Observation of a single protein by ultrafast X-ray diffraction.

The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.

Exploring fingerprints of ultrafast structural dynamics in molecular solutions with an X-ray laser.

We apply ultrashort X-ray laser pulses to track optically excited structural dynamics of [Ir2(dimen)4]2+ molecules in solution. In our exploratory study we determine angular correlations in the scattered X-rays, which comprise a complex fingerprint of the ultrafast dynamics. Model-assisted analysis of the experimental correlation data allows us to elucidate various aspects of the photoinduced changes in the excited molecular ensembles. We unambiguously identify that in our experiment the photoinduced transition dipole moments in [Ir2(dimen)4]2+ molecules are oriented perpendicular to the Ir-Ir bond. The analysis also shows that the ground state conformer of [Ir2(dimen)4]2+ with a larger Ir-Ir distance is mostly responsible for the formation of the excited state. We also reveal that the ensemble of solute molecules can be characterized with a substantial structural heterogeneity due to solvent influence. The proposed X-ray correlation approach offers an alternative path for studies of ultrafast structural dynamics of molecular ensembles in the liquid and gas phases.

The structure of tick-borne encephalitis virus determined at X-ray free-electron lasers. Simulations.

The study of virus structures by X-ray free-electron lasers (XFELs) has attracted increased attention in recent decades. Such experiments are based on the collection of 2D diffraction patterns measured at the detector following the application of femtosecond X-ray pulses to biological samples. To prepare an experiment at the European XFEL, the diffraction data for the tick-borne encephalitis virus (TBEV) was simulated with different parameters and the optimal values were identified. Following the necessary steps of a well established data-processing pipeline, the structure of TBEV was obtained. In the structure determination presented, a priori knowledge of the simulated virus orientations was used. The efficiency of the proposed pipeline was demonstrated.

Statistical analysis of hard X-ray radiation at the PAL-XFEL facility performed by Hanbury Brown and Twiss interferometry.

A Hanbury Brown and Twiss interferometry experiment based on second-order correlations was performed at the PAL-XFEL facility. The statistical properties of the X-ray radiation were studied within this experiment. Measurements were performed at the NCI beamline at 10 keV photon energy under various operation conditions: self-amplified spontaneous emission (SASE), SASE with a monochromator, and self-seeding regimes at 120 pC, 180 pC and 200 pC electron bunch charge. Statistical analysis showed short average pulse duration from 6 fs to 9 fs depending on the operational conditions. A high spatial degree of coherence of about 70-80% was determined in the spatial domain for the SASE beams with the monochromator and self-seeding regime of operation. The obtained values describe the statistical properties of the beams generated at the PAL-XFEL facility.

Angular X-ray cross-correlation analysis applied to the scattering data in 3D reciprocal space from a single crystal.

An application of angular X-ray cross-correlation analysis (AXCCA) to the scattered intensity distribution measured in 3D reciprocal space from a single-crystalline sample is proposed in this work. Contrary to the conventional application of AXCCA, when averaging over many 2D diffraction patterns collected from different randomly oriented samples is required, the proposed approach provides an insight into the structure of a single specimen. This is particularly useful in studies of defect-rich samples that are unlikely to have the same structure. The application of the method is shown on an example of a qualitative structure determination of a colloidal crystal from simulated as well as experimentally measured 3D scattered intensity distributions.

Classification of diffraction patterns using a convolutional neural network in single-particle-imaging experiments performed at X-ray free-electron lasers.

Single particle imaging (SPI) at X-ray free-electron lasers is particularly well suited to determining the 3D structure of particles at room temperature. For a successful reconstruction, diffraction patterns originating from a single hit must be isolated from a large number of acquired patterns. It is proposed that this task could be formulated as an image-classification problem and solved using convolutional neural network (CNN) architectures. Two CNN configurations are developed: one that maximizes the F1 score and one that emphasizes high recall. The CNNs are also combined with expectation-maximization (EM) selection as well as size filtering. It is observed that the CNN selections have lower contrast in power spectral density functions relative to the EM selection used in previous work. However, the reconstruction of the CNN-based selections gives similar results. Introducing CNNs into SPI experiments allows the reconstruction pipeline to be streamlined, enables researchers to classify patterns on the fly, and, as a consequence, enables them to tightly control the duration of their experiments. Incorporating non-standard artificial-intelligence-based solutions into an existing SPI analysis workflow may be beneficial for the future development of SPI experiments.

Mitigating the photodegradation of all-inorganic mixed-halide perovskite nanocrystals by ligand exchange.

We show that the decomposition of caesium lead halide perovskite nanocrystals under continuous X-ray illumination depends on the surface ligand. For oleic acid/oleylamine, we observe a fast decay accompanied by the formation of elemental lead and halogen. Upon surface functionalization with a metal porphyrin derivative, the decay is markedly slower and involves the disproportionation of lead to Pb0 and Pb3+. In both cases, the decomposition is preceded by a contraction of the atomic lattice, which appears to initiate the decay. We find that the metal porphyrin derivative induces a strong surface dipole on the nanocrystals, which we hold responsible for the altered and slower decomposition pathway. These results are important for application of lead halide perovskite nanocrystals in X-ray scintillators.

Erratum: An advanced workflow for single-particle imaging with the limited data at an X-ray free-electron laser. Corrigendum.

[This corrects the article DOI: 10.1107/S2052252520012798.].

Spatially resolved fluorescence of caesium lead halide perovskite supercrystals reveals quasi-atomic behavior of nanocrystals.

We correlate spatially resolved fluorescence (-lifetime) measurements with X-ray nanodiffraction to reveal surface defects in supercrystals of self-assembled cesium lead halide perovskite nanocrystals and study their effect on the fluorescence properties. Upon comparison with density functional modeling, we show that a loss in structural coherence, an increasing atomic misalignment between adjacent nanocrystals, and growing compressive strain near the surface of the supercrystal are responsible for the observed fluorescence blueshift and decreased fluorescence lifetimes. Such surface defect-related optical properties extend the frequently assumed analogy between atoms and nanocrystals as so-called quasi-atoms. Our results emphasize the importance of minimizing strain during the self-assembly of perovskite nanocrystals into supercrystals for lighting application such as superfluorescent emitters.

In situ characterization of crystallization and melting of soft, thermoresponsive microgels by small-angle X-ray scattering.

Depending on the volume fraction and interparticle interactions, colloidal suspensions can form different phases, ranging from fluids, crystals, and glasses to gels. For soft microgels that are made from thermoresponsive polymers, the volume fraction can be tuned by temperature, making them excellent systems to experimentally study phase transitions in dense colloidal suspensions. However, investigations of phase transitions at high particle concentration and across the volume phase transition temperature in particular, are challenging due to the deformability and possibility for interpenetration between microgels. Here, we investigate the dense phases of composite core-shell microgels that have a small gold core and a thermoresponsive microgel shell. Employing Ultra Small-Angle X-ray Scattering, we make use of the strong scattering signal from the gold cores with respect to the almost negligible signal from the shells. By changing the temperature we study the freezing and melting transitions of the system in situ. Using Bragg peak analysis and the Williamson-Hall method, we characterize the phase transitions in detail. We show that the system crystallizes into an rhcp structure with different degrees of in-plane and out-of-plane stacking disorder that increase upon particle swelling. We further find that the melting process is distinctly different, where the system separates into two different crystal phases with different melting temperatures and interparticle interactions.

Angular structure factor of the hexatic-B liquid crystals: bridging theory and experiment.

We report results of X-ray scattering studies of the angular structure factor of liquid crystal hexatic-B films. According to the sixfold rotational symmetry of the hexatic-B phase, its characteristic scattering splits into six reflections. The shape of the radial and angular cross-sections of these reflections and their temperature evolution are analyzed. We find that over a wide temperature range of the hexatic-B phase existence the angular profiles of the in-plane X-ray scattering are well fitted by the Voigt function, which is a convolution of the Gaussian and Lorentzian functions. This result is supported by the known theoretical considerations of the hexatic structure factor below the smectic-hexatic phase transition temperature. Similar predictions for the angular shape of the hexatic peak in the vicinity of the smectic-hexatic phase transition temperature follow from the multicritical scaling theory of the hexatic-B phase in three dimensions. We find that the specific shape of the hexatic structure factor can be explained by the interplay of two distinct contributions to the free energy of the system, a liquid-like density term and a coupling term between the bond-orientational order and short-range density fluctuations.

High spatial coherence and short pulse duration revealed by the Hanbury Brown and Twiss interferometry at the European XFEL.

Second-order intensity interferometry was employed to study the spatial and temporal properties of the European X-ray Free-Electron Laser (EuXFEL). Measurements were performed at the soft x-ray Self-Amplified Spontaneous Emission (SASE3) undulator beamline at a photon energy of 1.2 keV in the Self-Amplified Spontaneous Emission (SASE) mode. Two high-power regimes of the SASE3 undulator settings, i.e., linear and quadratic undulator tapering at saturation, were studied in detail and compared with the linear gain regime. The statistical analysis showed an exceptionally high degree of spatial coherence up to 90% for the linear undulator tapering. Analysis of the measured data in spectral and spatial domains provided an average pulse duration of about 10 fs in our measurements. The obtained results will be valuable for the experiments requiring and exploiting short pulse duration and utilizing high coherence properties of the EuXFEL.

Correction: Exploring the 3D structure and defects of a self-assembled gold mesocrystal by coherent X-ray diffraction imaging.

Correction for 'Exploring the 3D structure and defects of a self-assembled gold mesocrystal by coherent X-ray diffraction imaging' by Jerome Carnis et al., Nanoscale, 2021, DOI: 10.1039/D1NR01806J.

Exploring the 3D structure and defects of a self-assembled gold mesocrystal by coherent X-ray diffraction imaging.

Mesocrystals are nanostructured materials consisting of individual nanocrystals having a preferred crystallographic orientation. On mesoscopic length scales, the properties of mesocrystals are strongly affected by structural heterogeneity. Here, we report the detailed structural characterization of a faceted mesocrystal grain self-assembled from 60 nm sized gold nanocubes. Using coherent X-ray diffraction imaging, we determined the structure of the mesocrystal with the resolution sufficient to resolve each gold nanoparticle. The reconstructed electron density of the gold mesocrystal reveals its intrinsic structural heterogeneity, including local deviations of lattice parameters, and the presence of internal defects. The strain distribution shows that the average superlattice obtained by angular X-ray cross-correlation analysis and the real, "multidomain" structure of a mesocrystal are very close to each other, with a deviation less than 10%. These results will provide an important impact to understanding the fundamental principles of structuring and self-assembly including ensuing properties of mesocrystals.

An advanced workflow for single-particle imaging with the limited data at an X-ray free-electron laser.

An improved analysis for single-particle imaging (SPI) experiments, using the limited data, is presented here. Results are based on a study of bacteriophage PR772 performed at the Atomic, Molecular and Optical Science instrument at the Linac Coherent Light Source as part of the SPI initiative. Existing methods were modified to cope with the shortcomings of the experimental data: inaccessibility of information from half of the detector and a small fraction of single hits. The general SPI analysis workflow was upgraded with the expectation-maximization based classification of diffraction patterns and mode decomposition on the final virus-structure determination step. The presented processing pipeline allowed us to determine the 3D structure of bacteriophage PR772 without symmetry constraints with a spatial resolution of 6.9 nm. The obtained resolution was limited by the scattering intensity during the experiment and the relatively small number of single hits.

Diffraction data from aerosolized Coliphage PR772 virus particles imaged with the Linac Coherent Light Source.

Single Particle Imaging (SPI) with intense coherent X-ray pulses from X-ray free-electron lasers (XFELs) has the potential to produce molecular structures without the need for crystallization or freezing. Here we present a dataset of 285,944 diffraction patterns from aerosolized Coliphage PR772 virus particles injected into the femtosecond X-ray pulses of the Linac Coherent Light Source (LCLS). Additional exposures with background information are also deposited. The diffraction data were collected at the Atomic, Molecular and Optical Science Instrument (AMO) of the LCLS in 4 experimental beam times during a period of four years. The photon energy was either 1.2 or 1.7 keV and the pulse energy was between 2 and 4 mJ in a focal spot of about 1.3 µm x 1.7 µm full width at half maximum (FWHM). The X-ray laser pulses captured the particles in random orientations. The data offer insight into aerosolised virus particles in the gas phase, contain information relevant to improving experimental parameters, and provide a basis for developing algorithms for image analysis and reconstruction.

Femtosecond laser produced periodic plasma in a colloidal crystal probed by XFEL radiation.

With the rapid development of short-pulse intense laser sources, studies of matter under extreme irradiation conditions enter further unexplored regimes. In addition, an application of X-ray Free-Electron Lasers (XFELs) delivering intense femtosecond X-ray pulses, allows to investigate sample evolution in IR pump - X-ray probe experiments with an unprecedented time resolution. Here we present a detailed study of the periodic plasma created from the colloidal crystal. Both experimental data and theory modeling show that the periodicity in the sample survives to a large extent the extreme excitation and shock wave propagation inside the colloidal crystal. This feature enables probing the excited crystal, using the powerful Bragg peak analysis, in contrast to the conventional studies of dense plasma created from bulk samples for which probing with Bragg diffraction technique is not possible. X-ray diffraction measurements of excited colloidal crystals may then lead towards a better understanding of matter phase transitions under extreme irradiation conditions.

Structure-Transport Correlation Reveals Anisotropic Charge Transport in Coupled PbS Nanocrystal Superlattices.

The assembly of colloidal semiconductive nanocrystals into highly ordered superlattices predicts novel structure-related properties by design. However, those structure-property relationships, such as charge transport depending on the structure or even directions of the superlattice, have remained unrevealed so far. Here, electric transport measurements and X-ray nanodiffraction are performed on self-assembled lead sulfide nanocrystal superlattices to investigate direction-dependent charge carrier transport in microscopic domains of these materials. By angular X-ray cross-correlation analysis, the structure and orientation of individual superlattices is determined, which are directly correlated with the electronic properties of the same microdomains. By that, strong evidence for the effect of superlattice crystallinity on the electric conductivity is found. Further, anisotropic charge transport in highly ordered monocrystalline domains is revealed, which is attributed to the dominant effect of shortest interparticle distance. This implies that transport anisotropy should be a general feature of weakly coupled nanocrystal superlattices.

Soft X-ray diffraction patterns measured by a LiF detector with sub-micrometre resolution and an ultimate dynamic range.

The unique diagnostic possibilities of X-ray diffraction, small X-ray scattering and phase-contrast imaging techniques applied with high-intensity coherent X-ray synchrotron and X-ray free-electron laser radiation can only be fully realized if a sufficient dynamic range and/or spatial resolution of the detector is available. In this work, it is demonstrated that the use of lithium fluoride (LiF) as a photoluminescence (PL) imaging detector allows measuring of an X-ray diffraction image with a dynamic range of ∼107 within the sub-micrometre spatial resolution. At the PETRA III facility, the diffraction pattern created behind a circular aperture with a diameter of 5 µm irradiated by a beam with a photon energy of 500 eV was recorded on a LiF crystal. In the diffraction pattern, the accumulated dose was varied from 1.7 × 105 J cm-3 in the central maximum to 2 × 10-2 J cm-3 in the 16th maximum of diffraction fringes. The period of the last fringe was measured with 0.8 µm width. The PL response of the LiF crystal being used as a detector on the irradiation dose of 500 eV photons was evaluated. For the particular model of laser-scanning confocal microscope Carl Zeiss LSM700, used for the readout of the PL signal, the calibration dependencies on the intensity of photopumping (excitation) radiation (λ = 488 nm) and the gain have been obtained.

Revealing Grain Boundaries and Defect Formation in Nanocrystal Superlattices by Nanodiffraction.

X-ray nanodiffraction is applied to study the formation and correlation of domain boundaries in mesocrystalline superlattices of PbS nanocrystals with face-centered cubic structure. Each domain of the superlattice can be described with one of two mesocrystalline polymorphs with different orientational orders. Close to a grain boundary, the lattice constant decreases and the superlattice undergoes an out-of-plane rotation, while the orientation of the nanocrystals with respect to the superlattice remains unchanged. These findings are explained with the release of stress on the expense of specific nanocrystal-substrate interactions. The fact that correlations between adjacent nanocrystals are found to survive the structural changes at most grain boundaries implies that the key to nanocrystal superlattices with macroscopic domain sizes are strengthened interactions with the substrate.