Shaped Laser Pulses for Microsecond Time-Resolved Cryo-EM: Outrunning Crystallization During Flash Melting

TL;DR

This paper demonstrates how shaped microsecond laser pulses can achieve ultra-high heating rates to prevent crystallization in cryo-EM samples, enabling more accurate nanosecond time-resolved studies of biomolecules.

Contribution

It introduces a method using shaped laser pulses to surpass the critical heating rate, preventing crystallization during cryo-EM sample preparation.

Findings

Critical heating rate for crystallization is about 10^8 K/s.

Shaped laser pulses can increase heating rates by over two orders of magnitude.

The method enables nanosecond time-resolved cryo-EM experiments.

Abstract

Water vitrifies if cooled at rates above $3 \cdot 10^5$ K/s. Surprisingly, this process cannot simply be reversed by heating the resulting amorphous ice at a similar rate. Instead, we have recently shown that the sample transiently crystallizes even if the heating rate is more than one order of magnitude higher. This may present an issue for microsecond time-resolved cryo-electron microscopy experiments, in which vitreous ice samples are briefly flash melted with a laser pulse, since transient crystallization could potentially alter the dynamics of the embedded proteins. Here, we demonstrate how shaped microsecond laser pulses can be used to increase the heating rate and outrun crystallization during flash melting of amorphous solid water (ASW) samples. We use time-resolved electron diffraction experiments to determine that the critical heating rate is about $10^8$ K/s, more than two orders of magnitude higher than the critical cooling rate. Our experiments add to the toolbox of the emerging field of microsecond time-resolved cryo-electron microscopy by demonstrating a straightforward approach for avoiding crystallization during laser melting and for achieving significantly higher heating rates, which paves the way for nanosecond time-resolved experiments.