip with the MMA graphene side facing up and allow the grids to air dry for one to two minutes. Using flat tip tweezers, transfer the coverslip with the grids to a hot plate set at 130 degrees Celsius.

Incubate the grids after covering them with the top of a glass Petri dish for 20 minutes. Next, for washing MMA with acetone, transfer the entire coverslip into a Petri dish filled with 15 milliliters of acetone and incubate for 30 minutes. Then using a clean glass serological pipette, transfer the acetone to a waste container.

With another clean glass serological pipette, add 15 milliliters of fresh acetone to the Petri dish and incubate for 30 minutes. After three acetone washes, remove the acetone. And using a clean glass serological pipette, replace it with 15 milliliters of isopropanol.

Then using tweezers, carefully remove the grids from isopropanol to air dry them on a clean coverslip. Next, place the coverslip with graphene-coated grids on a hot plate set to 100 degrees Celsius for 10 minutes to evaporate the residual organics. Using reverse action tweezers, gently place the grids into a clean illumination area of a UV ozone cleaner with the graphene-coated side facing up.

Slide the illumination area closed and turn the machine on. Set the time dial to the designated treatment time to start UV ozone treating the grids. Finally, proceed to plunge a cryo-electron microscopy sample on the treated grids.