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Nature Communications 11(1), 4511 (2020). doi:10.1038/s41467-020-18156-7, Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported., Published by Nature Publishing Group UK, [London]

This article focuses on the challenges of hit finding and data reduction in serial crystallography (SX). An effective and reliable Bragg-peak-finding method, called robust peak finder (RPF), has been developed. RPF is based on the principle of robust statistics and can be used for SX data analysis., A peak-finding algorithm for serial crystallography (SX) data analysis based on the principle of ‘robust statistics’ has been developed. Methods which are statistically robust are generally more insensitive to any departures from model assumptions and are particularly effective when analysing mixtures of probability distributions. For example, these methods enable the discretization of data into a group comprising inliers (i.e. the background noise) and another group comprising outliers (i.e. Bragg peaks). Our robust statistics algorithm has two key advantages, which are demonstrated through testing using multiple SX data sets. First, it is relatively insensitive to the exact value of the input parameters and hence requires minimal optimization. This is critical for the algorithm to be able to run unsupervised, allowing for automated selection or ‘vetoing’ of SX diffraction data. Secondly, the processing of individual diffraction patterns can be easily parallelized. This means that it can analyse data from multiple detector modules simultaneously, making it ideally suited to real-time data processing. These characteristics mean that the robust peak finder (RPF) algorithm will be particularly beneficial for the new class of MHz X-ray free-electron laser sources, which generate large amounts of data in a short period of time.

Next generation pulsed neutron sources and wavelength dispersive imaging detectors are creating new opportunities for strain analysis. One such technique is Bragg edge transmission analysis in which projected measurements of the crystallographic properties of bulk polycrystalline samples are recorded on a time-of-flight area detector. The ability to measure the elastic strain field poses the question of whether it is possible to reconstruct a three-dimensional map of the elastic strain tensor from a set of lower order projection data. Here we present a fundamental exploration of the validity of axisymmetric strain reconstruction algorithms available for inverting Bragg edge data. The results demonstrate that the compatibility of the elastic strain field under investigation is critical in determining which algorithm may be successfully applied. Finally, a more robust approach to Radon transform strain tomography is presented based on the condition of zero total strain.

18 pags, 11 figs, 5 tabs, Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research., This work was supported by the National Science Foundation Science and Technology Center 'BioXFEL' through award STC-1231306, and in part by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DESC0002164 (AO, algorithm design and development) and by the National Science Foundation under contract Nos. 1551489 (AO, underlying analytical models) and DBI-2029533 (AO, functional conformations). This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1450681 to JLO. The work was also supported by funds from the National Institutes of Health grant R01 GM117342-0404. Funding and support are also acknowledged from the National Institutes of Health grant R01 GM095583, from the Biodesign Center for Applied Structural Discovery at ASU, from National Science Foundation award No. 1565180 and the US Department of Energy through Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. KAZ was supported by the Cornell Molecular Biophysics Training Program (NIH T32-GM008267). This work was also supported by the Cluster of Excellence 'CUI: Advanced Imaging of Matter' of the Deutsche Forschungsgemeinschaft (DFG), EXC 2056, project ID 390715994. CFEL is supported by the Gottfried Wilhelm Leibniz Program of the DFG, the 'X-probe' project funded by the European Union 2020 Research and Innovation Program under Marie Sklodowska-Curie grant agreement 637295, the European Research Council, 'Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS)', ERC-2013-SyG 609920, and the Human Frontiers Science Program grant RGP0010 2017. This work is also supported by the AXSIS project funded by the European Research Council under the European Union Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement No. 609920.

Optica 8(1), 15 - 23 (2021). doi:10.1364/OPTICA.410851, Single particle imaging at x-ray free electron lasers (XFELs) has the potential to determine the structure and dynamics of single biomolecules at room temperature. Two major hurdles have prevented this potential from being reached, namely, the collection of sufficient high-quality diffraction patterns and robust computational purification to overcome structural heterogeneity. We report the breaking of both of these barriers using gold nanoparticle test samples, recording around 10 million diffraction patterns at the European XFEL and structurally and orientationally sorting the patterns to obtain better than 3-nm-resolution 3D reconstructions for each of four samples. With these new developments, integrating advancements in x-ray sources, fast-framing detectors, efficient sample delivery, and data analysis algorithms, we illuminate the path towards sub-nanometer biomolecular imaging. The methods developed here can also be extended to characterize ensembles that are inherently diverse to obtain their full structural landscape., Published by OSA, Washington, DC

Communications Physics 3(1), 97 (2020). doi:10.1038/s42005-020-0362-y, The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate., Published by Springer Nature, London

Optica 7(6), 716 - (2020). doi:10.1364/OPTICA.396728, A critical challenge of pump-probe experiments with x-ray free-electron lasers (XFELs) is accurate synchronization of x-ray and optical pulses. At the European XFEL we observed megahertz rate timing jitter of 24.0±12.4fs., Published by OSA, Washington, DC

Interactions between neighboring grains influence the macroscale behavior of polycrystalline materials, particularly their deformation behavior, damage initiation and propagation mechanisms. However, mapping all of the critical material properties normally requires that several independent measurements are performed. Here we report the first grain mapping of a polycrystalline foil using a pixelated energy-dispersive X-ray area detector, simultaneously measuring X-ray fluorescence and diffraction with the Maia detector in order to determine grain orientation and estimate lattice strain. These results demonstrate the potential of the next generation of X-ray area detectors for materials characterization. By scanning the incident X-ray energy we investigate these detectors as a complete solution for simultaneously mapping the crystallographic and chemical properties of the sample. The extension of these techniques to broadband X-ray sources is also discussed.

Elemental, chemical, and structural analysis of polycrystalline materials at the micron scale is frequently carried out using microfocused synchrotron X-ray beams, sometimes on multiple instruments. The Maia pixelated energy-dispersive X-ray area detector enables the simultaneous collection of X-ray fluorescence (XRF) and diffraction because of the relatively large solid angle and number of pixels when compared with other systems. The large solid angle also permits extraction of surface topography because of changes in self-absorption. This work demonstrates the capability of the Maia detector for simultaneous measurement of XRF and diffraction for mapping the short- and long-range order across the grain structure in a Ni polycrystalline foil.

Optics letters 44(7), 1650 - 1653 (2019). doi:10.1364/OL.44.001650, Intense, ultrashort, and high-repetition-rate X-ray pulses, combined with a femtosecond optical laser, allow pump-probe experiments with fast data acquisition and femtosecond time resolution. However, the relative timing of the X-ray pulses and the optical laser pulses can be controlled only to a level of the intrinsic error of the instrument which, without characterization, limits the time resolution of experiments. This limitation inevitably calls for a precise determination of the relative arrival time, which can be used after measurement for sorting and tagging the experimental data to a much finer resolution than it can be controlled to. The observed root-mean-square timing jitter between the X-ray and the optical laser at the SPB/SFX instrument at European XFEL was 308 fs. This first measurement of timing jitter at the European XFEL provides an important step in realizing ultrafast experiments at this novel X-ray source. A method for determining the change in the complex refractive index of samples is also presented., Published by OSA, Washington, DC

The elastic properties of materials, either under external load or in a relaxed state, influence their mechanical behaviour. Conventional optical approaches based on techniques such as photoelasticity or thermoelasticity can be used for full-field analysis of the stress distribution within a specimen. The circular polariscope in combination with holographic photoelasticity allows the sum and difference of principal stress components to be determined by exploiting the temporary birefringent properties of materials under load. Phase stepping and interferometric techniques have been proposed as a method for separating the in-plane stress components in two-dimensional photoelasticity experiments. In this paper we describe and demonstrate an alternative approach based on photoelastic ptychography which is able to obtain quantitative stress information from far fewer measurements than is required for interferometric based approaches. The complex light intensity equations based on Jones calculus for this setup are derived. We then apply this approach to the problem of a disc under diametrical compression. The experimental results are validated against the analytical solution derived by Hertz for the theoretical displacement fields for an elastic disc subject to point loading.

Journal of synchrotron radiation 24(1), 83 - 94(2017). doi:10.1107/S1600577516017525, The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals (Helliwell, 1984). Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to the diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. A possible mechanism to account for these observations is proposed., Published by IUCr, Chester

Using recently developed microchannel plate (MCP) neutron area detectors at a pulsed neutron source provides a method of fast non-destructive imaging and characterisation of advanced materials at unprecedented spatial resolution. The energy resolved neutron transmission spectrum can be found from the neutron time of flight (ToF), this provides valuable information about the crystallographic structure within the sample such as the average residual elastic strain in the incident beam direction. By measuring this spectrum at a number of sample orientations information is built up about the strain distribution throughout the specimen. The energy-resolved neutron transmission spectrum of an additively manufactured turbine blade was measured at the Engin-X instrument, ISIS, UK. A high resolution three-dimensional reconstruction of the neutron attenuation coefficient of the blade was recovered, then analysis of the energy resolved spectrum gave insight into the underlying residual elastic strain profile imparted by the advanced manufacturing process.

Three-dimensional imaging of protein crystals during x-ray diffraction experiments opens up a range of possibilities for optimizing crystal quality and gaining new insights into the fundamental processes that drive radiation damage. Obtaining this information at the appropriate length-scales however is extremely challenging. One approach that has been recently demonstrated as a promising avenue for characterizing the size and shape of protein crystals at nanometre length-scales is Bragg coherent diffractive imaging (BCDI). BCDI is a recently developed technique that is able to recover the phase of the continuous diffraction intensity signal around individual Bragg peaks. When data is collected at multiple points on a rocking curve, a reciprocal space map (RSM) can be assembled and then inverted using BCDI to obtain a three-dimensional image of the crystal. The first demonstration of two-dimensional biological BCDI was reported by Boutet et al on holoferritin, recently this work was extended to the study of radiation damage in micron-sized protein crystals. Here we present the first three-dimensional reconstructions of a Lysozyme protein crystal using BDI. The results are validated against RSM and transmission electron microscopy data and have implications for both radiation damage studies and for developing new approaches for structure retrieval from micron-sized protein crystals.