Evaporation during preparation of unsupported thin vitrified aqueous layers for cryo-electron microscopy

Evaporation of water cannot be fully avoided when an unsupported thin vitrified film of an aqueous suspension is prepared for cryo-electron microscopy. This results in increasing concentration of solute which could affect the observed material. We have quantitatively studied this effect by measuring the contrast of polystyrene spheres in a metrizamide solution. The drying effect is generally negligible when specimens are prepared on a hydrophilic perforated support but it is frequently important when hydrophobic films are used instead. A flow of humid air, double blotting with minimal exposure of the thin liquid film to the atmosphere, or an automatic plunger optimizing the blotting conditions are simple methods for reducing drying effects. With this third device acting on a hydrophilic supporting film, the increase of solute concentration is limited to less than 20%.