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

Abstract 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.