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Trypanosoma brucei inosine-5′-monophosphate dehydrogenase (IMPDH) is an enzyme in the guanine nucleotide biosynthesis pathway and of interest as a drug target. Here the authors present the 2.8 Å room temperature structure of TbIMPDH determined by utilizing X-ray free-electron laser radiation and crystals that were grown in insect cells and find that ATP and GMP are bound at the canonical sites of the Bateman domains.

The design and optimization of laser-Compton x-ray systems based on compact distributed charge accelerator structures can enable micron-scale imaging of disease and the concomitant production of beams of Very High Energy Electrons (VHEEs) capable of producing FLASH-relevant dose rates. The physics of laser-Compton x-ray scattering ensures that the scattered x-rays follow exactly the trajectory of the incident electrons, thus providing a route to image-guided, VHEE FLASH radiotherapy. The keys to a compact architecture capable of producing both laser-Compton x-rays and VHEEs are the use of X-band RF accelerator structures which have been demonstrated to operate with over 100 MeV/m acceleration gradients. The operation of these structures in a distributed charge mode in which each radiofrequency (RF) cycle of the drive RF pulse is filled with a low-charge, high-brightness electron bunch is enabled by the illumination of a high-brightness photogun with a train of UV laser pulses synchronized to the frequency of the underlying accelerator system. The UV pulse trains are created by a patented pulse synthesis approach which utilizes the RF clock of the accelerator to phase and amplitude modulate a narrow band continuous wave (CW) seed laser. In this way it is possible to produce up to 10 μA of average beam current from the accelerator. Such high current from a compact accelerator enables production of sufficient x-rays via laser-Compton scattering for clinical imaging and does so from a machine of "clinical" footprint. At the same time, the production of 1000 or greater individual micro-bunches per RF pulse enables > 10 nC of charge to be produced in a macrobunch of < 100 ns. The design, construction, and test of the 100-MeV class prototype system in Irvine, CA is also presented.

A numerical analysis of spatially chirped beams produced by single-pass grating pairs is presented. It is shown that focused pulse structures can deviate significantly relative to standard linear spatial chirp approximations depending upon the pulse bandwidth, the angle of incidence, and groove density of the gratings used.

An ultra-broadband beam splitter arrangement, with a spectral amplitude response that is over seven orders-of-magnitude more uniform than broadband, multi-layer dielectric beam splitters, can be created by the combination of multiple Fresnel events on an uncoated, optical flat when used at a specific angle-of-incidence. This beam splitter arrangement produces three, spectral copies of the original, two of which have identical spectral phase. In this manuscript we derive the precise angle at which this maximally flat spectral amplitude response occurs for any material and present this angle’s material and polarization dependence.

On December 5, 2022, an indirect drive fusion implosion on the National Ignition Facility (NIF) achieved a target gain G_{target} of 1.5. This is the first laboratory demonstration of exceeding "scientific breakeven" (or G_{target}>1) where 2.05 MJ of 351 nm laser light produced 3.1 MJ of total fusion yield, a result which significantly exceeds the Lawson criterion for fusion ignition as reported in a previous NIF implosion [H. Abu-Shawareb et al. (Indirect Drive ICF Collaboration), Phys. Rev. Lett. 129, 075001 (2022)PRLTAO0031-900710.1103/PhysRevLett.129.075001]. This achievement is the culmination of more than five decades of research and gives proof that laboratory fusion, based on fundamental physics principles, is possible. This Letter reports on the target, laser, design, and experimental advancements that led to this result.

Electron beams used in laser-Compton X-ray sources can be utilized as ultrahigh dose rate (UHDR) ionizing radiation sources if the architecture provides sufficient peak current and beam energy. Using a multi-step simulation workflow, we identify commensurate X-ray and electron beam profiles for image-guided UHDR (FLASH) radiotherapy experiments.

A single energy tuning, K-edge subtraction imaging method using laser-Compton sources (LCS) is presented. The narrow spectral bandwidths that LCSs provide can improve clinical dual-energy mammography by 1000x in contrast enhancement or dose reduction.

Nuclear resonance fluorescence is the process by which atomic nuclei absorb and emit radiation. We discuss the possibility of using laser-Compton sources to interrogate these transitions for isotope selective biological functional imaging.

We present a photogun laser (PGL) architecture that generates 11.424 GHz repetition rate, 2.0 ps FWHM, 249 nm pulses for injecting electron bunches into sequential RF buckets of a pulsed X-band linac.

The intensity of nuclear resonant transitions is largely dominated by atomic motion. Laser-Compton sources have the capability to measure these Doppler spectrum shifts and a method of atomic velocity measurements is presented.

Nuclear resonance fluorescence is the process by which atomic nuclei absorb and emit radiation. Around these resonances, the refractive index changes according to the Kramers-Kronig relations. We discuss how to measure such physics using laser-Compton sources.

The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters. When using X-ray crystallography as a probe of ultrafast dynamics, the origin of the observed nuclear motions is not known. Now, high-resolution pump-probe X-ray crystallography reveals complex sub-ångström, ultrafast motions and hydrogen-bonding rearrangements in the active site of a fluorescent protein. However, we demonstrate that the measured motions are not part of the photoisomerization reaction but instead arise from impulsively driven coherent vibrational processes in the electronic ground state. A coherent-control experiment using a two-colour and two-pulse optical excitation strongly amplifies the X-ray crystallographic difference density, while it fully depletes the photoisomerization process. A coherent control mechanism was tested and confirmed the wave packets assignment.

A novel method of measuring an electron beam's energy spectrum and divergence using Compton-scattered x rays created by colliding a laser with an electron beam has been developed and tested using the compact laser-Compton x-ray source at LLNL. The method only requires an x-ray imaging device and a filter material whose K-edge energy matches that of the Compton-scattered x rays. K-edge filtering of energy-angle correlated Compton x rays causes large variations in intensity over the viewing angle. These intensity variations contain information about the electron beam's properties. By using simulation tools and adjusting the beam parameters to match the shape of the acquired image, the electron beam can be characterized. As a demonstration of this technique, a 75-μm Sn foil was used to filter 30-keV Compton x rays created from Compton scattering of a 532-μm laser beam by a 30-MeV electron beam. The measured parameters were mean energy E=28.51±0.06 MeV, energy spread σE

Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.

Intense femtosecond x-ray pulses from free-electron laser sources allow the imaging of individual particles in a single shot. Early experiments at the Linac Coherent Light Source (LCLS) have led to rapid progress in the field and, so far, coherent diffractive images have been recorded from biological specimens, aerosols, and quantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLS held a workshop to discuss the scientific and technical challenges for reaching the ultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap toward reaching atomic resolution, 3D imaging at free-electron laser sources.

Laser-Compton x-ray sources have many advantages over traditional x-ray tubes for use in medical imaging due to their monoenergetic energy spectrum, tunability, high-flux, and low-dose potential. These properties can specifically be taken advantage of in the context of K-edge subtraction (KES) imaging. Previous optimization approaches are time-consuming by scanning over high-dimensional parameter spaces. Here, we show how a Bayesian optimization routine optimizes LCS source parameters in only a fraction of the computational time. Using this approach, we found a configuration that produces non-inferior image quality in KES mammography compared to a previously optimized direct energy tuning technique. Moreover, a successfully optimized implementation of scanning K-edge subtraction imaging was realized applying this Bayesian approach.

This paper presents the ideal spatio-temporal pulse structure that is required to produce exawatt-scale pulses based on simultaneous chirped beam and chirped pulse amplification in a Nd:Mixed-glass laser system. It is shown, that a 100 fs Fourier transform-limited pulse is created from a 20 ns duration stretched beam-pulse after propagating through an appropriate six-grating compressor arrangement. Quantitative results, from a ray-tracing model of the six-grating compressor, provide the detailed spatio-spectral and spatio-temporal pulse distributions of the stretched pulse along with the higher-order phase distortions compensated by this pulse compression scheme.

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion.

The development of compact quasimonoenergetic x-ray radiation sources based on laser Compton scattering (LCS) offers opportunities for novel approaches to medical imaging. However, careful experimental design is required to fully utilize the angle-correlated x-ray spectra produced by LCS sources. Direct simulations of LCS x-ray spectra are computationally expensive and difficult to employ in experimental optimization. In this manuscript, we present a computational method that fully characterizes angle-correlated LCS x-ray spectra at any end point energy within a range defined by three direct simulations. With this approach, subsequent LCS x-ray spectra can be generated with up to 200 times less computational overhead.

Conventional x-ray sources for medical imaging utilize bremsstrahlung radiation. These sources generate large bandwidth (BW) x-ray spectra with large fractions of photons that impart a dose, but do not contribute to image production. X-ray sources based on laser-Compton scattering can have inherently small energy BWs and can be tuned to low dose-imparting energies, allowing them to take advantage of atomic K-edge contrast enhancement. This paper investigates the use of gadolinium-based K-edge subtraction imaging in the context of mammography using a laser-Compton source through simulations quantifying contrast and dose in such imaging systems as a function of laser-Compton source parameters. Our simulations indicate that a K-edge subtraction image generated with a 0.5% BW (FWHM) laser-Compton x-ray source can obtain an equal contrast to a bremsstrahlung image with only 3% of the dose.

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.

Coherent diffractive imaging of single particles using the single-shot “diffract and destroy” approach with an x-ray free electron laser (FEL) was recently demonstrated. A high-resolution low-noise coherent diffraction pattern, representative of the object before it turns into a plasma and explodes, results from the interaction of the FEL with the particle. Iterative phase retrieval algorithms are used to reconstruct two-dimensional projection images of the object from the recorded intensities alone. Here we describe the first single-shot diffraction data set that mimics the data proposed for obtaining 3D structure from identical particles. Ellipsoidal iron oxide nanoparticles (250 nm×50 nm) were aerosolized and injected through an aerodynamic lens stack into a soft x-ray FEL. Particle orientation was not controlled with this injection method. We observed that, at the instant the x-ray pulse interacts with the particle, a snapshot of the particle’s orientation is encoded in the diffraction pattern. The results give credence to one of the technical concepts of imaging individual nanometer and subnanometer-sized objects such as single molecules or larger clusters of molecules using hard x-ray FELs and will be used to help develop robust algorithms for determining particle orientations and 3D structure.

The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate.

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

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.