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We have produced hard x-ray free-electron laser (FEL) pulses, which are chirped both in photon energy and in spatial position. The experiments have been carried out at the hard x-ray beamline Aramis at SwissFEL, located at the Paul Scherrer Institute in Switzerland. The FEL beamline was operated without any external focusing and with a tilted, energy-chirped electron bunch, whose properties are then transferred to the photon beam. The resulting FEL pulses were used for a single-shot absorption x-ray spectroscopy experiment at the Alvra endstation. But, as will be outlined, applications of this type of tailored FEL pulse go beyond such experiments, allowing for x-ray pulse compression and control of the transverse coherence within the pulse.

Transition metals are at the basis of key processes in chemistry and biology but their complex electronic structures make understanding of their properties a challenge. Here the authors resolve individual spectral lines of Cu2 in the deep UV region by two-colour resonant four-wave mixing.

“Probe-before-destroy” methodology permitted diffraction and imaging measurements of intact specimens using ultrabright but highly destructive X-ray free-electron laser (XFEL) pulses. The methodology takes advantage of XFEL pulses ultrashort duration to outrun the destructive nature of the X-rays. Atomic movement, generally on the order of >50 fs, regulates the maximum pulse duration for intact specimen measurements. In this contribution, we report the electronic structure damage of a molecule with ultrashort X-ray pulses under preservation of the atoms' positions. A detailed investigation of the X-ray induced processes revealed that X-ray absorption events in the solvent produce a significant number of solvated electrons within attosecond and femtosecond timescales that are capable of coulombic interactions with the probed molecules. The presented findings show a strong influence on the experimental spectra coming from ionization of the probed atoms' surroundings leading to electronic structure modification much faster than direct absorption of photons. This work calls for consideration of this phenomenon in cases focused on samples embedded in, e.g., solutions or in matrices, which in fact concerns most of the experimental studies.

Time-resolved serial crystallography at X-ray Free Electron Lasers offers the opportunity to observe ultrafast photochemical reactions at the atomic level. The technique has yielded exciting molecular insights into various biological processes including light sensing and photochemical energy conversion. However, to achieve sufficient levels of activation within an optically dense crystal, high laser power densities are often used, which has led to an ongoing debate to which extent photodamage may compromise interpretation of the results. Here we compare time-resolved serial crystallographic data of the bacteriorhodopsin K-intermediate collected at laser power densities ranging from 0.04 to 2493 GW/cm 2 and follow energy dissipation of the absorbed photons logarithmically from picoseconds to milliseconds. Although the effects of high laser power densities on the overall structure are small, in the upper excitation range we observe significant changes in retinal conformation and increased heating of the functionally critical counterion cluster. We compare light-activation within crystals to that in solution and discuss the impact of the observed changes on bacteriorhodopsin biology., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The ability to freely control the polarization of X-rays enables measurement techniques relying on circular or linear dichroism, which have become indispensable tools for characterizing the properties of chiral molecules or magnetic structures. Therefore, the demand for polarization control in X-ray free-electron lasers is increasing to enable polarization-sensitive dynamical studies on ultrafast time scales. The soft X-ray branch Athos of SwissFEL was designed with the aim of providing freely adjustable and arbitrary polarization by building its undulator solely from modules of the novel Apple X type. In this paper, the magnetic model of the linear inclined and circular Apple X polarization schemes are studied. The polarization is characterized by measuring the angular electron emission distributions of helium for various polarizations using cold target recoil ion momentum spectroscopy. The generation of fully linear polarized light of arbitrary angle, as well as elliptical polarizations of varying degree, are demonstrated., (open access.)

To fully exploit ultra-short X-ray pulse durations routinely available at X-ray free-electron lasers to follow out-of-equilibrium dynamics, inherent arrival time fluctuations of the X-ray pulse with an external perturbing laser pulse need to be measured. In this work, two methods of arrival time measurement were compared to measure the arrival time jitter of hard X-ray pulses. The methods were photoelectron streaking by a THz field and a transient refractive index change of a semiconductor. The methods were validated by shot-to-shot correction of a pump-probe transient reflectivity measurement. An ultimate shot-to-shot full width at half-maximum error between the devices of 19.2 ± 0.1 fs was measured., (open access.)

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 transfer 1,2 . However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore 3-17 . As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern 18,19 whether this experimental approach 20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions 18,19 . 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 dynamics 21 ) 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 experiments 20 such that mechanistically relevant insight emerges., (© 2024. The Author(s).)

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

The evolution of charge carriers in photoexcited room temperature ZnO nanoparticles in solution is investigated using ultrafast ultraviolet photoluminescence spectroscopy, ultrafast Zn K-edge absorption spectroscopy, and ab initio molecular dynamics (MD) simulations. The photoluminescence is excited at 4.66 eV, well above the band edge, and shows that electron cooling in the conduction band and exciton formation occur in <500 fs, in excellent agreement with theoretical predictions. The x-ray absorption measurements, obtained upon excitation close to the band edge at 3.49 eV, are sensitive to the migration and trapping of holes. They reveal that the 2 ps transient largely reproduces the previously reported transient obtained at 100 ps time delay in synchrotron studies. In addition, the x-ray absorption signal is found to rise in ∼1.4 ps, which we attribute to the diffusion of holes through the lattice prior to their trapping at singly charged oxygen vacancies. Indeed, the MD simulations show that impulsive trapping of holes induces an ultrafast expansion of the cage of Zn atoms in <200 fs, followed by an oscillatory response at a frequency of ∼100 cm -1 , which corresponds to a phonon mode of the system involving the Zn sub-lattice., Competing Interests: The authors have no conflicts to disclose., (© 2023 Author(s).)

X-ray free-electron lasers (FELs) are state-of-the-art scientific tools capable to study matter on the scale of atomic processes. Since the initial operation of X-ray FELs more than a decade ago, several facilities with upgraded performance have been put in operation. Here we present the first lasing results of Athos, the soft X-ray FEL beamline of SwissFEL at the Paul Scherrer Institute in Switzerland. Athos features an undulator layout based on short APPLE-X modules providing full polarisation control, interleaved with small magnetic chicanes. This versatile configuration allows for many operational modes, giving control over many FEL properties. We show, for example, a 35% reduction of the required undulator length to achieve FEL saturation with respect to standard undulator configurations. We also demonstrate the generation of more powerful pulses than the ones obtained in typical undulators. Athos represents a fundamental step forward in the design of FEL facilities, creating opportunities in FEL-based sciences., (© 2023. Springer Nature Limited.)

We report femtosecond Fe K-edge absorption (XAS) and nonresonant X-ray emission (XES) spectra of ferric cytochrome C (Cyt c) upon excitation of the haem (>300 nm) or mixed excitation of the haem and tryptophan (<300 nm). The XAS and XES transients obtained in both excitation energy ranges show no evidence for electron transfer processes between photoexcited tryptophan (Trp) and the haem, but rather an ultrafast energy transfer, in agreement with previous ultrafast optical fluorescence and transient absorption studies. The reported ( J. Phys. Chem. B 2011 , 115 (46), 13723-13730) decay times of Trp fluorescence in ferrous (∼350 fs) and ferric (∼700 fs) Cyt c are among the shortest ever reported for Trp in a protein. The observed time scales cannot be rationalized in terms of Förster or Dexter energy transfer mechanisms and call for a more thorough theoretical investigation.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs) 1 . A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation 2 , thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature 3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation., (© 2023. The Author(s).)

Ultrafast single-particle imaging with intense x-ray pulses from free-electron laser sources provides a new approach for visualizing structure and dynamics on the nanoscale. After a short introduction to the novel free-electron laser sources and methods, we highlight selected applications and discuss how ultrafast imaging flourishes from method development to early applications in physics and biology to opportunities for chemical sciences., (Copyright 2022 Zhibin Sun, Andre Al Haddad, Sven Augustin, Gregor Knopp, Jonas Knurr, Kirsten Schnorr, Christoph Bostedt. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 Å resolution when exposed to XFEL radiation, which is an improvement of 0.15 Å over previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile Q B pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand., (open access.)

A highly excited electronic state of dicopper is observed and characterized for the first time. The [39.6]0 u + -X 1 Σ g + (0 g + ) system is measured at rotational resolution by using degenerate and two-color resonant four-wave-mixing, as well as laser induced fluorescence spectroscopy. Double-resonance experiments are performed by labeling selected rotational levels of the ground state by tuning the probe laser wavelength to transitions in the well-known (1-0) band of the B0 u + -X 1 Σ g + (0 g + ) electronic system. Spectra obtained by scans of the pump laser in the UV wavelength range were then assigned unambiguously by the stringent double-resonance selection rules. The absence of a Q-band suggests a parallel transition (ΔΩ = 0) and determines the term symbol of the state as 0 u + in Hund's case (c) notation. The equilibrium constants for 63 Cu 2 are T e = 39 559.921(92) cm -1 , ω e = 277.70(14) cm -1 , B e = 0.104 942(66) cm -1 , and r e = 2.2595(11) Å. These findings are supported by high-level ab initio calculations at the MRCI+Q level, which clearly identifies this state as resulting from a 4p ← 3d transition. In addition, three dark perturber states are found in the v = 1 and v = 2 vibrational levels of the new state. A deperturbation analysis characterizes the interaction and rationalizes the anomalous dips in the excitation spectrum of the [39.6]0 u + -X 1 Σ g + (0 g + ) system.

Chloride transport by microbial rhodopsins is an essential process for which molecular details such as the mechanisms that convert light energy to drive ion pumping and ensure the unidirectionality of the transport have remained elusive. We combined time-resolved serial crystallography with time-resolved spectroscopy and multiscale simulations to elucidate the molecular mechanism of a chloride-pumping rhodopsin and the structural dynamics throughout the transport cycle. We traced transient anion-binding sites, obtained evidence for how light energy is used in the pumping mechanism, and identified steric and electrostatic molecular gates ensuring unidirectional transport. An interaction with the π-electron system of the retinal supports transient chloride ion binding across a major bottleneck in the transport pathway. These results allow us to propose key mechanistic features enabling finely controlled chloride transport across the cell membrane in this light-powered chloride ion pump.

X-ray free-electron lasers (FELs) deliver ultrabright X-ray pulses, but not the sequences of phase-coherent pulses required for time-domain interferometry and control of quantum states. For conventional split-and-delay schemes to produce such sequences, the challenge stems from extreme stability requirements when splitting Ångstrom wavelength beams, where the tiniest path-length differences introduce phase jitter. We describe an FEL mode based on selective electron-bunch degradation and transverse beam shaping in the accelerator, combined with a self-seeded photon emission scheme. Instead of splitting the photon pulses after their generation by the FEL, we split the electron bunch in the accelerator, prior to photon generation, to obtain phase-locked X-ray pulses with subfemtosecond duration. Time-domain interferometry becomes possible, enabling the concomitant program of classical and quantum optics experiments with X-rays. The scheme leads to scientific benefits of cutting-edge FELs with attosecond and/or high-repetition rate capabilities, ranging from the X-ray analog of Fourier transform infrared spectroscopy to damage-free measurements., Competing Interests: Competing interest statement: J.H. and G.A., who have never had a joint research project, participated in a workshop that resulted in a report [B. Adams et al. arXiv [Preprint] (2019). https://arxiv.org/abs/1903.09317] co-authored by all participants and were co-authors of an invited perspective paper [J. B. Hastings, L. Rivkin, G. Aeppli, “Present and future accelerator-based X-ray sources: A perspective” in The Future of Accelerators, A. W. Chao, W. Chou, Eds. (2019), vol. 10, pp. 33–48] on accelerator-based light sources, which reported no original research. J.H. has served on advisory panels at the Paul Scherrer Institute, and G.A. has served on advisory panels at the SLAC National Accelerator Laboratory, where J.H. is employed. S.R. was a postdoctoral fellow and assistant/associate researcher of C. Pellegrini at the University of California Los Angeles from 2000 to 2008. S.G. was a postdoctoral fellow at Stanford University and SLAC from 2013 to 2015, which has led to one shared publication with J.H. [H. Jang et al., Proc. Natl. Acad. Sci. U.S.A. 113, 14645–14650 (2016)]., (Copyright © 2022 the Author(s). Published by PNAS.)

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables essentially radiation-damage-free macromolecular structure determination using microcrystals that are too small for synchrotron studies. However, SFX experiments often require large amounts of sample in order to collect highly redundant data where some of the many stochastic errors can be averaged out to determine accurate structure-factor amplitudes. In this work, the capability of the Swiss X-ray free-electron laser (SwissFEL) was used to generate large-bandwidth X-ray pulses [Δλ/λ = 2.2% full width at half-maximum (FWHM)], which were applied in SFX with the aim of improving the partiality of Bragg spots and thus decreasing sample consumption while maintaining the data quality. Sensitive data-quality indicators such as anomalous signal from native thaumatin micro-crystals and de novo phasing results were used to quantify the benefits of using pink X-ray pulses to obtain accurate structure-factor amplitudes. Compared with data measured using the same setup but using X-ray pulses with typical quasi-monochromatic XFEL bandwidth (Δλ/λ = 0.17% FWHM), up to fourfold reduction in the number of indexed diffraction patterns required to obtain similar data quality was achieved. This novel approach, pink-beam SFX, facilitates the yet underutilized de novo structure determination of challenging proteins at XFELs, thereby opening the door to more scientific breakthroughs., (© Karol Nass et al. 2021.)

We discuss our recently reported femtosecond (fs) X-ray emission spectroscopy results on the ligand dissociation and recombination in nitrosylmyoglobin (MbNO) in the context of previous studies on ferrous haem proteins. We also present a preliminary account of femtosecond X-ray absorption studies on MbNO, pointing to the presence of more than one species formed upon photolysis.

Perturbation facilitated double-resonant four-wave mixing is applied to access high-lying vibrational levels of the X  1 Σ g +  (0 g + ) ground state of Cu 2 . Rotationally resolved transitions up to v ″ = 102 are measured. The highest observed level is at 98% of the dissociation energy. The range and accuracy of previous measurements are significantly extended. By applying the near dissociation equation developed by Le Roy [R. J. Le Roy, J. Quant. Spectrosc. Radiat. Transfer 186, 197 (2017)], a dissociation energy of D e = 16 270(7) hc cm -1 is determined, and an accurate potential energy function for the X  1 Σ g +  (0 g + ) ground state is obtained. Molecular constants are determined from the measured transitions and by solving the radial Schrödinger equation using this function and are compared with results from earlier measurements. In addition, benchmark multi-reference configuration interaction computations are performed using the Douglas-Kroll-Hess Hamiltonian and the appropriate basis of augmented valence quadruple ζ type. Coupled-cluster single, double, and perturbative triple calculations were performed for comparison.

Wastewater treatment plants are major point sources of (micro)pollutant emissions and advanced wastewater treatment technologies can improve their removal capacity. While abundant data on individual advanced treatment technologies is available, there is limited knowledge regarding the removal performance of ozonation combined with multiple post-treatments and stand-alone membrane bioreactors. This is especially true for the removal of in vitro and in vivo toxicity. Therefore, we investigated the removal of 40 micropollutants and toxicity by a pilot-scale ozonation with four post-treatments: non-aerated and aerated granular activated carbon and biological filtration. In addition, two stand-alone membrane bioreactors fed with untreated wastewater and one MBR operating with ozonated partial flow recirculation were analysed. Aqueous and extracted samples were analysed in vitro for (anti)estrogenic, (anti)androgenic and mutagenic effects. To assess in vivo effects, the mudsnail Potamopyrgus antipodarum was exposed in an on-site flow-through system. Multiple in vitro effects were detected in conventionally treated wastewater including estrogenic and anti-androgenic activity. Ozonation largely removed these effects, while anti-estrogenic and mutagenic effects increased suggesting the formation of toxic transformation products. These effects were significantly reduced by granular activated carbon being more effective than biological filtration. The membrane bioreactor performed similarly to the conventional treatment while the membrane bioreactor with ozonation had a comparable removal performance like ozonation. Conventionally treated wastewater increased the growth of P. antipodarum. Ozonation reduced the reproduction indicating a potential formation of toxic transformation products. In the post-treatments, these effects were compensated or remained unaffected. The effluents of the membrane bioreactors induced reproductive toxicity. Our results show that ozonation is effective in further reducing toxicity and micropollutant concentrations. However, the formation of toxicity requires a post-treatment. Here, ozonation coupled to granular activated carbon filtration seemed the most promising treatment process., 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 © 2020 Elsevier Ltd. All rights reserved.)

We present an approach to determine the absolute thickness profile of flat liquid jets, which takes advantage of the information of thin film interference combined with light absorption, both captured in a single microscopic image. The feasibility of the proposed method is demonstrated on our compact experimental setup used to generate micrometer thin, free-flowing liquid jet sheets upon collision of two identical laminar cylindrical jets. Stable operation was achieved over several hours of the flat jet in vacuum (10 -4 mbar), making the system ideally suitable for soft x-ray photon spectroscopy of liquid solutions. We characterize the flat jet size and thickness generated with two solvents, water and ethanol, employing different flow rates and nozzles of variable sizes. Our results show that a gradient of thickness ranging from a minimal thickness of 2 µm to over 10 µm can be found within the jet surface area. This enables the tunability of the sample thickness in situ, allowing the optimization of the transmitted photon flux for the chosen photon energy and sample. We demonstrate the feasibility of x-ray absorption spectroscopy experiments in transmission mode by measuring at the oxygen K-edge of ethanol. Our characterization method and the description of the experimental setup and its reported performance are expected to expand the range of applications and facilitate the use of flat liquid jets for spectroscopy experiments.