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

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

Electron Diffraction of Water in No Man's Land

A generally accepted understanding of the anomalous properties of water will only emerge if it becomes possible to systematically characterize water in the deeply supercooled regime, from where the anomalies appear to emanate. This has largely remained elusive because water crystallizes rapidly between 160 K and 232 K. Here, we present an experimental approach to rapidly prepare deeply supercooled water at a well-defined temperature and probe it with electron diffraction before crystallization occurs. We show that as water is cooled from room temperature to cryogenic temperature, its structure evolves smoothly, approaching that of amorphous ice just below 200 K. Our experiments narrow down the range of possible explanations of the origin for the water anomalies and open up new avenues for studying supercooled water

Visualizing Nanoscale Dynamics with Time-resolved Electron Microscopy

The large number of interactions in nanoscale systems leads to the emergence of complex behavior. Understanding such complexity requires atomic-resolution observations with a time resolution that is high enough to match the characteristic timescale of the system. Our laboratory’s method of choice is time-resolved electron microscopy. In particular, we are interested in the development of novel methods and instrumentation for high-speed observations with atomic resolution. Here, we present an overview of the activities in our laboratory

Flash Melting Amorphous Ice

Water can be vitrified if it is cooled at rates exceeding $3*10^5$ K/s. This makes it possible to outrun crystallization in so-called no man's land, a range of deeply supercooled temperatures where water crystallizes rapidly. One would naively assume that the process can simply be reversed by heating the resulting amorphous ice at a similar rate. We demonstrate that this is not the case. When amorphous ice samples are flash melted with a microsecond laser pulse, time-resolved electron diffraction reveals that the sample transiently crystallizes despite a heating rate of more than $5*10^6$ K/s, demonstrating that the critical heating rate for outrunning crystallization is significantly higher than the critical cooling rate during vitrification. Moreover, we observe different crystallization kinetics for amorphous solid water (ASW) and hyperquenched glassy water (HGW), which suggests that the supercooled liquids formed during laser heating transiently retain distinct non-equilibrium structures that are associated with different nucleation rates. These experiments open up new avenues for elucidating the crystallization mechanism of water and studying its dynamics in no man's land. They also add important mechanistic details to the laser melting and revitrification process that is integral to the emerging field of microsecond time-resolved cryo-electron microscopy.Comment: arXiv admin note: text overlap with arXiv:2211.0441

In Situ Melting and Revitrification as an Approach to Microsecond Time-Resolved Cryo-Electron Microscopy

Proteins typically undergo conformational dynamics on the microsecond to millisecond timescale as they perform their function, which is much faster than the time-resolution of cryo-electron microscopy and has thus prevented real-time observations. Here, we propose a novel approach for microsecond time-resolved cryo-electron microscopy that involves rapidly melting a cryo specimen in situ with a laser beam. The sample remains liquid for the duration of the laser pulse, offering a tunable time window in which the dynamics of embedded particles can be induced in their native liquid environment. After the laser pulse, the sample vitrifies in just a few microseconds, trapping particles in their transient configurations, so that they can subsequently be characterized with conventional cryo-electron microscopy. We demonstrate that our melting and revitrification approach is viable and affords microsecond time resolution. As a proof of principle, we study the disassembly of particles after they incur structural damage and trap them in partially unraveled configurations

EUV ionization of pure He nanodroplets: Mass-correlated photoelectron imaging, Penning ionization and electron energy-loss spectra

The ionization dynamics of pure He nanodroplets irradiated by EUV radiation is studied using Velocity-Map Imaging PhotoElectron-PhotoIon COincidence (VMI-PEPICO) spectroscopy. We present photoelectron energy spectra and angular distributions measured in coincidence with the most abundant ions He+, He2+, and He3+. Surprisingly, below the autoionization threshold of He droplets we find indications for multiple excitation and subsequent ionization of the droplets by a Penning-like process. At high photon energies we evidence inelastic collisions of photoelectrons with the surrounding He atoms in the droplets

Communication: Nucleation of quantized vortex rings in 4He nanodroplets

Whereas most of the phenomena associated with superfluidity have been observed in finite-size helium systems, the nucleation of quantized vortices has proven elusive. Here we show using time-dependent density functional simulations that the solvation of a Ba+ ion created by photoionization of neutral Ba at the surface of a 4He nanodroplet leads to the nucleation of a quantized ring vortex. The vortex is nucleated on a 10 ps timescale at the equator of a solid-like solvation structure that forms around the Ba+ ion. The process is expected to be quite general and very efficient under standard experimental conditions

Repurposing HLA genotype data of renal transplant patients to prevent severe drug hypersensitivity reactions

Introduction: Specific alleles in human leukocyte antigens (HLAs) are associated with an increased risk of developing drug hypersensitivity reactions induced by abacavir, allopurinol, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, or flucloxacillin. Transplant patients are genotyped for HLA as a routine practice to match a potential donor to a recipient. This study aims to investigate the feasibility and potential impact of repurposing these HLA genotype data from kidney transplant patients to prevent drug hypersensitivity reactions.Methods: A cohort of 1347 kidney transplant recipients has been genotyped in the Leiden University Medical Center (LUMC) using next-generation sequencing (NGS). The risk alleles HLA-A*31:01, HLA-B*15:02, HLA-B*15:11, HLA-B*57:01, and HLA-B*58:01 were retrieved from the NGS data. Medical history, medication use, and allergic reactions were obtained from the patient's medical records. Carrier frequencies found were compared to a LUMC blood donor population.Results: A total of 13.1% of transplant cohort patients carried at least one of the five HLA risk alleles and therefore had an increased risk of drug-induced hypersensitivity for specific drugs. HLA-A*31:01, HLA-B*15:02, HLA-B*57:01, and HLA-B*58:01 were found in carrier frequencies of 4.61%, 1.19%, 4.46%, and 3.35% respectively. No HLA-B*15:11 carrier was found. In total nine HLA-B*57:01 carriers received flucloxacillin and seven HLA-B*58:01 carriers within our cohort received allopurinol.Discussion: Our study shows that repurposing HLA genotype data from transplantation patients for the assignment of HLA risk alleles associated with drug hypersensitivity is feasible. The use of these data by physicians while prescribing drugs or by the pharmacist when dispensing drugs holds the potential to prevent drug hypersensitivity reactions. The utility of this method was highlighted by 13.1% of the transplant cohort patients carrying an actionable HLA allele. </p

Real-time dynamics of the formation of hydrated electrons upon irradiation of water clusters with extreme ultraviolet light

Free electrons in a polar liquid can form a bound state via interaction with the molecular environment. This so-called hydrated electron state in water is of fundamental importance e.g.~in cellular biology or radiation chemistry. Hydrated electrons are highly reactive radicals that can either directly interact with DNA or enzymes, or form highly excited hydrogen (H∗) after being captured by protons. Here, we investigate the formation of the hydrated electron in real-time employing XUV femtosecond pulses from a free electron laser, in this way observing the initial steps of the hydration process. Using time-resolved photoelectron spectroscopy we find formation timescales in the low picosecond range and resolve the prominent dynamics of forming excited hydrogen states

Critical Landau Velocity in Helium Nanodroplets

The best-known property of superfluid helium is the vanishing viscosity that objects experience while moving through the liquid with speeds below the so-called critical Landau velocity. This critical velocity is generally considered a macroscopic property as it is related to the collective excitations of the helium atoms in the liquid. In the present work we determine to what extent this concept can still be applied to nanometer-scale, finite size helium systems. To this end, atoms and molecules embedded in helium nanodroplets of various sizes are accelerated out of the droplets by means of optical excitation, and the speed distributions of the ejected particles are determined. The measurements reveal the existence of a critical velocity in these systems, even for nanodroplets consisting of only a thousand helium atoms. Accompanying theoretical simulations based on a time-dependent density functional description of the helium confirm and further elucidate this experimental finding