A computer-controlled spraying-freezing apparatus for millisecond time-resolution electron cryomicroscopy

摘要

Apparatus is described for the kinetic investigation of biological reactions by electron cryomicroscopy with time resolution on the order of milliseconds. This involves layering a grid with one reactant and then spraying on a second reactant immediately before freezing. Two-stage mixing can be achieved by mixing two solutions, holding them in a delay line for a preset interval, and then spraying the aged solution onto a grid carrying a third reactant. The individual steps of these procedures are under software control and can be adjusted independently. Spray-freezing is widely applicable since solutions of small molecules, proteins, and protein assemblies can be delivered as aerosols. Thus the method can be used to study both the effects of small molecules on macromolecules and for monitoring protein-protein interactions. It may also be useful in other situations, for instance in light microscopy.