A fast-freezing device with a retractable environmental chamber, suitable for kinetic cryo-electron microscopy studies

Abstract

The design and construction of a fast-freezing device are described. A polycarbonate chamber, in which the humidity and temperature are controlled by microprocessors, slides on a robust chassis guided by ball or Teflon bushings. In its freezing position, the chamber rests on top of the cryogen vessel. The specimen is therefore frozen directly from the experimental conditions within the chamber without exposure to the external environment. After freezing, the chamber, but not the specimen, rises automatically, vacating space for handling the specimen. The chamber, the shutter, and the specimen are all driven pneumatically at an adjustable speed of up to approximately 10 m s-1 and coordinated by either pneumatic or electronic logical gates. Provisions are made for automatic blotting, spraying, and flashing. The chamber is compatible with a liquid nitrogen-cooled copper block assembly for impact (slam) freezing for freeze-substitution and freeze-fracture.