Cryo-EM grid nanofabrication

Rubinstein Laboratory's nanofabrication protocol for making holey-carbon coated grids for cryo-EM. Evaporation of carbon onto the formvar layer can be substituted for evaporation of gold to nanofabricate holey-gold coated grids. Described in Marr, Benlekbir, and Rubinstein (2014). Fabrication of carbon films with ∼ 500nm holes for cryo-EM with a direct detector device. Journal of Structural Biology 185, 42-7. Starring Chelsea Marr as narrator.

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Members of the laboratory now inspect the plastic pattern regularly during baking to ensure desired hole sizes are obtained.

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Our current protocol for stamping is ~4-5 lbs for 2 minutes (with the 1.2 um pattern)

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I tend to let PDMS polymerize for a few days before removing stamps from the silicon master pattern. The surface of the PDMS should not be tacky at all. We attach stamps to the pressure test stand with double sided tape.

[@rubinsteinlaboratory2935]

We now use a release agent on the silicon master patter (TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL)-1-TRICHLOROSILANE [WARNING: dangerous substance - use appropriate precautions]. This only has to be done once per master pattern. Deposit ~ 30 uL into an open top container inside a dedicated desiccator. Apply vacuum with wafer in chamber for ~ 15 min to apply release agent to wafer. Too much release agent will inhibit PDMS binding to glass during stamp making. (NB: the release agent is not added directly on top of the master pattern, it transfers through the atmosphere in the dessicator)

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Most people in the laboratory now use a sputter coater to deposit ~35 nm of gold on grids, rather than evaporating gold (or carbon)

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We attach the PDMS stamp to the pressure test stand with double sided tape.

[@rubinsteinlaboratory2935]

We now let the PDMS cure for several days before removing it from the silicon wafer. It should not be at all tacky when removed.

[@lindseyfiddes459]

What is the height of the features on the silicon master?

[@rubinsteinlaboratory2935]

Sorry, didn't see this question here. The e-beam resist is ZEP-520A, ~350 nm thick. The features should be the same height. The features are made of resist, not etched into the silicon.