Pulsed IR Melting and Revitrification of Cryo-EM Specimens for Time-Resolved Experiments

作者：Ioana T. Grigoras, Biplob Nandy, Mathew James Peet, Christopher J. Russo · 发表于：Microscopy and Microanalysis · 年份：2026 · DOI：10.1093/mam/ozag053.441 · 研究领域：Advanced Electron Microscopy Techniques and Applications、Electron Spin Resonance Studies、Quantum, superfluid, helium dynamics

Laser melting and revitrification techniques offer a way of extending the time resolution of cryo-EM from milliseconds to microseconds, and thus observing transient conformational states of biological molecules that are too short-lived to be trapped by mixing followed by plunge-freezing [1, 2]. We present an apparatus for controlling the phase of the water in cryo-EM specimens using 3 μm laser pulses (Figure 1), which match the highest resonance peak in the infrared spectrum of water. We demonstrate rapid melting and revitrification using phase changes and the motion of ice-embedded gold nanoparticles (Figure 2), and we show that proteins are not destroyed by irradiation with 3 μm laser pulses. Cryo-EM specimens are submerged in liquid ethane during pulsing and multiple pulses are applied per location to increase the total reaction time. The pulsing process currently allows total reaction times of tens of microseconds. We also present an instrument for preparing an initial, out-of-equilibrium state using vapour deposition of small molecules. Cryo-EM specimens under vacuum are coated with thin films of ligand molecules and a cryogenically cooled crystal sensor is used to measure the deposition rate so that target ligand concentrations can be obtained upon subsequent pulsing. We describe experiments performed to explore the formation of various protein-ligand complexes using vapour deposition followed by laser pulsing. Further improvements in the methods and instruments promise...