Fabrication of Monolayer Graphene-Coated Grids for Cryoelectron Microscopy

Currently optimally preserving biological samples is a major challenge in obtaining high resolution cryo-EM structures. During the sample preparation process, specimens are exposed to hydrophobic air-water interface, which has denaturing effects on the samples and can cause stable samples to adopt a preferential orientation in the ice, which prevents high resolution structure determination. Coating cryo-EM grids with a thin layer of carbon of like soft graphene oxide has been commonly used to address issues related to interactions with air-water interface.

However, these layers often increase background noise in our images, which compromise the resolutions of our structure. Coating the grids with a monolayer of graphene accomplishes similar protection without introducing significant background noise. Using graphene coated cryo-EM grids can help overcome issues related to interactions with air-water interface.

However, commercial graphene grids are expensive and many labs consider graphene grids to be too complicated to prepare in-house. Researchers in the cryo-EM field can now use this step-by-step protocol to reproducefully produce dozens of high quality graphenes in one day. To begin, inside a fume hood, place a cover slip with a taped graphene sheet on a homemade spin coater.

Wearing safety goggles, use a glass pipette to add two drops of methylmethacrylate or MMA onto the graphene sheet and immediately start spinning at maximum speed. While spinning, add two more drops in the center and spin for one minute. After cleaning and drying three crystallizing dishes, pour 200 milliliters of chloroform, acetone, and isopropyl alcohol into each dish.

Place the base of the clamping TEM grid holder block at the bottom of the first dish containing 200 milliliters of chloroform. Ensuring the fenestrated film side is facing up, transfer each grid individually from the grid box to a well in the grid holder block. Cover the dish with aluminum foil and place it on an orbital shaker to gently shake for 30 minutes.

Next, place the metal lid on the grid holder block to secure the grids for transfer to the next crystallizing dish. Using a bent fork and a long tweezer, carefully lift the grid holder block out of the dish and place it at the bottom of a second dish containing 200 milliliters of acetone. Remove the lid from the grid holder block and gently shake it on an orbital shaker.

After 30 minutes, place the lid on the grid holder block and transfer it to a dish containing 200 milliliters of isopropyl alcohol to remove acetone residue. Remove the lid from the grid holder block and gently shake it for 20 minutes. Individually transfer the grids with the fenestrated film side up from the grid holder block to a glass Petri dish covered with blotting paper.

Transfer the cleaned grids with the fenestrated film side facing up onto the wet blotting paper. Submerge the hydrophobic grids vertically into the water to prevent bending due to water tension. Arrange the submerged grids in a square array, keeping them close but not overlapping.

Fill the grid coating trough with more deionized water, ensuring the water surface is at least five millimeters above the grids. To scoop out the MMA graphene film from the dish, slowly lower the cover slip into the dish at an angle some distance away from the graphene film. Position the cover slip underneath the graphene MMA sheet and align its edges parallelly with the sheet.

Then lift the cover slip vertically from the water, bringing the MMA graphene film with it. Transfer the MMA graphene film to the trough by lowering the cover slip into the water at a 45 degree angle so that the film detaches from the cover slip and floats on the water surface. Position the MMA graphene film above the grids before the water level decreases.

Using a Pasteur pipette whose tip has been melted closed, carefully manipulate the MMA graphene film's position. Using a syringe, slowly lower the water level such that the MMA graphene film perfectly lands on the grids and completely covers their surfaces. Using a pair of clean dry tweezers, lift the blotting paper holding the grids to a clean dust-free Petri dish.

Place the base of the clamping TEM grid holder block at the bottom of the first crystallizing dish containing 200 milliliters of fresh acetone. Individually transfer each grid with the MMA side facing up from the blotting paper to a well in the grid holder block. Cover the dish with foil and gently shake on an orbital shaker for 30 minutes.

Secure the grids by placing the metal lid on the grid holder block. Then, using a bent fork or long tweezers, carefully lift the block out of the dish and place it at the bottom of the second dish containing 200 milliliters of fresh acetone. After removing the lid from the block, gently shake it on an orbital shaker for 30 minutes.

Place the lid back on the grid holder block and transfer it to the dish containing 200 milliliters of isopropyl alcohol. Remove the lid before gently shaking for 20 minutes. Transfer the grids to a small glass Petri dish covered with blotting paper and air dry the grids for 10 minutes.

In TEM diffraction modes, EM grids covered with a monolayer of graphene show Bragg peaks corresponding to the hexagonal lattice of the graphene. Graphene grids have a concentrating effect on a sample, as observed when comparing apoferritin applied to wholly gold grids with and without the graphene support. A fast Fourier transform of the cryo-EM micrograph of apoferritin on graphene covered gold grids also showed Bragg peaks corresponding to the hexagonal graphene lattice.