Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.

• Keywords
• Mathematical transforms, Multiplexing, Protein dynamics, Time-resolved, X-ray crystallography,
• MeSH
• Databases, Protein MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray methods   MeSH
• Models, Molecular MeSH
• Proteins * chemistry   MeSH
• Synchrotrons MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Proteins * MeSH

PURPOSE: The time structures of proton spot delivery in proton pencil beam scanning (PBS) radiation therapy are essential in many clinical applications. This study aims to characterize the time structures of proton PBS delivered by both synchrotron and synchrocyclotron accelerators using a non-invasive technique based on scattered particle tracking. METHODS: A pixelated semiconductor detector, AdvaPIX-Timepix3, with a temporal resolution of 1.56 ns, was employed to measure time of arrival of secondary particles generated by a proton beam. The detector was placed laterally to the high-flux area of the beam in order to allow for single particle detection and not interfere with the treatment. The detector recorded counts of radiation events, their deposited energy and the timestamp associated with the single events. Individual recorded events and their temporal characteristics were used to analyze beam time structures, including energy layer switch time, magnet switch time, spot switch time, and the scanning speeds in the x and y directions. All the measurements were repeated 30 times on three dates, reducing statistical uncertainty. RESULTS: The uncertainty of the measured energy layer switch times, magnet switch time, and the spot switch time were all within 1% of average values. The scanning speeds uncertainties were within 1.5% and are more precise than previously reported results. The measurements also revealed continuous sub-milliseconds proton spills at a low dose rate for the synchrotron accelerator and radiofrequency pulses at 7 µs and 1 ms repetition time for the synchrocyclotron accelerator. CONCLUSION: The AdvaPIX-Timepix3 detector can be used to directly measure and monitor time structures on microseconds scale of the PBS proton beam delivery. This method yielded results with high precision and is completely independent of the machine log files.

• Keywords
• dose rate, pencil beam scanning, proton therapy, scanning speeds, semiconductor detectors, time structure,
• MeSH
• Time Factors MeSH
• Particle Accelerators * instrumentation   MeSH
• Radiotherapy Dosage * MeSH
• Humans MeSH
• Neoplasms radiotherapy   MeSH
• Radiotherapy Planning, Computer-Assisted * methods   MeSH
• Semiconductors * MeSH
• Proton Therapy * instrumentation   MeSH
• Protons MeSH
• Synchrotrons instrumentation   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Protons MeSH

ELI Beamlines is one of the pillars of the Extreme Light Infrastructure European Research Infrastructure Consortium (ELI ERIC), the European project aiming at building the next generation of high power lasers for fundamental research and industrial applications. Several high-power lasers are hosted by the ELI Beamlines facility. Even at a power lower than the nominal one, when interacting with a target, the laser can generate mixed ionizing radiation fields of unique nature. One of the major laser systems, High-repetition-rate advanced petawatt laser system (HAPLS) was already used in commissioning experiments. Detecting the neutrons generated during these experiments has been a challenging task, since certain difficulties were faced. First, the experimental conditions were frequently altered during the commissioning phase (such as laser beam parameters, experimental geometry or target type). Next, the extremely short duration of the ionizing radiation pulse generated by the laser (~10-14 s) complicated the correct interpretation of the data provided by the detectors designed and calibrated in standard fields. Here, one commissioning experiment is described, together with the means of addressing the problem of the detection of the ionizing radiation and the lessons learned in this endeavour.

• MeSH
• Radiation, Ionizing * MeSH
• Lasers MeSH
• Neutrons MeSH
• Synchrotrons * MeSH
• Publication type
• Journal Article MeSH

Iron is accumulated symplastically in kelp in a non-ferritin core that seems to be a general feature of brown algae. Microprobe studies show that Fe binding depends on tissue type. The sea is generally an iron-poor environment and brown algae were recognized in recent years for having a unique, ferritin-free iron storage system. Kelp (Laminaria digitata) and the filamentous brown alga Ectocarpus siliculosus were investigated using X-ray microprobe imaging and nanoprobe X-ray fluorescence tomography to explore the localization of iron, arsenic, strontium, and zinc, and micro-X-ray absorption near-edge structure (μXANES) to study Fe binding. Fe distribution in frozen hydrated environmental samples of both algae shows higher accumulation in the cortex with symplastic subcellular localization. This should be seen in the context of recent ultrastructural insight by cryofixation-freeze substitution that found a new type of cisternae that may have a storage function but differs from the apoplastic Fe accumulation found by conventional chemical fixation. Zn distribution co-localizes with Fe in E. siliculosus, whereas it is chiefly located in the L. digitata medulla, which is similar to As and Sr. Both As and Sr are mostly found at the cell wall of both algae. XANES spectra indicate that Fe in L. digitata is stored in a mineral non-ferritin core, due to the lack of ferritin-encoding genes. We show that the L. digitata cortex contains mostly a ferritin-like mineral, while the meristoderm may include an additional component.

• Keywords
• XANES, algae, ferritin, iron, strontium, tomography,
• MeSH
• Ferritins metabolism   MeSH
• Kelp * metabolism   MeSH
• Laminaria * metabolism   MeSH
• Minerals metabolism   MeSH
• Phaeophyceae * metabolism   MeSH
• X-Rays MeSH
• Trace Elements * metabolism   MeSH
• Synchrotrons MeSH
• Iron metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Ferritins MeSH
• Minerals MeSH
• Trace Elements * MeSH
• Iron MeSH

Trilobites are among the most iconic of fossils and formed a prominent component of marine ecosystems during most of their 270-million-year-long history from the early Cambrian period to the end Permian period1. More than 20,000 species have been described to date, with presumed lifestyles ranging from infaunal burrowing to a planktonic life in the water column2. Inferred trophic roles range from detritivores to predators, but all are based on indirect evidence such as body and gut morphology, modes of preservation and attributed feeding traces; no trilobite specimen with internal gut contents has been described3,4. Here we present the complete and fully itemized gut contents of an Ordovician trilobite, Bohemolichas incola, preserved three-dimensionally in a siliceous nodule and visualized by synchrotron microtomography. The tightly packed, almost continuous gut fill comprises partly fragmented calcareous shells indicating high feeding intensity. The lack of dissolution of the shells implies a neutral or alkaline environment along the entire length of the intestine supporting digestive enzymes comparable to those in modern crustaceans or chelicerates. Scavengers burrowing into the trilobite carcase targeted soft tissues below the glabella but avoided the gut, suggesting noxious conditions and possibly ongoing enzymatic activity.

• MeSH
• Biological Evolution MeSH
• Arthropods * anatomy & histology   enzymology   physiology   MeSH
• Hydrogen-Ion Concentration MeSH
• Crustacea enzymology   MeSH
• Intestines * chemistry   enzymology   metabolism   MeSH
• Synchrotrons MeSH
• Aquatic Organisms enzymology   physiology   MeSH
• Fossils * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm-1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

• Keywords
• Modeling, Native MS, Protein complex structure, SPI, Simulation, Viral particles, X-ray,
• MeSH
• Electrons * MeSH
• Capsid * MeSH
• Computer Simulation MeSH
• X-Rays MeSH
• Synchrotrons MeSH
• Publication type
• Journal Article MeSH

A combination of synchrotron-based elemental analysis and acute toxicity tests was used to investigate the biodistribution and adverse effects in Daphnia magna exposed to uranium nanoparticle (UNP, 3-5 nm) suspensions or to uranium reference (Uref) solutions. Speciation analysis revealed similar size distributions between exposures, and toxicity tests showed comparable acute effects (UNP LC50: 402 μg L-1 [336-484], Uref LC50: 268 μg L-1 [229-315]). However, the uranium body burden was 3- to 5-fold greater in UNP-exposed daphnids, and analysis of survival as a function of body burden revealed a ∼5-fold higher specific toxicity from the Uref exposure. High-resolution X-ray fluorescence elemental maps of intact, whole daphnids from sublethal, acute exposures of both treatments revealed high uranium accumulation onto the gills (epipodites) as well as within the hepatic ceca and the intestinal lumen. Uranium uptake into the hemolymph circulatory system was inferred from signals observed in organs such as the heart and the maxillary gland. The substantial uptake in the maxillary gland and the associated nephridium suggests that these organs play a role in uranium removal from the hemolymph and subsequent excretion. Uranium was also observed associated with the embryos and the remnants of the chorion, suggesting uptake in the offspring. The identification of target organs and tissues is of major importance to the understanding of uranium and UNP toxicity and exposure characterization that should ultimately contribute to reducing uncertainties in related environmental impact and risk assessments.

• Keywords
• X-ray absorption spectroscopy, ecotoxicology, elemental distributions, model organism, synchrotron-based imaging, tomography, uranium nanoparticles,
• MeSH
• Water Pollutants, Chemical * chemistry   MeSH
• Daphnia chemistry   MeSH
• Optical Imaging MeSH
• X-Rays MeSH
• Synchrotrons MeSH
• Tissue Distribution MeSH
• Toxicokinetics MeSH
• Uranium * toxicity   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Water Pollutants, Chemical * MeSH
• Uranium * MeSH

A novel approach to the remote-control system for the compact multi-crystal energy-dispersive spectrometer for X-ray emission spectroscopy (XES) applications has been developed. This new approach is based on asynchronous communication between software components and on reactive design principles. In this paper, the challenges faced, their solutions, as well as the implementation and future development prospects are identified. The main motivation of this work was the development of a new holistic communication protocol that can be implemented to control various hardware components allowing both independent operation and easy integration into different SCADA systems.

• Keywords
• X-ray spectroscopy, control system, experiment control, physics facility, reactive system, system design,
• MeSH
• Software * MeSH
• Spectrometry, X-Ray Emission MeSH
• Synchrotrons * MeSH
• Publication type
• Journal Article MeSH

We report a strategy for sustainable development of pH-responsive cubic liquid crystalline nanoparticles (cubosomes), in which the structure-defining lyotropic nonlamellar lipid and the eventually encapsulated guest molecules can be protected by pH-sensitive polyelectrolyte shells with mucoadhesive properties. Bulk non-lamellar phases as well as pH-responsive polyelectrolyte-modified nanocarriers were formed by spontaneous assembly of the nonlamellar lipid monoolein and two biopolymers tailored in nanocomplexes with pH-dependent net charge. The mesophase particles involved positively charged N-arginine-modified chitosan (CHarg) and negatively charged alginate (ALG) chains assembled at different biopolymer concentrations and charge ratios into a series of pH-responsive complexes. The roles of Pluronic F127 as a dispersing agent and a stabilizer of the nanoscale dispersions were examined. Synchrotron small-angle X-ray scattering (SAXS) investigations were performed at several N-arginine-modified chitosan/alginate ratios (CHarg/ALG with 10, 15 and 20 wt% ALG relative to CHarg) and varying pH values mimicking the pH conditions of the gastrointestinal route. The structural parameters characterizing the inner cubic liquid crystalline organizations of the nanocarriers were determined as well as the particle sizes and stability on storage. The surface charge variations, influencing the measured zeta-potentials, evidenced the inclusion of the CHarg/ALG biopolymer complexes into the lipid nanoassemblies. The polyelectrolyte shells rendered the hybrid cubosome nanocarriers pH-sensitive and influenced the swelling of their lipid-phase core as revealed by the acquired SAXS patterns. The pH-responsiveness and the mucoadhesive features of the cubosomal lipid/polyelectrolyte nanocomplexes may be of interest for in vivo drug delivery applications.

• Keywords
• Cubic phase nanoparticles (cubosomes), Drug delivery systems for oral administration, Hybrid nonlamellar liquid crystalline nanostructures, N-arginine-modified chitosan, Self-assembled lipid/biopolymer complexes, Synchrotron small-angle X-ray scattering (SAXS),
• MeSH
• Biopolymers MeSH
• X-Ray Diffraction MeSH
• Liquid Crystals * MeSH
• Hydrogen-Ion Concentration MeSH
• Lipids MeSH
• Scattering, Small Angle MeSH
• Synchrotrons * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biopolymers MeSH
• Lipids MeSH

Algae are the main primary producers in aquatic environments and therefore of fundamental importance for the global ecosystem. Mid-infrared (IR) microspectroscopy is a non-invasive tool that allows in principle studying chemical composition on a single-cell level. For a long time, however, mid-infrared (IR) imaging of living algal cells in an aqueous environment has been a challenge due to the strong IR absorption of water. In this study, we employed multi-beam synchrotron radiation to measure time-resolved IR hyperspectral images of individual Thalassiosira weissflogii cells in water in the course of acclimation to an abrupt change of CO2 availability (from 390 to 5000 ppm and vice versa) over 75 min. We used a previously developed algorithm to correct sinusoidal interference fringes from IR hyperspectral imaging data. After preprocessing and fringe correction of the hyperspectral data, principal component analysis (PCA) was performed to assess the spatial distribution of organic pools within the algal cells. Through the analysis of 200,000 spectra, we were able to identify compositional modifications associated with CO2 treatment. PCA revealed changes in the carbohydrate pool (1200-950 cm[Formula: see text]), lipids (1740, 2852, 2922 cm[Formula: see text]), and nucleic acid (1160 and 1201 cm[Formula: see text]) as the major response of exposure to elevated CO2 concentrations. Our results show a local metabolism response to this external perturbation.

• MeSH
• Acclimatization MeSH
• Single-Cell Analysis methods   MeSH
• Time-Lapse Imaging MeSH
• Carbon Dioxide pharmacology   MeSH
• Diatoms drug effects   metabolism   MeSH
• Spectrophotometry, Infrared MeSH
• Synchrotrons MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Carbon Dioxide MeSH