Implementation of a flash-photolysis system for time-resolved cryo-electron microscopy.

ABSTRACT

We describe here the implementation of a flash-photolysis system for time-resolved cryo-electron microscopy. A previously designed computer-controlled cryo-plunging apparatus [White, H.D., Thirumurugan, K., Walker, M.L., Trinick, J., 2003. A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray. J. Struct. Biol. 144, 246-252] was used as a hardware platform, onto which a xenon flash lamp and liquid light pipe were mounted. The irradiation initiates a reaction through cleavage of the photolabile blocking group from a biologically active compound. The timespan between flashing and freezing in cryogen is on the order of milliseconds, and defines the fastest observable reaction. Blotting of excess fluid, which takes on the order of 1s, is done before irradiation and thus does not represent a rate-limiting step. A specimen-heating problem, identified by measurements with a thermocouple, was alleviated with the use of thick, aluminum-coated grids.