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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.

Diffraction before destruction using X-ray free-electron lasers (XFELs) has the potential to determine radiation-damage-free structures without the need for crystallization. This article presents the three-dimensional reconstruction of the Melbournevirus from single-particle X-ray diffraction patterns collected at the LINAC Coherent Light Source (LCLS) as well as reconstructions from simulated data exploring the consequences of different kinds of experimental sources of noise. The reconstruction from experimental data suffers from a strong artifact in the center of the particle. This could be reproduced with simulated data by adding experimental background to the diffraction patterns. In those simulations, the relative density of the artifact increases linearly with background strength. This suggests that the artifact originates from the Fourier transform of the relatively flat background, concentrating all power in a central feature of limited extent. We support these findings by significantly reducing the artifact through background removal before the phase-retrieval step. Large amounts of blurring in the diffraction patterns were also found to introduce diffuse artifacts, which could easily be mistaken as biologically relevant features. Other sources of noise such as sample heterogeneity and variation of pulse energy did not significantly degrade the quality of the reconstructions. Larger data volumes, made possible by the recent inauguration of high repetition-rate XFELs, allow for increased signal-to-background ratio and provide a way to minimize these artifacts. The anticipated development of three-dimensional Fourier-volume-assembly algorithms which are background aware is an alternative and complementary solution, which maximizes the use of data.

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 β-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.

We recently published a study concerning femtosecond pump–probe absorption edge spectroscopy of cubic GaN (fundamental bandgap: 3.23 eV), resulting in the transient dielectric function. In the present study, we continue our investigations of those pump–probe measurements by determining the time-dependent transition energy at the Fermi-vector between the conduction and valence bands. The generation of electron–hole pairs by the 266 nm pump-beam (4.66 eV) shifts the absorption edge by ≈ 500 meV within 1 ps due to many-body effects like band-filling and bandgap renormalization. Modeling this ultra-fast change is achieved by converting the transition energies into free-carrier concentrations, assuming the electron contributions to be dominant. We consider the relaxation, recombination, and diffusion of those free-carriers as well as either an additional gain-recombination or temperature effects. This allows for describing the transition energies on short time scales. Both models yield similar values for the characteristic relaxation time (≈ 0.21 ps), recombination time (≈ 25 ps), and diffusion coefficient (≈ 1 cm 2 /s). [ABSTRACT FROM AUTHOR]

An ultra-fast change of the absorption onset for zincblende gallium-nitride (zb-GaN) (fundamental bandgap: 3.23 eV) is observed by investigating the imaginary part of the dielectric function using time-dependent femtosecond pump–probe spectroscopic ellipsometry between 2.9 and 3.7 eV. The 266 nm (4.66 eV) pump pulses induce a large electron–hole pair concentration up to 4 × 10 20 cm − 3 , which shift the transition energy between conduction and valence bands due to many-body effects up to ≈ 500 meV. Here, the absorption onset increases due to band filling while the bandgap renormalization at the same time decreases the bandgap. Additionally, the absorption of the pump-beam creates a free-carrier profile within the 605 nm zb-GaN layer with high free-carrier concentrations at the surface, and low concentrations at the interface to the substrate. This leads to varying optical properties from the sample surface (high transition energy) to substrate (low transition energy), which are taken into account by grading analysis for an accurate description of the experimental data. For this, a model describing the time- and position-dependent free-carrier concentration is formulated by considering the relaxation, recombination, and diffusion of those carriers. We provide a quantitative analysis of optical experimental data (ellipsometric angles Ψ and Δ) as well as a plot for the time-dependent change of the imaginary part of the dielectric function. [ABSTRACT FROM AUTHOR]

This study explores the capabilities of the Coherent X-ray Imaging Instrument at the Linac Coherent Light Source to image small biological samples. The weak signal from small samples puts a significant demand on the experiment. Aerosolized Omono River virus particles of ∼40 nm in diameter were injected into the submicrometre X-ray focus at a reduced pressure. Diffraction patterns were recorded on two area detectors. The statistical nature of the measurements from many individual particles provided information about the intensity profile of the X-ray beam, phase variations in the wavefront and the size distribution of the injected particles. The results point to a wider than expected size distribution (from ∼35 to ∼300 nm in diameter). This is likely to be owing to nonvolatile contaminants from larger droplets during aerosolization and droplet evaporation. The results suggest that the concentration of nonvolatile contaminants and the ratio between the volumes of the initial droplet and the sample particles is critical in such studies. The maximum beam intensity in the focus was found to be 1.9 × 1012 photons per µm2 per pulse. The full-width of the focus at half-maximum was estimated to be 500 nm (assuming 20% beamline transmission), and this width is larger than expected. Under these conditions, the diffraction signal from a sample-sized particle remained above the average background to a resolution of 4.25 nm. The results suggest that reducing the size of the initial droplets during aerosolization is necessary to bring small particles into the scope of detailed structural studies with X-ray lasers.

With the emergence of ultrafast X‐ray sources, interest in following fast processes in small molecules and macromolecules has increased. Most of the current research into ultrafast structural dynamics of macromolecules uses X‐ray free‐electron lasers. In parallel, small‐scale laboratory‐based laser‐driven ultrafast X‐ray sources are emerging. Continuous development of these sources is underway, and as a result many exciting applications are being reported. However, because of their low flux, such sources are not commonly used to study the structural dynamics of macromolecules. This article examines the feasibility of time‐resolved powder diffraction of macromolecular microcrystals using a laboratory‐scale laser‐driven ultrafast X‐ray source. [ABSTRACT FROM AUTHOR]

Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms.

We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.