Microsecond time-resolved X-ray scattering by utilizing MHz repetition rate at second-generation XFELs.

Detecting microsecond structural perturbations in biomolecules has wide relevance in biology, chemistry and medicine. Here we show how MHz repetition rates at X-ray free-electron lasers can be used to produce microsecond time-series of protein scattering with exceptionally low noise levels of 0.001%. We demonstrate the approach by examining Jɑ helix unfolding of a light-oxygen-voltage photosensory domain. This time-resolved acquisition strategy is easy to implement and widely applicable for direct observation of structural dynamics of many biochemical processes.

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX.

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.

Time-resolved serial femtosecond crystallography at the European XFEL.

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

Online dynamic flat-field correction for MHz microscopy data at European XFEL.

The high pulse intensity and repetition rate of the European X-ray Free-Electron Laser (EuXFEL) provide superior temporal resolution compared with other X-ray sources. In combination with MHz X-ray microscopy techniques, it offers a unique opportunity to achieve superior contrast and spatial resolution in applications demanding high temporal resolution. In both live visualization and offline data analysis for microscopy experiments, baseline normalization is essential for further processing steps such as phase retrieval and modal decomposition. In addition, access to normalized projections during data acquisition can play an important role in decision-making and improve the quality of the data. However, the stochastic nature of X-ray free-electron laser sources hinders the use of standard flat-field normalization methods during MHz X-ray microscopy experiments. Here, an online (i.e. near real-time) dynamic flat-field correction method based on principal component analysis of dynamically evolving flat-field images is presented. The method is used for the normalization of individual X-ray projections and has been implemented as a near real-time analysis tool at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of EuXFEL.

Emergence of anomalous dynamics in soft matter probed at the European XFEL.

Dynamics and kinetics in soft matter physics, biology, and nanoscience frequently occur on fast (sub)microsecond but not ultrafast timescales which are difficult to probe experimentally. The European X-ray Free-Electron Laser (European XFEL), a megahertz hard X-ray Free-Electron Laser source, enables such experiments via taking series of diffraction patterns at repetition rates of up to 4.5 MHz. Here, we demonstrate X-ray photon correlation spectroscopy (XPCS) with submicrosecond time resolution of soft matter samples at the European XFEL. We show that the XFEL driven by a superconducting accelerator provides unprecedented beam stability within a pulse train. We performed microsecond sequential XPCS experiments probing equilibrium and nonequilibrium diffusion dynamics in water. We find nonlinear heating on microsecond timescales with dynamics beyond hot Brownian motion and superheated water states persisting up to 100 µs at high fluences. At short times up to 20 µs we observe that the dynamics do not obey the Stokes-Einstein predictions.

3D printed devices and infrastructure for liquid sample delivery at the European XFEL.

The Sample Environment and Characterization (SEC) group of the European X-ray Free-Electron Laser (EuXFEL) develops sample delivery systems for the various scientific instruments, including systems for the injection of liquid samples that enable serial femtosecond X-ray crystallography (SFX) and single-particle imaging (SPI) experiments, among others. For rapid prototyping of various device types and materials, sub-micrometre precision 3D printers are used to address the specific experimental conditions of SFX and SPI by providing a large number of devices with reliable performance. This work presents the current pool of 3D printed liquid sample delivery devices, based on the two-photon polymerization (2PP) technique. These devices encompass gas dynamic virtual nozzles (GDVNs), mixing-GDVNs, high-viscosity extruders (HVEs) and electrospray conical capillary tips (CCTs) with highly reproducible geometric features that are suitable for time-resolved SFX and SPI experiments at XFEL facilities. Liquid sample injection setups and infrastructure on the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument are described, this being the instrument which is designated for biological structure determination at the EuXFEL.

Shot-to-shot two-dimensional photon intensity diagnostics within megahertz pulse-trains at the European XFEL.

Characterizing the properties of X-ray free-electron laser (XFEL) sources is a critical step for optimization of performance and experiment planning. The recent availability of MHz XFELs has opened up a range of new opportunities for novel experiments but also highlighted the need for systematic measurements of the source properties. Here, MHz-enabled beam imaging diagnostics developed for the SPB/SFX instrument at the European XFEL are exploited to measure the shot-to-shot intensity statistics of X-ray pulses. The ability to record pulse-integrated two-dimensional transverse intensity measurements at multiple planes along an XFEL beamline at MHz rates yields an improved understanding of the shot-to-shot photon beam intensity variations. These variations can play a critical role, for example, in determining the outcome of single-particle imaging experiments and other experiments that are sensitive to the transverse profile of the incident beam. It is observed that shot-to-shot variations in the statistical properties of a recorded ensemble of radiant intensity distributions are sensitive to changes in electron beam current density. These changes typically occur during pulse-distribution to the instrument and are currently not accounted for by the existing suite of imaging diagnostics. Modulations of the electron beam orbit in the accelerator are observed to induce a time-dependence in the statistics of individual pulses - this is demonstrated by applying radio-frequency trajectory tilts to electron bunch-trains delivered to the instrument. We discuss how these modifications of the beam trajectory might be used to modify the statistical properties of the source and potential future applications.

Pump-probe capabilities at the SPB/SFX instrument of the European XFEL.

Pump-probe experiments at X-ray free-electron laser (XFEL) facilities are a powerful tool for studying dynamics at ultrafast and longer timescales. Observing the dynamics in diverse scientific cases requires optical laser systems with a wide range of wavelength, flexible pulse sequences and different pulse durations, especially in the pump source. Here, the pump-probe instrumentation available for measurements at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the European XFEL is reported. The temporal and spatial stability of this instrumentation is also presented.

Ultrafast nonthermal heating of water initiated by an X-ray Free-Electron Laser.

The bright ultrafast pulses of X-ray Free-Electron Lasers allow investigation into the structure of matter under extreme conditions. We have used single pulses to ionize and probe water as it undergoes a phase transition from liquid to plasma. We report changes in the structure of liquid water on a femtosecond time scale when irradiated by single 6.86 keV X-ray pulses of more than 106 J/cm2 These observations are supported by simulations based on molecular dynamics and plasma dynamics of a water system that is rapidly ionized and driven out of equilibrium. This exotic ionic and disordered state with the density of a liquid is suggested to be structurally different from a neutral thermally disordered state.

X-ray diffractive imaging of controlled gas-phase molecules: Toward imaging of dynamics in the molecular frame.

We report experimental results on the diffractive imaging of three-dimensionally aligned 2,5-diiodothiophene molecules. The molecules were aligned by chirped near-infrared laser pulses, and their structure was probed at a photon energy of 9.5 keV (λ ≈ 130 pm) provided by the Linac Coherent Light Source. Diffracted photons were recorded on the Cornell-SLAC pixel array detector, and a two-dimensional diffraction pattern of the equilibrium structure of 2,5-diiodothiophene was recorded. The retrieved distance between the two iodine atoms agrees with the quantum-chemically calculated molecular structure to be within 5%. The experimental approach allows for the imaging of intrinsic molecular dynamics in the molecular frame, albeit this requires more experimental data, which should be readily available at upcoming high-repetition-rate facilities.

Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser.

To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 µm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 µm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.

The Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography instrument of the European XFEL: initial installation.

The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation - in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described.

Initial observations of the femtosecond timing jitter at the European XFEL.

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.

Post-sample aperture for low background diffraction experiments at X-ray free-electron lasers.

The success of diffraction experiments from weakly scattering samples strongly depends on achieving an optimal signal-to-noise ratio. This is particularly important in single-particle imaging experiments where diffraction signals are typically very weak and the experiments are often accompanied by significant background scattering. A simple way to tremendously reduce background scattering by placing an aperture downstream of the sample has been developed and its application in a single-particle X-ray imaging experiment at FLASH is demonstrated. Using the concept of a post-sample aperture it was possible to reduce the background scattering levels by two orders of magnitude.

Single-shot determination of focused FEL wave fields using iterative phase retrieval.

Determining fluctuations in focus properties is essential for many experiments at Self-Amplified-Spontaneous-Emission (SASE) based Free-Electron-Lasers (FELs), in particular for imaging single non-crystalline biological particles. We report on a diffractive imaging technique to fully characterize highly focused, single-shot pulses using an iterative phase retrieval algorithm, and benchmark it against an existing Hartmann wavefront sensor. The results, both theoretical and experimental, demonstrate the effectiveness of this technique to provide a comprehensive and convenient shot-to-shot measurement of focused-pulse wave fields and source-point positional variations without the need for manipulative optics between the focus and the detector.

Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams.

Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with the presence of high-atomic-number elements and X-ray energy. To explore the effects of local damage in serial femtosecond crystallography (SFX), Clostridium ferredoxin was used as a model system. The protein contains two [4Fe-4S] clusters that serve as sensitive probes for radiation-induced electronic and structural changes. High-dose room-temperature SFX datasets were collected at the Linac Coherent Light Source of ferredoxin microcrystals. Difference electron density maps calculated from high-dose SFX and synchrotron data show peaks at the iron positions of the clusters, indicative of decrease of atomic scattering factors due to ionization. The electron density of the two [4Fe-4S] clusters differs in the FEL data, but not in the synchrotron data. Since the clusters differ in their detailed architecture, this observation is suggestive of an influence of the molecular bonding and geometry on the atomic displacement dynamics following initial photoionization. The experiments are complemented by plasma code calculations.

Coherent x-ray imaging of collagen fibril distributions within intact tendons.

The characterization of the structure of highly hierarchical biosamples such as collagen-based tissues at the scale of tens of nanometers is essential to correlate the tissue structure with its growth processes. Coherent x-ray Bragg ptychography is an innovative imaging technique that gives high resolution images of the ordered parts of such samples. Herein, we report how we used this method to image the collagen fibrillar ultrastructure of intact rat tail tendons. The images show ordered fibrils extending over 10-20 µm in length, with a quantifiable D-banding spacing variation of 0.2%. Occasional defects in the fibrils distribution have also been observed, likely indicating fibrillar fusion events.

Conformation sequence recovery of a non-periodic object from a diffraction-before-destruction experiment.

Knowledge of the sequence of different conformational states of a protein molecule is key to better understanding its biological function. A diffraction pattern from a single conformational state can be captured with an ultrafast X-ray Free-Electron Laser (XFEL) before the target is completely annihilated by the radiation. In this paper, we report the first experimental demonstration of conformation sequence recovery using diffraction patterns from randomly ordered conformations of a non-periodic object using the dimensional reduction technique Isomap and coherent diffraction imaging.

Explosion dynamics of sucrose nanospheres monitored by time of flight spectrometry and coherent diffractive imaging at the split-and-delay beam line of the FLASH soft X-ray laser.

We use a Mach-Zehnder type autocorrelator to split and delay XUV pulses from the FLASH soft X-ray laser for triggering and subsequently probing the explosion of aerosolised sugar balls. FLASH was running at 182 eV photon energy with pulses of 70 fs duration. The delay between the pump-probe pulses was varied between zero and 5 ps, and the pulses were focused to reach peak intensities above 10¹6W/cm² with an off-axis parabola. The direct pulse triggered the explosion of single aerosolised sucrose nano-particles, while the delayed pulse probed the exploding structure. The ejected ions were measured by ion time of flight spectrometry, and the particle sizes were measured by coherent diffractive imaging. The results show that sucrose particles of 560-1000 nm diameter retain their size for about 500 fs following the first exposure. Significant sample expansion happens between 500 fs and 1 ps. We present simulations to support these observations.

Computational study of diffraction image formation from XFEL irradiated single ribosome molecule.

Single particle imaging at atomic resolution is perhaps one of the most desired goals for ultrafast X-ray science with X-ray free-electron lasers. Such a capability would create great opportunity within the biological sciences, as high-resolution structural information of biosamples that may not crystallize is essential for many research areas therein. In this paper, we report on a comprehensive computational study of diffraction image formation during single particle imaging of a macromolecule, containing over one hundred thousand non-hydrogen atoms. For this study, we use a dedicated simulation framework, SIMEX, available at the European XFEL facility. Our results demonstrate the full feasibility of computational single-particle imaging studies for biological samples of realistic size. This finding is important as it shows that the SIMEX platform can be used for simulations to inform relevant single-particle-imaging experiments and help to establish optimal parameters for these experiments. This will enable more focused and more efficient single-particle-imaging experiments at XFEL facilities, making the best use of the resource-intensive XFEL operation.