Resolving Nonequilibrium Shape Variations among Millions of Gold Nanoparticles.

Nanoparticles, exhibiting functionally relevant structural heterogeneity, are at the forefront of cutting-edge research. Now, high-throughput single-particle imaging (SPI) with X-ray free-electron lasers (XFELs) creates opportunities for recovering the shape distributions of millions of particles that exhibit functionally relevant structural heterogeneity. To realize this potential, three challenges have to be overcome: (1) simultaneous parametrization of structural variability in real and reciprocal spaces; (2) efficiently inferring the latent parameters of each SPI measurement; (3) scaling up comparisons between 105 structural models and 106 XFEL-SPI measurements. Here, we describe how we overcame these three challenges to resolve the nonequilibrium shape distributions within millions of gold nanoparticles imaged at the European XFEL. These shape distributions allowed us to quantify the degree of asymmetry in these particles, discover a relatively stable "shape envelope" among nanoparticles, discern finite-size effects related to shape-controlling surfactants, and extrapolate nanoparticles' shapes to their idealized thermodynamic limit. Ultimately, these demonstrations show that XFEL SPI can help transform nanoparticle shape characterization from anecdotally interesting to statistically meaningful.

Direct Observation of a Roaming Intermediate and Its Dynamics.

Chemical reactions are often characterized by their transition state, which defines the critical geometry the molecule must pass through to move from reactants to products. Roaming provides an alternative picture, where in a dissociation reaction, the bond breaking is frustrated and a loosely bound intermediate is formed. Following bond breaking, the two partners are seen to roam around each other at distances of several Ångstroms, forming a loosely bound, and structurally ill-defined, intermediate that can subsequently lead to reactive or unreactive collisions. Here, we present a direct and time-resolved experimental measurement of roaming. By measuring the photoelectron spectrum of UV-excited acetaldehyde with a femtosecond extreme ultraviolet pulse, we captured spectral signatures of all of the key reactive structures, including that of the roaming intermediate. This provided a direct experimental measurement of the roaming process and allowed us to identify the time scales by which the roaming intermediate is formed and removed and the electronic potential surfaces upon which roaming proceeds.

Preparation of Tautomer-Pure Molecular Beams by Electrostatic Deflection.

Tautomers are ubiquitous throughout chemistry and typically considered inseparable in solution. Yet (bio)chemical activity is highly tautomer-specific, with common examples being the amino and nucleic acids. While tautomers exist in an equilibrium in solution, in the cold environment of a molecular beam the barrier to tautomerization is typically much too high for interconversion, and tautomers can be considered separate species. Here we demonstrate the first separation of tautomers within a molecular beam and the production of tautomerically pure gas-phase samples. We show this for the 2-pyridone/2-hydroxypyridine system, an important structural motif in both uracil and cytosine. Spatial separation of the tautomers is achieved via electrostatic deflection in strong inhomogeneous fields. We furthermore collect tautomer-resolved photoelectron spectra using femtosecond multiphoton ionization. This paves the way for studying the structure-function-dynamic relationship on the level of individual tautomers, using approaches that typically lack the resolution to do so, such as ultrafast dynamics experiments.

High-Throughput UV Photoionization and Fragmentation of Neutral Biomolecules as a Structural Fingerprint.

We present UV photofragmentation studies of the structural isomers paracetamol, 3-Pyridinepropionic acid (3-PPIA) and (R)-(-)-2-Phenylglycine. In particular, we utilized a new laser-based thermal desorption source in combination with femtosecond multiphoton ionization at 343 nm and 257 nm. The continuous nature of our molecule source, combined with the 50 kHz repetition rate of the laser, allowed us to perform these experiments at high throughput. In particular, we present detailed laser intensity dependence studies at both wavelengths, producing 2D mass spectra with highly differential information about the underlying fragmentation processes. We show that UV photofragmentation produces highly isomer-specific mass spectra, and assign all major fragmentation pathways observed. The intensity-dependence measurements, furthermore, allowed us to evaluate the appearance intensities for each fragmentation channel, which helped to distinguish competing from consecutive fragmentation pathways.

Vaporization of Intact Neutral Biomolecules Using Laser-Based Thermal Desorption.

The production of a clean neutral molecular sample is a crucial step in many gas-phase spectroscopy and reaction dynamics experiments investigating neutral species. Unfortunately, conventional methods based on heating cannot be used with most nonvolatile biomolecules due to their thermal instability. In this paper, we demonstrate the application of laser-based thermal desorption (LBTD) to produce neutral molecular plumes of biomolecules such as dipeptides and lipids. Specifically, we report mass spectra of glycylglycine, glycyl-l-alanine, and cholesterol obtained using LBTD vaporization, followed by soft femtosecond multiphoton ionization (fs-MPI) at 400 nm. For all molecules, the signal from the intact precursor ion was observed, highlighting the softness and applicability of the LBTD and fs-MPI approach. In more detail, cholesterol underwent hardly any fragmentation. Both dipeptides fragmented significantly, although mostly through only a single channel, which we attribute to the fs-MPI process.

High-throughput UV-photofragmentation studies of thymine and guanine.

High-throughput photofragmentation studies of thymine and guanine were performed at 257 and 343 nm and for a wide range of ionisation laser intensities. Combining a continuous laser-based thermal desorption source with femtosecond multiphoton ionisation using a 50 kHz repetition rate laser allowed us to produce detailed 2D maps of fragmentation as a function of incident laser intensity. The fragmentation was distinctly soft, the parent ions being at least an order of magnitude more abundant than fragment ions. For thymine there was a single dominant fragmentation channel, which involves consecutive HNCO and CO losses. In contrast, for guanine there were several competing ones, the most probable channel corresponding to CH2N2 loss through opening of the pyrimidine ring. The dependence of parent ion abundance on the ionisation laser intensity showed that at 257 nm the ionisation of thymine is a 1 + 1 resonance enhanced process through its open-shell singlet state.

Disentangling Multiphoton Ionization and Dissociation Channels in Molecular Oxygen Using Photoelectron-Photoion Coincidence Imaging.

Multiphoton excitation of molecular oxygen in the 392-408 nm region is studied using a tunable femtosecond laser coupled with a double velocity map imaging photoelectron-photoion coincidence spectrometer. The laser intensity is held at ≤∼1 TW/cm2 to ensure excitation in the perturbative regime, where the possibility of resonance enhanced multiphoton ionization (REMPI) can be investigated. O2+ production is found to be resonance enhanced around 400 nm via three-photon excitation to the e'3Δu(v = 0) state, similar to results from REMPI studies using nanosecond dye lasers. O+ production reaches 7% of the total ion yield around 405 nm due to two processes: autoionization following five-photon excitation of O2, producing O2+(X(v)) in a wide range of vibrational states followed by two- or three-photon dissociation, or six-photon excitation to a superexcited O2** state followed by neutral dissociation and subsequent ionization of the electronically excited O atom. Coincidence detection is shown to be crucial in identifying these competing pathways.

Single-Color Isomer-Resolved Spectroscopy.

Structural isomers, such as conformers or tautomers, are of significant importance across chemistry and biology, as they can have different functionalities. In gas-phase experiments using molecular beams, formation of many different isomers cannot be prevented, and their presence significantly complicates the assignment of spectral lines. Current isomer-resolved spectroscopic techniques heavily rely on theoretical calculations or make use of elaborate double-resonance schemes. We show here that isomer-resolved spectroscopy can also be performed using a single tunable laser. In particular, we demonstrate single-color isomer-resolved spectroscopy by utilizing electrostatic deflection to spatially separate the isomers. We show that for 3-aminophenol we can spatially separate the syn and anti conformers and use these pure samples to perform high-resolution REMPI spectroscopy, making the assignment of transitions to a particular isomer trivial, without any additional a priori information. This approach allows one to add isomer specificity to any molecular-beam-based experiment.

Unsupervised learning approaches to characterizing heterogeneous samples using X-ray single-particle imaging.

One of the outstanding analytical problems in X-ray single-particle imaging (SPI) is the classification of structural heterogeneity, which is especially difficult given the low signal-to-noise ratios of individual patterns and the fact that even identical objects can yield patterns that vary greatly when orientation is taken into consideration. Proposed here are two methods which explicitly account for this orientation-induced variation and can robustly determine the structural landscape of a sample ensemble. The first, termed common-line principal component analysis (PCA), provides a rough classification which is essentially parameter free and can be run automatically on any SPI dataset. The second method, utilizing variation auto-encoders (VAEs), can generate 3D structures of the objects at any point in the structural landscape. Both these methods are implemented in combination with the noise-tolerant expand-maximize-compress (EMC) algorithm and its utility is demonstrated by applying it to an experimental dataset from gold nanoparticles with only a few thousand photons per pattern. Both discrete structural classes and continuous deformations are recovered. These developments diverge from previous approaches of extracting reproducible subsets of patterns from a dataset and open up the possibility of moving beyond the study of homogeneous sample sets to addressing open questions on topics such as nanocrystal growth and dynamics, as well as phase transitions which have not been externally triggered.

Femtosecond 2 + 1 Resonance-Enhanced Multiphoton Ionization Spectroscopy of the C-State in Molecular Oxygen.

Coincidence electron-cation imaging is used to characterize the multiphoton ionization of O2 via the v = 4,5 levels of the 3s(3Πg) Rydberg state. A tunable 100 fs laser beam operating in the 271-263 nm region is found to cause a nonresonant ionization across this wavelength range, with an additional resonant ionization channel only observed when tuned to the 3Πg(v = 5) level. A distinct 3s → p wave character is observed in the photoelectron angular distribution for the v = 5 channel when on resonance.

Time resolved detection of the S(1D) product of the UV induced dissociation of CS2.

The products formed following the photodissociation of UV (200 nm) excited CS2 are monitored in a time resolved photoelectron spectroscopy experiment using femtosecond XUV (21.5 eV) photons. By spectrally resolving the electrons, we identify separate photoelectron bands related to the CS2 + hν → S(1D) + CS and CS2 + hν → S(3P) + CS dissociation channels, which show different appearance and rise times. The measurements show that there is no delay in the appearance of the S(1D) product contrary to the results of Horio et al. [J. Chem. Phys. 147, 013932 (2017)]. Analysis of the photoelectron yield associated with the atomic products allows us to obtain a S(3P)/S(1D) branching ratio and the rate constants associated with dissociation and intersystem crossing rather than the effective lifetime observed through the measurement of excited state populations alone.

Controlled beams of shock-frozen, isolated, biological and artificial nanoparticles.

X-ray free-electron lasers promise diffractive imaging of single molecules and nanoparticles with atomic spatial resolution. This relies on the averaging of millions of diffraction patterns of identical particles, which should ideally be isolated in the gas phase and preserved in their native structure. Here, we demonstrated that polystyrene nanospheres and Cydia pomonella granulovirus can be transferred into the gas phase, isolated, and very quickly shock-frozen, i.e., cooled to 4 K within microseconds in a helium-buffer-gas cell, much faster than state-of-the-art approaches. Nanoparticle beams emerging from the cell were characterized using particle-localization microscopy with light-sheet illumination, which allowed for the full reconstruction of the particle beams, focused to < 100 µ m , as well as for the determination of particle flux and number density. The experimental results were quantitatively reproduced and rationalized through particle-trajectory simulations. We propose an optimized setup with cooling rates for particles of few-nanometers on nanosecond timescales. The produced beams of shock-frozen isolated nanoparticles provide a breakthrough in sample delivery, e.g., for diffractive imaging and microscopy or low-temperature nanoscience.

Light-sheet imaging for the recording of transverse absolute density distributions of gas-phase particle-beams from nanoparticle injectors.

Imaging biological molecules in the gas-phase requires novel sample delivery methods, which generally have to be characterized and optimized to produce high-density particle beams. A non-destructive characterization method of the transverse particle beam profile is presented. It enables the characterization of the particle beam in parallel to the collection of, for instance, x-ray-diffraction patterns. As a rather simple experimental method, it requires the generation of a small laser-light sheet using a cylindrical telescope and a microscope. The working principle of this technique was demonstrated for the characterization of the fluid-dynamic-focusing behavior of 220 nm polystyrene beads as prototypical nanoparticles. The particle flux was determined and the velocity distribution was calibrated using Mie-scattering calculations.

Pure Molecular Beam of Water Dimer.

Spatial separation of water dimers from water monomers and larger water clusters through the electric deflector is presented. A beam of water dimers with 93% purity and a rotational temperature of 1.5 K was obtained. Following strong-field ionization using a 35 fs laser pulse with a wavelength centered around 800 nm and a peak intensity of 1014 W/cm2, we observed proton transfer and 46% of ionized water dimers broke apart into hydronium ions H3O+ and neutral OH.

Megahertz serial crystallography.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a ß-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.

Rapid sample delivery for megahertz serial crystallography at X-ray FELs.

Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s-1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments.

Spatially Separating the Conformers of a Dipeptide.

Atomic-resolution-imaging approaches for single molecules, such as coherent X-ray diffraction at free-electron lasers, require the delivery of high-density beams of identical molecules. However, even very cold beams of biomolecules typically have multiple conformational states populated. We demonstrate the production of very cold (Trot ≈2.3 K) molecular beams of intact dipeptide molecules, which were then spatially separated into the individual populated conformational states. This is achieved using the combination of supersonic expansion laser-desorption vaporization with electrostatic deflection in strong inhomogeneous fields. This represents the first demonstration of a conformer-separated and rotationally cold molecular beam of a peptide, which enables the investigation of conformer-specific chemistry using inherently non-conformer-specific techniques. It furthermore represents a milestone toward the direct structural imaging of individual biological molecules with atomic resolution by ultrafast diffractive-imaging methods.

Mapping the Complete Reaction Path of a Complex Photochemical Reaction.

We probe the dynamics of dissociating CS_{2} molecules across the entire reaction pathway upon excitation. Photoelectron spectroscopy measurements using laboratory-generated femtosecond extreme ultraviolet pulses monitor the competing dissociation, internal conversion, and intersystem crossing dynamics. Dissociation occurs either in the initially excited singlet manifold or, via intersystem crossing, in the triplet manifold. Both product channels are monitored and show that, despite being more rapid, the singlet dissociation is the minor product and that triplet state products dominate the final yield. We explain this by a consideration of accurate potential energy curves for both the singlet and triplet states. We propose that rapid internal conversion stabilizes the singlet population dynamically, allowing for singlet-triplet relaxation via intersystem crossing and the efficient formation of spin-forbidden dissociation products on longer timescales. The study demonstrates the importance of measuring the full reaction pathway for defining accurate reaction mechanisms.

Femtosecond X-ray diffraction from an aerosolized beam of protein nanocrystals.

High-resolution Bragg diffraction from aerosolized single granulovirus nanocrystals using an X-ray free-electron laser is demonstrated. The outer dimensions of the in-vacuum aerosol injector components are identical to conventional liquid-microjet nozzles used in serial diffraction experiments, which allows the injector to be utilized with standard mountings. As compared with liquid-jet injection, the X-ray scattering background is reduced by several orders of magnitude by the use of helium carrier gas rather than liquid. Such reduction is required for diffraction measurements of small macromolecular nanocrystals and single particles. High particle speeds are achieved, making the approach suitable for use at upcoming high-repetition-rate facilities.

Development and Characterization of a Laser-Induced Acoustic Desorption Source.

A laser-induced acoustic desorption source, developed for use at central facilities, such as free-electron lasers, is presented. It features prolonged measurement times and a fixed interaction point. A novel sample deposition method using aerosol spraying provides a uniform sample coverage and hence stable signal intensity. Utilizing strong-field ionization as a universal detection scheme, the produced molecular plume is characterized in terms of number density, spatial extend, fragmentation, temporal distribution, translational velocity, and translational temperature. The effect of desorption laser intensity on these plume properties is evaluated. While translational velocity is invariant for different desorption laser intensities, pointing to a nonthermal desorption mechanism, the translational temperature increases significantly and higher fragmentation is observed with increased desorption laser fluence.