  Proteins typically undergo conformational dynamics on the microsecond to
millisecond timescale as they perform their function, which is much faster than
the time-resolution of cryo-electron microscopy and has thus prevented
real-time observations. Here, we propose a novel approach for microsecond
time-resolved cryo-electron microscopy that involves rapidly melting a cryo
specimen in situ with a laser beam. The sample remains liquid for the duration
of the laser pulse, offering a tunable time window in which the dynamics of
embedded particles can be induced in their native liquid environment. After the
laser pulse, the sample vitrifies in just a few microseconds, trapping
particles in their transient configurations, so that they can subsequently be
characterized with conventional cryo-electron microscopy. We demonstrate that
our melting and revitrification approach is viable and affords microsecond time
resolution. As a proof of principle, we study the disassembly of particles
after they incur structural damage and trap them in partially unraveled
configurations.
