Direct measurement of the critical cooling rate for the vitrification of water
Mowry NJ, Constantin R. Krüger, Marcel Drabbels, Ulrich J. Lorenz
Abstract
The vitrification of aqueous solutions through rapid cooling is a remarkable achievement that launched the field of cryo-electron microscopy (cryo-EM) and has enabled the cryopreservation of biological specimens. For judging the feasibility of a vitrification experiment, the critical cooling rate of pure water is a frequently cited reference quantity. However, an accurate determination has remained elusive, with estimates varying by several orders of magnitude. Here, we employ time-resolved electron microscopy to obtain a precise measurement of this quantity. We use shaped microsecond laser pulses to briefly melt an amorphous ice sample before flash freezing it with a variable, well-defined cooling rate. This allows us to directly measure the critical cooling rate of water, which we determine to be <a:math xmlns:a="http://www.w3.org/1998/Math/MathML"> <a:mrow> <a:mn>6.4</a:mn> <a:mo>×</a:mo> <a:msup> <a:mrow> <a:mn>10</a:mn> </a:mrow> <a:mn>6</a:mn> </a:msup> <a:mspace width="0.16em"/> <a:mi mathvariant="normal">K</a:mi> <a:mo>/</a:mo> <a:mi mathvariant="normal">s</a:mi> </a:mrow> </a:math> . This result provides important insights into the question of how closely the conformational ensembles in plunge-frozen cryo samples reflect the structure of a protein at room temperature. Moreover, our experimental approach expands the toolkit of microsecond time-resolved cryo-EM, an emerging technique, in which a cryo sample is flash melted and revitrified with a laser pulse in order to enable time-resolved observations of protein dynamics.