High-pressure X-ray photon correlation spectroscopy at fourth-generation synchrotron sources.

A new experimental setup combining X-ray photon correlation spectroscopy (XPCS) in the hard X-ray regime and a high-pressure sample environment has been developed to monitor the pressure dependence of the internal motion of complex systems down to the atomic scale in the multi-gigapascal range, from room temperature to 600 K. The high flux of coherent high-energy X-rays at fourth-generation synchrotron sources solves the problems caused by the absorption of diamond anvil cells used to generate high pressure, enabling the measurement of the intermediate scattering function over six orders of magnitude in time, from 10-3 s to 103 s. The constraints posed by the high-pressure generation such as the preservation of X-ray coherence, as well as the sample, pressure and temperature stability, are discussed, and the feasibility of high-pressure XPCS is demonstrated through results obtained on metallic glasses.

Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Ultrafast and persistent photoinduced phase transition at room temperature monitored by streaming powder diffraction.

Ultrafast photoinduced phase transitions at room temperature, driven by a single laser shot and persisting long after stimuli, represent emerging routes for ultrafast control over materials' properties. Time-resolved studies provide fundamental mechanistic insight into far-from-equilibrium electronic and structural dynamics. Here we study the photoinduced phase transformation of the Rb0.94Mn0.94Co0.06[Fe(CN)6]0.98 material, designed to exhibit a 75 K wide thermal hysteresis around room temperature between MnIIIFeII tetragonal and MnIIFeIII cubic phases. We developed a specific powder sample streaming technique to monitor by ultrafast X-ray diffraction the structural and symmetry changes. We show that the photoinduced polarons expand the lattice, while the tetragonal-to-cubic photoinduced phase transition occurs within 100 ps above threshold fluence. These results are rationalized within the framework of the Landau theory of phase transition as an elastically-driven and cooperative process. We foresee broad applications of the streaming powder technique to study non-reversible and ultrafast dynamics.

X-ray beam quality after a mirror reflection: Experimental and simulated results for a toroidal mirror in a 4 th generation storage-ring beamline.

Background: The surface errors found in X-ray mirrors constitute a limiting factor for preserving beam quality. This is particularly important when the X-ray beam has low emittance and a significant coherence fraction, like in newly upgraded synchrotron storage rings. Methods: We studied the fringes observed in the image of an undulator-produced X-ray beam reflected by a high-quality toroidal mirror. The measurements and simulations were performed using different conditions: a photon beam either monochromatic or with large bandwidth, reflected by a mirror with variable curvature. Results: The experimental data are compared with up-to-date simulation including partial coherence. Conclusions: The observed fringes in the unfocused beam correlate with low spatial frequency structures in mirror profiles, irrespective of beam coherence. Both classical ray tracing and partially coherent simulations through coherent mode decomposition are confirmed as accurate methods for such simulations.

In this study, researchers focused on the surface errors found in X-ray mirrors and their impact on beam quality. These errors can be problematic, especially when dealing with X-ray beams coming from low emittance (a measure of beam size and divergence) electron beam sources and a significant coherence fraction (indicating the level of wavefront coherence). The researchers specifically investigated the fringes observed in the image of an X-ray beam produced by an undulator and reflected by a high-quality toroidal mirror. They conducted measurements and simulations under different conditions, such as using a monochromatic photon beam or one with a wide range of wavelengths, and varying the curvature of the mirror.

Single-shot X-ray absorption spectroscopy at X-ray free electron lasers.

X-ray Absorption Spectroscopy (XAS) is a widely used X-ray diagnostic method for studying electronic and structural properties of matter. At first glance, the relatively narrow bandwidth and the highly fluctuating spectral structure of X-ray Free Electron Lasers (XFEL) sources seem to require accumulation over many shots to achieve high data quality. To date the best approach to implementing XAS at XFEL facilities has been using monochromators to scan the photon energy across the desired spectral range. While this is possible for easily reproducible samples such as liquids, it is incompatible with many important systems. Here, we demonstrate collection of single-shot XAS spectra over 10s of eV using an XFEL source, with error bars of only a few percent. We additionally show how to extend this technique over wider spectral ranges towards Extended X-ray Absorption Fine Structure measurements, by concatenating a few tens of single-shot measurements. Our results pave the way for future XAS studies at XFELs, in particular those in the femtosecond regime. This advance is envisioned to be especially important for many transient processes that can only be initiated at lower repetition rates, for difficult to reproduce excitation conditions, or for rare samples, such as those encountered in high-energy density physics.

Revitalizing an important field in biophysics: The new frontiers of molecular crowding.

Taking into account the presence of the crowded environment of a macromolecule has been an important goal of biology over the past 20 years. Molecular crowding affects the motions, stability and the kinetic behaviour of proteins. New powerful approaches have recently been developed to study molecular crowding, some of which make use of the synchrotron radiation light. The meeting "New Frontiers in Molecular Crowding" was organized in July 2022at the European Synchrotron Radiation facility of Grenoble to discuss the new frontiers of molecular crowding. The workshop brought together researchers from different disciplines to highlight the new developments of the field, including areas where new techniques allow the scientists to gain unprecedently expected information. A key conclusion of the meeting was the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives that could let the molecular crowding field flourish further.

Tilting refractive x-ray lenses for fine-tuning of their focal length.

In this work, we measure and model tilted x-ray refractive lenses to investigate their effects on an x-ray beam. The modelling is benchmarked against at-wavelength metrology obtained with x-ray speckle vector tracking experiments (XSVT) at the BM05 beamline at the ESRF-EBS light source, showing very good agreement. This validation permits us to explore possible applications of tilted x-ray lenses in optical design. We conclude that while tilting 2D lenses does not seem interesting from the point of view of aberration-free focusing, tilting 1D lenses around their focusing direction can be used for smoothly fine-tuning their focal length. We demonstrate experimentally this continuous change in the apparent lens radius of curvature R: a reduction up to a factor of two and beyond is achieved and possible applications in beamline optical design are proposed.

A fast and lightweight tool for partially coherent beamline simulations in fourth-generation storage rings based on coherent mode decomposition.

A new algorithm to perform coherent mode decomposition of undulator radiation is proposed. It is based on separating the horizontal and vertical directions, reducing the problem by working with one-dimension wavefronts. The validity conditions of this approximation are discussed. Simulations require low computer resources and run interactively on a laptop. The focusing with lenses of the radiation emitted by an undulator in a fourth-generation storage ring (EBS-ESRF) is studied. Results are compared against multiple optics packages implementing a variety of methods for dealing with partial coherence: full two-dimension coherent mode decomposition, Monte Carlo combination of wavefronts from electrons entering the undulator with different initial conditions, and hybrid ray-tracing correcting geometrical optics with wave optics.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Impact of the terahertz and optical pump penetration depths on generated strain waves temporal profiles in a V2O3 thin film.

Triggering new stable macroscopic orders in materials by ultrafast optical or terahertz pump pulses is a difficult challenge, complicated by the interplay between multiscale microscopic mechanisms, and macroscopic excitation profiles in samples. In particular, the differences between the two types of excitations are still unclear. In this article, we compare the optical response on acoustic timescale of a V2O3 Paramagnetic Metallic (PM) thin film excited by a terahertz (THz) pump or an optical pump, at room temperature. We show that the penetration depth of the deposited energy has a strong influence on the shape of the optical transmission signal, consistent with the modulation of permittivity by the superposition of depth-dependent static strain, and dynamical strain waves travelling back and forth in the sample layer. In particular, the temporal modulation of the optical transmission directly reflects the excitation profile as a function of depth, as well as the sign of the acoustic reflection coefficient between the film and the substrate. The acoustic mismatch between the V2O3 layer and the substrate was also measured. The raw data were interpreted with a one-dimensional analytical model, using three fitting parameters only. These results are discussed in the context of triggering phase transitions by ultrafast pump pulses. To the best of our knowledge, this is the first report of the modulation of the optical transmission of V2O3 with a THz pump within the acoustic timescale.

Ultrafast coherent motion and helix rearrangement of homodimeric hemoglobin visualized with femtosecond X-ray solution scattering.

Ultrafast motion of molecules, particularly the coherent motion, has been intensively investigated as a key factor guiding the reaction pathways. Recently, X-ray free-electron lasers (XFELs) have been utilized to elucidate the ultrafast motion of molecules. However, the studies on proteins using XFELs have been typically limited to the crystalline phase, and proteins in solution have rarely been investigated. Here we applied femtosecond time-resolved X-ray solution scattering (fs-TRXSS) and a structure refinement method to visualize the ultrafast motion of a protein. We succeeded in revealing detailed ultrafast structural changes of homodimeric hemoglobin involving the coherent motion. In addition to the motion of the protein itself, the time-dependent change of electron density of the hydration shell was tracked. Besides, the analysis on the fs-TRXSS data of myoglobin allows for observing the effect of the oligomeric state on the ultrafast coherent motion.

Interplays of electron and nuclear motions along CO dissociation trajectory in myoglobin revealed by ultrafast X-rays and quantum dynamics calculations.

Ultrafast structural dynamics with different spatial and temporal scales were investigated during photodissociation of carbon monoxide (CO) from iron(II)-heme in bovine myoglobin during the first 3 ps following laser excitation. We used simultaneous X-ray transient absorption (XTA) spectroscopy and X-ray transient solution scattering (XSS) at an X-ray free electron laser source with a time resolution of 80 fs. Kinetic traces at different characteristic X-ray energies were collected to give a global picture of the multistep pathway in the photodissociation of CO from heme. In order to extract the reaction coordinates along different directions of the CO departure, XTA data were collected with parallel and perpendicular relative polarizations of the laser pump and X-ray probe pulse to isolate the contributions of electronic spin state transition, bond breaking, and heme macrocycle nuclear relaxation. The time evolution of the iron K-edge X-ray absorption near edge structure (XANES) features along the two major photochemical reaction coordinates, i.e., the iron(II)-CO bond elongation and the heme macrocycle doming relaxation were modeled by time-dependent density functional theory calculations. Combined results from the experiments and computations reveal insight into interplays between the nuclear and electronic structural dynamics along the CO photodissociation trajectory. Time-resolved small-angle X-ray scattering data during the same process are also simultaneously collected, which show that the local CO dissociation causes a protein quake propagating on different spatial and temporal scales. These studies are important for understanding gas transport and protein deligation processes and shed light on the interplay of active site conformational changes and large-scale protein reorganization.

Charge transfer driven by ultrafast spin transition in a CoFe Prussian blue analogue.

Photoinduced charge-transfer is an important process in nature and technology and is responsible for the emergence of exotic functionalities, such as magnetic order for cyanide-bridged bimetallic coordination networks. Despite its broad interest and intensive developments in chemistry and material sciences, the atomic-scale description of the initial photoinduced process, which couples intermetallic charge-transfer and spin transition, has been debated for decades; it has been beyond reach due to its extreme speed. Here we study this process in a prototype cyanide-bridged CoFe system by femtosecond X-ray and optical absorption spectroscopies, enabling the disentanglement of ultrafast electronic and structural dynamics. Our results demonstrate that it is the spin transition that occurs first on the Co site within ~50 fs, and it is this that drives the subsequent Fe-to-Co charge-transfer within ~200 fs. This study represents a step towards understanding and controlling charge-transfer-based functions using light.

Femtosecond electronic structure response to high intensity XFEL pulses probed by iron X-ray emission spectroscopy.

We report the time-resolved femtosecond evolution of the K-shell X-ray emission spectra of iron during high intensity illumination of X-rays in a micron-sized focused hard X-ray free electron laser (XFEL) beam. Detailed pulse length dependent measurements revealed that rapid spectral energy shift and broadening started within the first 10 fs of the X-ray illumination at intensity levels between 1017 and 1018 W cm-2. We attribute these spectral changes to the rapid evolution of high-density photoelectron mediated secondary collisional ionization processes upon the absorption of the incident XFEL radiation. These fast electronic processes, occurring at timescales well within the typical XFEL pulse durations (i.e., tens of fs), set the boundary conditions of the pulse intensity and sample parameters where the widely-accepted 'probe-before-destroy' measurement strategy can be adopted for electronic-structure related XFEL experiments.

Photoswitchable 11 nm CsCoFe Prussian Blue Analogue Nanocrystals with High Relaxation Temperature.

Photoswitchable 11 nm nanocrystals with the coordination network Cs{Co[Fe(CN)6]} were obtained using a template-free method. The nanocrystals were recovered from the colloidal solutions as solid materials surrounded by cetyltrimethylammonium (CTA) cations or embedded in the organic polymer polyvinylpyrrolidone (PVP). Complementary magnetic, spectroscopic, and structural techniques, including EPR spectroscopy, reveal a majority (∼70%) of the low-spin and photoactive diamagnetic CoIIIFeII pairs located in the core of the nanocrystals and a mixture of CoIIFeII and CoIIFeIII species present mainly within the shell of the objects. While bulk compounds with similar vacancy concentration do not exhibit noticeable photoinduced charge transfer, the observed photoactivity of the nanocrystals is ascribed to their nanometric size. The relaxation temperature of the photoinduced state shifts upward by ∼55 K when PVP is replaced by CTA. This is ascribed to the larger rigidity of the dense CsCoFe_CTA material, whose metastable state is lower than that for CsCoFe_PVP, leading to a larger relaxation energy barrier and, therefore, to a higher relaxation temperature.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the µs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.

Experimental station Bernina at SwissFEL: condensed matter physics on femtosecond time scales investigated by X-ray diffraction and spectroscopic methods.

The Bernina instrument at the SwissFEL Aramis hard X-ray free-electron laser is designed for studying ultrafast phenomena in condensed matter and material science. Ultrashort pulses from an optical laser system covering a large wavelength range can be used to generate specific non-equilibrium states, whose subsequent temporal evolution can be probed by selective X-ray scattering techniques in the range 2-12 keV. For that purpose, the X-ray beamline is equipped with optical elements which tailor the X-ray beam size and energy, as well as with pulse-to-pulse diagnostics that monitor the X-ray pulse intensity, position, as well as its spectral and temporal properties. The experiments can be performed using multiple interchangeable endstations differing in specialization, diffractometer and X-ray analyser configuration and load capacity for specialized sample environment. After testing the instrument in a series of pilot experiments in 2018, regular user operation begins in 2019.

MHz data collection of a microcrystalline mixture of different jack bean proteins.

We provide a detailed description of a serial femtosecond crystallography (SFX) dataset collected at the European X-ray free-electron laser facility (EuXFEL). The EuXFEL is the first high repetition rate XFEL delivering MHz X-ray pulse trains at 10 Hz. The short spacing (<1 µs) between pulses requires fast flowing microjets for sample injection and high frame rate detectors. A data set was recorded of a microcrystalline mixture of at least three different jack bean proteins (urease, concanavalin A, concanavalin B). A one megapixel Adaptive Gain Integrating Pixel Detector (AGIPD) was used which has not only a high frame rate but also a large dynamic range. This dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development and for data analysis training for prospective XFEL users.

Light-induced structural changes in a full-length cyanobacterial phytochrome probed by time-resolved X-ray scattering.

Phytochromes are photoreceptor proteins that transmit a light signal from a photosensory region to an output domain. Photoconversion involves protein conformational changes whose nature is not fully understood. Here, we use time-resolved X-ray scattering and optical spectroscopy to study the kinetics of structural changes in a full-length cyanobacterial phytochrome and in a truncated form with no output domain. X-ray and spectroscopic signals on the µs/ms timescale are largely independent of the presence of the output domain. On longer time-scales, large differences between the full-length and truncated proteins indicate the timeframe during which the structural transition is transmitted from the photosensory region to the output domain and represent a large quaternary motion. The suggested independence of the photosensory-region dynamics on the µs/ms timescale defines a time window in which the photoreaction can be characterized (e.g. for optogenetic design) independently of the nature of the engineered output domain.

Dynamic multiple-scattering treatment of X-ray absorption: Parameterization of a new molecular dynamics force field for myoglobin.

We present a detailed analysis of the X-ray absorption near-edge structure (XANES) data on the Fe K-edge of CO Myoglobin based on a combined procedure of Molecular Dynamics (MD) calculations and MXAN (Minuit XANes) data analysis that we call D-MXAN. The ability of performing quantitative XANES data analysis allows us to refine classical force field MD parameters, thus obtaining a reliable tool for the atomic investigation of this important model system for biological macromolecules. The iterative procedure here applied corrects the greatest part of the structural discrepancy between classical MD sampling and experimental determinations. Our procedure, moreover, is able to discriminate between different heme conformational basins visited during the MD simulation, thus demonstrating the necessity of a sampling on the order of tens of nanoseconds, even for an application such X-ray absorption spectroscopy data analysis.