Laser Flash Melting Cryo-EM Samples to Overcome Preferred Orientation.

Sample preparation remains a bottleneck for protein structure determination by cryo-electron microscopy. A frequently encountered issue is that proteins adsorb to the air-water interface of the sample in a limited number of orientations. This makes it challenging to obtain high-resolution reconstructions or may even cause projects to fail altogether.

Microsecond time-resolved cryo-electron microscopy.

Microsecond time-resolved cryo-electron microscopy has emerged as a novel approach for directly observing protein dynamics. By providing microsecond temporal and near-atomic spatial resolution, it has the potential to elucidate a wide range of dynamics that were previously inaccessible and therefore, to significantly advance our understanding of protein function. This review summarizes the properties of the laser melting and revitrification process that underlies the technique and describes different experimental implementations.

Flash melting amorphous ice.

Water can be vitrified if it is cooled at high rates, which makes it possible to outrun crystallization in so-called no man's land, a range of deeply supercooled temperatures where water crystallizes rapidly. Here, we study the reverse process in pure water samples by flash melting amorphous ice with microsecond laser pulses. Time-resolved electron diffraction reveals that the sample transiently crystallizes despite a heating rate of more than 5 × 106 K/s, even though under the same conditions, vitrification can be achieved with a similar cooling rate of 107 K/s.

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

Water vitrifies if cooled at rates above 3 × 10 K/s. In contrast, when the resulting amorphous ice is flash heated, crystallization occurs even at a more than 10 times higher heating rate, as we have recently shown. This may present an issue for microsecond time-resolved cryo-electron microscopy experiments, in which vitreous ice samples are briefly melted with a laser pulse because transient crystallization could potentially alter the dynamics of the embedded proteins.