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The routine atomic resolution structure determination of single particles is expected to have profound implications for probing structure–function relationships in systems ranging from energy-storage materials to biological molecules. Extremely bright ultrashort-pulse X-ray sources – X-ray free-electron lasers (XFELs) – provide X-rays that can be used to probe ensembles of nearly identical nanoscale particles. When combined with coherent diffractive imaging, these objects can be imaged; however, as the resolution of the images approaches the atomic scale, the measured data are increasingly difficult to obtain and, during an X-ray pulse, the number of photons incident on the 2D detector is much smaller than the number of pixels. This latter concern, the signal `sparsity', materially impedes the application of the method. An experimental analog using a conventional X-ray source is demonstrated and yields signal levels comparable with those expected from single biomolecules illuminated by focused XFEL pulses. The analog experiment provides an invaluable cross check on the fidelity of the reconstructed data that is not available during XFEL experiments. Using these experimental data, it is established that a sparsity of order 1.3 × 10−3 photons per pixel per frame can be overcome, lending vital insight to the solution of the atomic resolution XFEL single-particle imaging problem by experimentally demonstrating 3D coherent diffractive imaging from photon-sparse random projections.

We report on recent developments that enable megahertz hard X-ray phase contrast imaging (MHz XPCI) experiments at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the European XFEL facility (EuXFEL). We describe the technical implementation of the key components, including an MHz fast camera and a modular indirect X-ray microscope system based on fast scintillators coupled through a high-resolution optical microscope, which enable full-field X-ray microscopy with phase contrast of fast and irreversible phenomena. The image quality for MHz XPCI data showed significant improvement compared with a pilot demonstration of the technique using parallel beam illumination, which also allows access to up to 24 keV photon energies at the SPB/SFX instrument of the EuXFEL. With these developments, MHz XPCI was implemented as a new method offered for a broad user community (academic and industrial) and is accessible via standard user proposals. Furthermore, intra-train pulse diagnostics with a high few-micrometre spatial resolution and recording up to 128 images of consecutive pulses in a train at up to 1.1 MHz repetition rate is available upstream of the instrument. Together with the diagnostic camera upstream of the instrument and the MHz XPCI setup at the SPB/SFX instrument, simultaneous two-plane measurements for future beam studies and feedback for machine parameter tuning are now possible., (open access.)

Hydrodynamic cavitation is useful in many processing applications, for example, in chemical reactors, water treatment and biochemical engineering. An important type of hydrodynamic cavitation that occurs in a Venturi tube is vortex cavitation known to cause luminescence whose intensity is closely related to the size and number of cavitation events. However, the mechanistic origins of bubbles constituting vortex cavitation remains unclear, although it has been concluded that the pressure fields generated by the cavitation collapse strongly depends on the bubble geometry. The common view is that vortex cavitation consists of numerous small spherical bubbles. In the present paper, aspects of vortex cavitation arising in a Venturi tube were visualized using high-speed X-ray imaging at SPring-8 and European XFEL. It was discovered that vortex cavitation in a Venturi tube consisted of angulated rather than spherical bubbles. The tangential velocity of the surface of vortex cavitation was assessed considering the Rankine vortex model., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (open access.)

The characterisation of fast phenomena at the microscopic scale is required for the understanding of catastrophic responses of materials to loads and shocks, the processing of materials by optical or mechanical means, the processes involved in many key technologies such as additive manufacturing and microfluidics, and the mixing of fuels in combustion. Such processes are usually stochastic in nature and occur within the opaque interior volumes of materials or samples, with complex dynamics that evolve in all three dimensions at speeds exceeding many meters per second. There is therefore a need for the ability to record three-dimensional X-ray movies of irreversible processes with resolutions of micrometers and frame rates of microseconds. Here we demonstrate a method to achieve this by recording a stereo phase-contrast image pair in a single exposure. The two images are combined computationally to reconstruct a 3D model of the object. The method is extendable to more than two simultaneous views. When combined with megahertz pulse trains of X-ray free-electron lasers (XFELs) it will be possible to create movies able to resolve 3D trajectories with velocities of kilometers per second.

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., (open access.)

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., (open access.)

Here a direct comparison is made between various X-ray wavefront sensing methods with application to optics alignment and focus characterization at X-ray free-electron lasers (XFELs). Focus optimization at XFEL beamlines presents unique challenges due to high peak powers as well as beam pointing instability, meaning that techniques capable of single-shot measurement and that probe the wavefront at an out-of-focus location are desirable. The techniques chosen for the comparison include single-phase-grating Talbot interferometry (shearing interferometry), dual-grating Talbot interferometry (moiré deflectometry) and speckle tracking. All three methods were implemented during a single beam time at the Linac Coherent Light Source, at the X-ray Pump Probe beamline, in order to make a direct comparison. Each method was used to characterize the wavefront resulting from a stack of beryllium compound refractive lenses followed by a corrective phase plate. In addition, difference wavefront measurements with and without the phase plate agreed with its design to within λ/20, which enabled a direct quantitative comparison between methods. Finally, a path toward automated alignment at XFEL beamlines using a wavefront sensor to close the loop is presented., (open access.)

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., (open access.)

We present a fast and accurate method for wave propagation through a set of inclined reflecting planes. It is based on the coordinate transformation in reciprocal space leading to a diffraction integral, which can be calculated only by using two 2D Fast Fourier Transforms and one 2D interpolation. The method is numerically tested, and comparisons with standard methods show its superiority in both computational speed and accuracy. The direct application of this method is found in the X-ray phase contrast imaging using the Bragg magnifier-an optics consisting of crystals asymmetrically diffracting in Bragg geometry.

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.

We present an improved, single-distance phase retrieval algorithm applicable for holographic X-ray imaging of biological objects for an in-line germanium Bragg Magnifier Microscope (BMM). The proposed algorithm takes advantage of a modified shrink-wrap algorithm for phase objects, robust unwrapping algorithm as well as other reasonable constraints applied to the wavefield at the object and the detector plane. The performance of the algorithm is analyzed on phantom objects and the results are shown and discussed. We demonstrated the suitability of the algorithm for the phase retrieval on a more complex biological specimen Tardigrade, where we achieved successful phase retrieval from only a single hologram. The spatial resolution obtained by Fourier spectral power method for biological objects is ∼ 300 nm, the same value as obtained from the reconstructed test pattern. Our results achieved using the new algorithm confirmed the potential of BMM for in-vivo, dose-efficient single-shot imaging of biological objects.

The thorax morphology, especially the muscles and the tracheal system of three flightless species of Cryptorhynchinae is examined by digital 3D reconstructions based on synchrotron X-ray microtomography and compared to other Curculionidae. Wings, metanepisternites, and muscles functional in flight are fully reduced in the species examined: Kyklioacalles roboris (Curtis), Trigonopterus scharfi Riedel and Trigonopterus vandekampi Riedel. All three share the same set of thoracic muscles, but differences exist in the shape and size of muscles. Both Trigonopterus species examined have a conspicuous fan-shaped branch of Musculus mesosterni primus contracting pro- and mesothorax, interpreted as an adaption to their thanatosis defense strategy. Trigonopterus vandekampi furthermore shows a marked increase in the size of two metacoxal muscles, which may be functional in this species' thanatosis blocking mechanisms. The metathoracic spiracle of all Trigonopterus species is located at the side of the metaventrite externally and not in the subelytral space as in other beetles. It is hypothesized that this translocation was triggered by the need to improve oxygen supply during thanatosis, when both the mesothoracic spiracle and the subelytral cavity are tightly sealed from the outside., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

X-ray grating interferometry has been highlighted in the last decade as a multi-modal X-ray phase-imaging technique for providing absorption, differential phase, and visibility-contrast images. It has been mainly reported that the visibility contrast in the visibility-contrast image originates from unresolvable random microstructures. In this paper, we show that the visibility contrast is even reduced by a uniform sample with flat surfaces due to the so-called "beam-hardening effect", which has to be taken into account when X-rays with a continuous spectrum is used. We drive a criterion for determining whether the beam-hardening effect occurs or not, and propose a method for correcting the effect of beam hardening on a visibility-contrast image.

We report on the successful demonstration of X-ray phase contrast microscopy and micro computed tomography (CT) with a Bragg magnifier microscope (BMM) in a laboratory setup. The Bragg magnifiers, constituted by two channel-cut crystals in asymmetric diffraction, produced a 15X magnification of the X-ray beam, thus enabling high resolution imaging to be attained. The angular sensitivity of the crystals was used to implement analyzer-based phase contrast imaging: acquiring images at different angular positions and the three parametric images (apparent absorption, differential phase and scattering) have been obtained. Micro-CT, with resolution of about 5 μm is demonstrated with the same system. The main limitations, as well as the ways to mitigate them, are discussed with the aid of the experimental data. The technique demonstrated herein extends high-resolution, multi-modal, x-ray imaging and micro-CT to compact laboratory setups, with the potential of broadening the reach of these techniques outside the community of synchrotron users.

We present the theoretical description of the image formation with the in-line germanium Bragg Magnifier Microscope (BMM) and the first successful phase retrieval of X-ray holograms recorded with this imaging system. The conditions under which the BMM acts as a linear shift invariant system are theoretically explained and supported by the experiment. Such an approach simplifies the mathematical treatment of the image formation and reconstruction as complicated propagation of the wavefront onto inclined planes can be avoided. Quantitative phase retrieval is demonstrated using a test sample and a proof of concept phase imaging of a spider leg is also presented.

Digital surface mesh models based on segmented datasets have become an integral part of studies on animal anatomy and functional morphology; usually, they are published as static images, movies or as interactive PDF files. We demonstrate the use of animated 3D models embedded in PDF documents, which combine the advantages of both movie and interactivity, based on the example of preserved Trigonopterus weevils. The method is particularly suitable to simulate joints with largely deterministic movements due to precise form closure. We illustrate the function of an individual screw-and-nut type hip joint and proceed to the complex movements of the entire insect attaining a defence position. This posture is achieved by a specific cascade of movements: Head and legs interlock mutually and with specific features of thorax and the first abdominal ventrite, presumably to increase the mechanical stability of the beetle and to maintain the defence position with minimal muscle activity. The deterministic interaction of accurately fitting body parts follows a defined sequence, which resembles a piece of engineering.

Dinosaurs undoubtedly produced huge quantities of excrements. But who cleaned up after them? Dung beetles and flies with rapid development were rare during most of the Mesozoic. Candidates for these duties are extinct cockroaches (Blattulidae), whose temporal range is associated with herbivorous dinosaurs. An opportunity to test this hypothesis arises from coprolites to some extent extruded from an immature cockroach preserved in the amber of Lebanon, studied using synchrotron X-ray microtomography. 1.06% of their volume is filled by particles of wood with smooth edges, in which size distribution directly supports their external pre-digestion. Because fungal pre-processing can be excluded based on the presence of large particles (combined with small total amount of wood) and absence of damages on wood, the likely source of wood are herbivore feces. Smaller particles were broken down biochemically in the cockroach hind gut, which indicates that the recent lignin-decomposing termite and cockroach endosymbionts might have been transferred to the cockroach gut upon feeding on dinosaur feces.