An instrument for physical vapor deposition onto cryo-electron microscopy samples for microsecond time-resolved cryo-electron microscopy

Laser flash melting and revitrification experiments have recently improved the time resolution of cryo-electron microscopy (cryo-EM) to the microsecond timescale, making it fast enough to observe many of the protein motions that are associated with function. The technique has also opened up a new dimension for cryo-EM sample preparation, making it possible to deposit compounds onto a cryo-EM sample while it is frozen, so that upon flash melting, the embedded particles experience an altered environment. For example, we have recently shown that depositing ultrathin silicon dioxide membranes onto a cryo-EM sample causes particles to detach from the interface upon flash melting, removing preferred particle orientation. Here, we describe an apparatus for physical vapor deposition of compounds onto cryo-EM samples, detailing its design and operation. As a demonstration, we determine that the minimum thickness of silicon dioxide sealing membranes in a laser flash melting experiment is just over two monolayers. Moreover, we show that it is possible to deposit reagents onto the cryo-EM and induce mixing during flash melting, which points toward a new strategy for initiating protein dynamics in time resolved experiments. We propose that the design of our instrument can form the basis for an integrated platform for microsecond time-resolved cryo-EM experiments.