Nanosecond Hyperquenching for Electron Cryo-Microscopy Without Air-Water Interface Artifacts

A major challenge in electron cryo-microscopy (ECM) imaging is preparing the protein specimen without the artifacts caused by the surface tension at the air-water interface (AWI). Here, we report nanosecond hyperquenching (NHQ) - a method of preparing ECM samples without AWI-bound protein macromolecules. The fast narrow sample jet impinges the eutectic propane-ethane (PET) liquid cryogen at 77 K and breaks up, forming 30-150-nm-thick vitrified films. NHQ films with the protein particles are formed directly in the PET cryogen, precluding AWI tension-driven protein adsorption, preferred orientation, subunit dissociation and denaturation. The formed film surfaces are essentially specimen-free, with a 2.7-nm-thick protein depleted layer of hyperquenched glassy water (HGW). This "surface sealing" appears to be the first essential stage of vitrification at NHQ conditions; it occurs in about 35 ps on cryogen encounter. We outline the depletion mechanism, where the growing HGW layer displaces protein particles from the surface inwards the film.