An Instrument for Physical Vapor Deposition onto Cryo-EM Samples for Microsecond Time-Resolved Cryo-EM

TL;DR

This paper introduces a new apparatus for depositing compounds onto cryo-EM samples to enable microsecond time-resolved experiments, enhancing the ability to observe rapid protein dynamics.

Contribution

The paper presents a novel instrument for physical vapor deposition onto cryo-EM samples, facilitating time-resolved studies by enabling reagent deposition during sample preparation.

Findings

Minimum silicon dioxide membrane thickness is just over two monolayers.

The apparatus can be used to deposit ultrathin membranes for cryo-EM.

Proposed platform aims to enable microsecond time-resolved cryo-EM experiments.

Abstract

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. These experiments also point towards a new strategy for initiating protein dynamics in time resolved experiments by depositing reagents, which will then mix with the sample upon flash melting. 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. We propose that our design can form the basis for an integrated platform for microsecond time-resolved cryo-EM experiments.