Microsecond melting and revitrification of cryo samples with a correlative light-electron microscopy approach

Abstract

We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that affords microsecond time resolution. It involves melting a cryo sample with a laser beam to allow dynamics of the embedded particles to occur. Once the laser beam is switched off, the sample revitrifies within just a few microseconds, trapping the particles in their transient configurations, which can subsequently be imaged to obtain a snap shot of the dynamics at this point in time. While we have previously performed such experiments with a modified transmission electron microscope, we here demonstrate a simpler implementation that uses an optical microscope. We believe that this will make our technique more easily accessible and hope that it will encourage other groups to apply microsecond time-resolved cryo-EM to study the fast dynamics of a variety of proteins.

Near-atomic resolution reconstructions from in situ revitrified cryo samples

“…Our experiments demonstrate that near-atomic resolution reconstructions can be obtained from in situ melted and revitrified cryo samples, in agreement with previous results for samples that we revitrified in an optical microscope (Bongiovanni et al, 2022). This strongly suggests that the revitrification process does not impose a fundamental limit on the obtainable spatial resolution.…”

“…We have previously described two different implementations of our technique. Melting and revitrification experiments can be performed in an optical microscope using a correlative light-electron microscopy approach (Bongiovanni et al, 2022). Alternatively, such experiments can be carried out in situ (Voss et al, 2021a,b;Harder et al, 2022) with a modified transmission electron microscope (Olshin et al, 2020).…”

Fast Viral Dynamics Revealed by Microsecond Time-Resolved Cryo-EM

“…This has largely remained elusive, which has left our understanding of protein function fundamentally incomplete (12). In order to enable such observations, we have recently proposed microsecond time-resolved cryo-EM (13)(14)(15)(16)(17), which affords a time resolution of about 5 µs or better (13) and enables near-atomic resolution reconstructions (16,17).…”

A simple flash and freeze system for cryogenic time-resolved electron microscopy

“…Furthermore, as modern computational approaches in cryoEM can 3D classify particles, branched conformational change pathways can be quantified and resolved. While the practical lower limit of 1 ms is governed by the cooling rate of the plunge freezing method ( Dubochet, 2007 ; Frank, 2017 ), in-situ rapid heating and cooling methods may provide access to microsecond timescales ( Voss et al, 2021 ; Bongiovanni et al, 2022 ).…”