Here we present improved protocols to prepare cryo-EM samples with support films using a novel flotation block that facilitates handling of such delicate films. The key advantage of this method is that the sample is protected from the air water interface where damaging effects like denaturation can occur. One of the protocols presented here avoids any kind of expensive treatment to render graphing hydrophilic, therefore increasing its accessibility to the whole EM community.
Small flotation devices are not yet widely used by electro microscopy for good preparation. So these visual demonstrations can provide a benchmark for their use. To begin, wash the TEM grids with double distilled water and ethel acetate followed by plasma cleaning.
Then add 10 to 12 microliters of the sample into the buffer exchange well, the flotation block and 10 to 12 microliters of 2%urinal acetate solution into the adjacent non-buffer exchange well for negative staining. It is essential to take appropriate safety precautions when working with stain. Carefully cut two small pieces of mica with predeposited carbon film on top.
Ensure that the mica fragments are wide enough to fit into the well and longer than the well length, such that the fragment can sit on the well while the carbon is floating and there is enough space to handle the fragment with the tweezers. Immerse the mica into the well with an approximate angle of 45 degrees until the mica sits on the ramp of the well and the carbon layer is observed at the surface of the liquid sample. After incubating the carbon with sample, withdraw the mica sheet very slowly to recover the carbon film and to minimize the residual viscus sample retention.
Carefully blot the mica by tapping the lower non-carbon surface with a filter paper to remove the excess liquid and immerse the mica fragment into the opposing well containing 2%urinal acetate solution to exchange the carbon layer with the negative stain. Recover the floating carbon layer with the holy carbon covered side of the washed and plasma cleaned EM grid. Then leave the grids to air dry until imaging on the TEM, preferably covering them to avoid airborne contamination.
After cleaning the TEM grids as previously demonstrated, prepare the graphene oxide suspension just before use and vortex thoroughly. Then add 10 to 12 microliters of graphene oxide suspension into the four non buffer exchange wells. along the flotation block.
Add 10 to 12 microliters of double distilled water or ultra pure water into the remaining four buffer exchange wells of the block. Drop four grids gently onto the graphene oxide suspension of each well and incubate for one minute to ensure that the holy carbon covered side is in contact with the solution. After one minute, recover each grid carefully by sliding the tweezers into the tweezer groove of each non buffer exchange well.
Gently and briefly nudge the copper non-carbon covered side of each grid with the double distilled water in the adjacent well until the drop of water is visible on the grid. Then carefully hold the grid water drop upside down against a piece of filter paper. Cover the grids and leave them in the tweezers to air dry.
Place four wash grids on top of a copper graphing sheet deposited on a glass slide, and cover each grid with a drop of isopropanol to allow intimate contact between the monolayer graphene and the grid. After the complete evaporation of the isopropanol, float the copper graphene sheet with grids onto the ferric chloride solution in the glass Petri dish. Then cover the dish to avoid airborne contamination and leave it to etch at room temperature overnight.
Use a loop with the diameter larger than the TEM grid size to fish out the grids floating on the graphene monolayer and carefully transfer them to a glass Petri dish containing double distilled water for washing. Wash the grids twice in water and transfer the grids into a Petri dish containing sample buffer until sample preparation and plunge freezing. Add 10 to 12 microliters of the sample into a well of the flotation block.
When the sample is ready in the block, pick the graphene coated grid from the buffer solution using a pair of clean tweezers and place it onto the surface of the sample containing well. After an appropriate incubation period, pick up the grid with a pair of clean freezing tweezers and proceed with blotting and vitrification. Negatively stained grids prepared with the support flotation block are typically covered with amorphous carbon across the entire grid surface.
While breakage of the carbon film occurs in some instances, a large number of grid squares are pristine and therefore applicable for negative staining purposes. Similarly, good film coverage is routinely achieved across the entire grid using the graphene oxide protocol. Graphene oxide grids can be prepared quickly from raw materials and are highly protective of the sample.
Keeping the graphene wet and sample application in situ just before freezing is sufficient to generate suitable ice layers for cryo-EM. This protocol does not require equipment to render the graphene hydrophilic and provides an advantage for sample recovery. The support flotation block has two needle ports at the site of each well that allow buffer extend in C2 which is very useful for samples derived from glycerol gradients.
The flotation block has enabled exploration of on grid affinity purification methods by utilizing controllable buffer flow of small volumes, as well as functionalized support films.
