Synchrotron Beam Time Research Articles

X-ray wavefront sensing and optics metrology using a microfocus x-ray grating interferometer with electromagnetic phase stepping

A metrology method of x-ray optical elements based on an electromagnetic phase stepping x-ray grating interferometer with high slope accuracy is presented in this study. The device consists of an x-ray tube, a phase grating G1 for modulating the incoming wavefront, and an absorption grating G2 as a transmission mask to produce a broader moiré pattern for the x-ray camera. The focal spot of the microfocus x-ray source is shifted by a magnetic field from a solenoid coil. Electromagnetic phase stepping analysis is used to obtain a pixel-wise map of the wavefront phase distortion to replace the traditional precision mechanical scanning system, improving stability, speed, accuracy, and flexibility. The x-ray grating interferometer can be used as a feedback tool for evaluating the quality of optical elements and detecting defects caused by the x-ray beam or the x-ray optical elements in ordinary laboratories and mirror-processing centers without the need of scheduling synchrotron beam time.

Characterization and Speciation of Marine Materials Using Synchrotron Probes: Guidelines for New Users

Synchrotron instruments are useful for marine studies because they make nondestructive measurements of chemical composition and speciation on small sample volumes and at low concentrations. Synchrotron beamtime is available without cost using a peer-reviewed proposal system. New users do not have to be synchrotron radiation experts to design a good experiment, but some guidance is needed to design and propose appropriate experiments. Here we present some of that guidance to encourage and increase access to synchrotron facilities for marine science. We provide advice and examples from experts on how to access these instruments, choose the optimal sample preparation, and avoid common pitfalls. We then present some examples of successful marine studies that use these techniques.

Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures.

Traditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structure-function studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Furthermore, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that can be directly related to biological function. Given this capability, the next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperature. This challenge is addressed herein. The approach described provides complete diffraction data sets with total collection times as short as ∼5 s from single protein crystals, dramatically increasing the quantity of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09-1.54 Å resolution data over a temperature range of 293-363 K for proteinase K, thaumatin and lysozyme crystals at BL14-1 at the Stanford Synchrotron Radiation Lightsource. The analyses presented here indicate that the diffraction data are of high quality and do not suffer from excessive dehydration or radiation damage.

X-Ray Flow Visualization in Multiphase Flows

The use of X-ray flow visualization has brought a powerful new tool to the study of multiphase flows. Penetrating radiation can probe the spatial concentration of the different phases without the refraction, diffraction, or multiple scattering that usually produce image artifacts or reduce the signal-to-noise ratio below reliable values in optical visualization of multiphase flows; hence, X-ray visualization enables research into the three-dimensional (3D) structure of multiphase flows characterized by complex interfaces. With the commoditization of X-ray laboratory sources and wider access to synchrotron beam time for fluid mechanics, this novel imaging technique has shed light onto many multiphase flows of industrial and environmental interest under realistic 3D configurations and at realistic operating conditions (high Reynolds numbers and high volume fractions) that had defied study for decades. We present a broad survey of the most commonly studied multiphase flows (e.g., sprays, fluidized beds, bubble columns) in order to highlight the progress X-ray imaging has made in understanding the internal structure and multiphase coupling of these flows, and we discuss the potential of advanced tomography and time-resolved and particle tracking radiography for further study of multiphase flows.

Open-Source Tool Kit Uses 3D Printing for Micromodel Generation

This article, written by JPT Technology Editor Chris Carpenter, contains highlights of paper SPE 190852, “Open-Source Tool Kit for Micromodel Generation Using 3D Printing,” by Thomas D. Seers and Nayef Alyafei, SPE, Texas A&M University at Qatar, prepared for the 2018 SPE Europec featured at the 80th EAGE Conference and Exhibition, Copenhagen, Denmark, 11–14 June. The paper has not been peer reviewed. In this paper, the authors present an open-source tool kit for the generation of microfabricated transparent models of porous media (micromodels) from image data sets using optically transparent 3D polymer additive manufacturing (3D printing or sintering). These micromodels serve as research and pedagogical tools that facilitate the direct visualization of drainage and imbibition within quasi-2D porous media, generated from a range of image modalities [e.g., thin section micrographs, µ-computed tomography (µCT) orthoslices, and conventional digital photography]. Introduction Though recent advances in 3D X-ray imaging, such as X-ray microtomography and µCT, permit volumetric imaging of microcore flood experiments with-in geological samples at the pore scale, experimental observations of dynamic (time-resolved) multiphase flow with-in pore networks still are obtained conventionally using transparent etched or molded synthetic porous media commonly referred to as micromodels. Typically, video footage of fluid imbibition and drainage experiments conducted across these quasi-2D pore networks is used to understand fluid distributions and displacement mechanisms within an equivalent 3D porous media. Contrary to state-of-the-art dynamic µCT coreflood experiments, which require synchrotron beam time to be conducted, micromodel studies can be undertaken routinely within a laboratory-based setting with a relatively simple experimental setup. However, the facilities required to fabricate micromodels typically are highly specialized, with production often outsourced to third-party manufacturers. In this work, the authors consider the potential of using additive manufacturing (3D printing or sintering) as an alternative to conventional micromodel-fabrication techniques. With the advent of light-transmissible 3D-printable materials, flow experiments conducted with these 3D-printed physical models can be captured using widely available optical imaging techniques (i.e., standalone digital cameras and trinocular microscopes). However, a major obstacle encountered in harnessing 3D-printing technologies for the goal of micromodels fabrication is the paucity in software tools capable of processing and converting raster-based images to mesh-based file formats parsable by commercially available 3D-printing systems. The paper aims to provide an open-source platform through which 3D-printable mesh-based representations of micromodels can be generated from raster imagery. Using this tool, standard micromodel components are fully integrated into the fabricated model. The availability of such a tool kit could act as an enabler for community research into fluid-transport phenomena in porous media. The authors suggest that 3D-printed pore networks may provide an effective pedagogical tool for teaching multiphase flow, enabling petroleum- and chemical-engineering and geology students to visualize directly often obtuse immiscible-fluid-flow processes within the classroom. Software-Implementation Description The tool kit is developed in the popular MATLAB language and is executed by graphical user interface. The generation of 3D-printable micromodels from raster datasets comprises three main stages: Image processing Micromodel construction Mesh post-processing

Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument.

Coupling of size-exclusion chromatography with biological solution small-angle X-ray scattering (SEC-SAXS) on dedicated synchrotron beamlines enables structural analysis of challenging samples such as labile proteins and low-affinity complexes. For this reason, the approach has gained increased popularity during the past decade. Transportation of perishable samples to synchrotrons might, however, compromise the experiments, and the limited availability of synchrotron beamtime renders iterative sample optimization tedious and lengthy. Here, the successful setup of laboratory-based SEC-SAXS is described in a proof-of-concept study. It is demonstrated that sufficient quality data can be obtained on a laboratory instrument with small sample consumption, comparable to typical synchrotron SEC-SAXS demands. UV/vis measurements directly on the SAXS exposure cell ensure accurate concentration determination, crucial for direct molecular weight determination from the scattering data. The absence of radiation damage implies that the sample can be fractionated and subjected to complementary analysis available at the home institution after SEC-SAXS. Laboratory-based SEC-SAXS opens the field for analysis of biological samples at the home institution, thus increasing productivity of biostructural research. It may further ensure that synchrotron beamtime is used primarily for the most suitable and optimized samples.

Synchrotron Big Data Science.

The rapid development of synchrotrons has massively increased the speed at which experiments can be performed, while new techniques have increased the amount of raw data collected during each experiment. While this has created enormous new opportunities, it has also created tremendous challenges for national facilities and users. With the huge increase in data volume, the manual analysis of data is no longer possible. As a result, only a fraction of the data collected during the time- and money-expensive synchrotron beam-time is analyzed and used to deliver new science. Additionally, the lack of an appropriate data analysis environment limits the realization of experiments that generate a large amount of data in a very short period of time. The current lack of automated data analysis pipelines prevents the fine-tuning of beam-time experiments, further reducing their potential usage. These effects, collectively known as the "data deluge," affect synchrotrons in several different ways including fast data collection, available local storage, data management systems, and curation of the data. This review highlights the Big Data strategies adopted nowadays at synchrotrons, documenting this novel and promising hybridization between science and technology, which promise a dramatic increase in the number of scientific discoveries.

Syris: a flexible and efficient framework for X-ray imaging experiments simulation

An open-source framework for conducting a broad range of virtual X-ray imaging experiments, syris, is presented. The simulated wavefield created by a source propagates through an arbitrary number of objects until it reaches a detector. The objects in the light path and the source are time-dependent, which enables simulations of dynamic experiments, e.g. four-dimensional time-resolved tomography and laminography. The high-level interface of syris is written in Python and its modularity makes the framework very flexible. The computationally demanding parts behind this interface are implemented in OpenCL, which enables fast calculations on modern graphics processing units. The combination of flexibility and speed opens new possibilities for studying novel imaging methods and systematic search of optimal combinations of measurement conditions and data processing parameters. This can help to increase the success rates and efficiency of valuable synchrotron beam time. To demonstrate the capabilities of the framework, various experiments have been simulated and compared with real data. To show the use case of measurement and data processing parameter optimization based on simulation, a virtual counterpart of a high-speed radiography experiment was created and the simulated data were used to select a suitable motion estimation algorithm; one of its parameters was optimized in order to achieve the best motion estimation accuracy when applied on the real data. syris was also used to simulate tomographic data sets under various imaging conditions which impact the tomographic reconstruction accuracy, and it is shown how the accuracy may guide the selection of imaging conditions for particular use cases.

Optimizing buffer conditions to improve crystallizability and SAXS measurement

The Sample Preparation and Characterization (SPC) facility at EMBL Hamburg is situated next to the PETRA3 beamlines for protein crystallography and small angle scattering (SAXS) that are operated by EMBL. The facility is equipped with molecular biology and biophysical instrumentation to carry out purification and characterization of macromolecular samples. It serves a mixed community of local EMBL scientists, beamline visitors and scientists from the European Union research area. Most incoming samples are destined for high-throughput crystallization at the facility or characterization by SAXS. The facility offers standardized quality control reports on each incoming sample, including characterization by mass spectrometry and Thermofluor. Based on this report, local staff can suggest and perform optimization protocols that increase the stability of the sample. For instance, Thermofluor screens(1) were developed that probe the effect of buffers and additives that are commonly used in sample preparation. These systematic screens provide a high-throughput method to identify stabilizing conditions for sample purification, storage and structural characterization. This technique has been also a valuable asset providing a high-throughput method for assessing the crystallizability of proteins by screening for conditions which contribute to the protein sample homogeneity, stability and solubility. The home-made screens have been tested on more than 200 different protein constructs at SPC facility. The aim of the SPC facility is to integrate off-line biophysical techniques with synchrotron beamlines, to offer the European user community a full package for sample characterization. The facility is especially geared towards cell biologists with little experience in structure determination. Expert staff is available to help to plan, perform and interpret biophysical experiments. It is possible for users to book SPC equipment together with their synchrotron beamtime, for protein purification, circular dichroism and isothermal calorimetry. Funded access to the facility is currently provided by the European Community's Seventh Framework Programme Biostruct-X. For further information, please contact spc@embl-hamburg.de.

When protein crystal dehydration helps

A typical protein crystal contains 30-60% solvent. For a naked crystal, this solvent is distributed between solvent shells, where water and solvent molecules make specific interactions with the crystalline protein, and solvent channels filled with disordered solvent molecules. This internal solvent map of the crystal can be modified by placing the crystal in a dehydrating environment. This may in turn induce changes to the crystal lattice and affect mosaicity, resolution and quality of diffraction data. A dehydrating environment can be generated around a crystal in several ways with various degrees of precision and complexity. In this study we have used the HC1 device (Maatel) to mount crystals an air stream of known relative humidity – a precise yet hassle-free approach to altering crystal hydration. We set out to analyse a range of different crystals to establish usable protocols that will allow one to explore to crystal hydration space, either by preparing samples before synchrotron beamtime or by undertaking the experiments during beamtime. Our results, considered in the light of the literature surrounding crystal dehydration, provide guidance for when dehydration can help diffraction.

A compact tool for coaxial laser alignment on a time-sharing beamline at Taiwan Light Source.

A simple design and easily installed tool for alignment has been developed for time-sharing undulator beamlines. A laser beam is directed onto a beam splitter inside the vacuum chamber, then reflects 90° along the synchrotron beam path; this beam serves as a reference to mimic the synchrotron beam path. Use of this tool greatly abbreviated alignment of an end-station after beamline switching; both beamline diagnosis and end-station development can be completed before the synchrotron beam time begins.

10 years of protein crystallography at AR-NW12A beamline

The exponential growth of protein crystallography can be observed in the continuously increasing demand for synchrotron beam time, both from academic and industrial users. Nowadays, the screening of a profusion of sample crystals for more and more projects is being implemented by taking advantage of fully automated procedures at every level of the experiments. The insertion device AR-NW12A beamline is one of the five macromolecular crystallography (MX) beamlines at the Photon Factory (PF). Currently the oldest MX beamline operational at the High Energy Accelerator Research Organization (KEK), the end-station was launched in 2001 as part of an upgrade of the PF Advanced Ring. Since its commissioning, AR-NW12A has been operating as a high-throughput beamline, slowly evolving to a multipurpose end-station for MX experiments. The development of the beamline took place about a decade ago, in parallel with a drastic development of protein crystallography and more general synchrotron technology. To keep the beamline up-to-date and competitive with other MX stations in Japan and worldwide, new features have been constantly added, with the goal of user friendliness of the various beamline optics and other instruments. Here we describe the evolution of AR-NW12A for its tenth anniversary. We also discuss the plans for upgrades for AR-NW12A, the future objectives in terms of the beamline developments, and especially the strong desire to open the beamline to a larger user community.

The application of hybrid pixel detectors for in-house SAXS instrumentation with a view to combined chromatographic operation

This study analyses the potential for laboratory-based size-exclusion chromatography (SEC) integrated small-angle X-ray scattering (SAXS) instrumentation to characterize protein complexes. Using a high-brilliance home source in conjunction with a hybrid pixel X-ray detector, the efficacy of SAXS data collection at pertinent protein concentrations and exposure times has been assessed. Scattering data from SOD1 and from the complex of SOD1 with its copper chaperone, using 10 min exposures, provided data quality in the range 0.03 < q < 0.25 Å(-1) that was sufficient to accurately assign radius of gyration, maximum dimension and molecular mass. These data demonstrate that a home source with integrated SEC-SAXS technology is feasible and would enable structural biologists studying systems containing transient protein complexes, or proteins prone to aggregation, to make advanced preparations in-house for more effective use of limited synchrotron beam time.

Advances in exploiting preferred orientation in the structure analysis of polycrystalline materials

In an attempt to overcome the reflection overlap problem, which is the primary hindrance to structure determination from powder diffraction data, an experimental approach that exploits preferred orientation was developed some years ago. Now both the experimental setup and the data analysis procedure have been optimized, with the result that the quality of the extracted reflection intensities has been improved significantly and the synchrotron beamtime required for the data collection reduced. The one-dimensional Si microstrip detector Mythen II on the Materials Science beamline at the Swiss Light Source and new features in the data analysis softwareMAUD[Lutterotti, Matthies &amp; Wenk (1999).IUCr Commission on Powder Diffraction Newsletter, No. 21, pp. 14–15] have made these improvements possible. The main changes in the experimental setup are (1) using an optimized set of sample orientations, (2) placing the detector such that both positive and negative 2θ angles are measured simultaneously, and (3) introducing an additional sample tilt angle, to measure data that cannot be accessed otherwise. On the data analysis side, the programMAUDis now used for both the determination of the orientation of the crystallites and the intensity extraction. The evaluation of data obtained from a textured zirconium phosphate pyridinium sample shows a significant improvement in the reliability of the structure factor amplitudes derived from overlapping reflections.

UV LED lighting for automated crystal centring.

A direct outcome of the exponential growth of macromolecular crystallography is the continuously increasing demand for synchrotron beam time, both from academic and industrial users. As more and more projects entail screening a profusion of sample crystals, fully automated procedures at every level of the experiments are being implemented at all synchrotron facilities. One of the major obstacles to achieving such automation lies in the sample recognition and centring in the X-ray beam. The capacity of UV light to specifically react with aromatic residues present in proteins or with DNA base pairs is at the basis of UV-assisted crystal centring. Although very efficient, a well known side effect of illuminating biological samples with strong UV sources is the damage induced on the irradiated samples. In the present study the effectiveness of a softer UV light for crystal centring by taking advantage of low-power light-emitting diode (LED) sources has been investigated. The use of UV LEDs represents a low-cost solution for crystal centring with high specificity.

Power to Advocate for Health

<h3>Abstract</h3> Traditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structure-function studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Further, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that is directly related to biological function. The next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperatures and was addressed herein. This approach provides complete diffraction datasets with total collection times as short as ~5 sec from single protein crystals, dramatically increasing the amount of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09-1.54 Å resolution data over a temperature range of 293–363 K for proteinase K, thaumatin, and lysozyme crystals. Our analyses indicate that the diffraction data is of high-quality and do not suffer from excessive dehydration or damage.

Technical Report: Automatic Experiments at the European Synchrotron Radiation Facility MX Beam-lines

During the last twenty years macromolecular crystallography (MX) has become the predominant tool for the investigation of structure/function relationships in biology. This is due to many technological advances and, in particular, improved access to synchrotron radiation (SR) sources, data from which has lead to ∼80% of the macromolecular crystal structures currently being deposited in the Protein Data Bank (PDB) (Figure 1, http://www.rcsb.org). The demand for synchrotron beam-time is also being fuelled by the funding of a number of structural genomics programmes [1], which, following an initial investment in protein production technology, have now contributed ∼1000 new structures to the PDB.

An overview of informatics developments for managing target selection to X-ray data collection

The drive toward high throughput in structural biology has resulted inmany technical innovations and developments. From ligation-independent cloning through nanolitre-scale crystallization to automated data collection the effects of these developments are changing the process of macromolecular structure determination for all laboratories, large and small, and dramatically extending our ability to address challenging scientific problems. Informatics and data management systems have also seen rapid development, and indeed for true high throughput they have become absolutely essential. Areas covered by these developments include: annotation of potential targets and target selection; construct and primer design; laboratory information management systems (LIMS) for tracking experimental work in the laboratory; managing setup and monitoring of crystallization trials; requesting and scheduling of resources such as synchrotron beam time; tracking storage and exchange of samples (such as shipping crystals to synchrotrons); controlling beam lines for both manual and automated data collection; data reduction and formulation of data collection strategies; techniques for automated structure solution, refinement, annotation and deposition; software for tracking progress of targets along the pipeline. The Oxford Protein Production Facility (OPPF) has been at the forefront of many of the developments in Europe, and through collaborative projects such as SPINE (www.spineurope.org), eHTPX (www.e-htpx.ac.uk) and PiMS (www.pimslims.org) it is endeavouring tomake these developments relevant to other structural biology groups. This presentation provides an overview (with a European perspective) of the current state of informatics developments for the first part of this pipeline as far as x-ray data collection by describing the informatics techniques developed by these collaborations and regularly employed at the OPPF. Areas that will be covered in more detail include target selection, the current state of development of the PiMS LIMS, crystallization and developments toward automated/remote synchrotron data collection. m03.o02

Advanced beamline automation for biological crystallography experiments

An automated crystal-mounting/alignment system has been developed at Lawrence Berkeley National Laboratory and has been installed on three of the protein-crystallography experimental stations at the Advanced Light Source (ALS); it is currently being implemented at synchrotron crystallography beamlines at CHESS, NSLS and the APS. The benefits to using an automounter system include (i) optimization of the use of synchrotron beam time, (ii) facilitation of advanced data-collection techniques, (iii) collection of higher quality data, (iv) reduction of the risk to crystals and (v) exploration of systematic studies of experimental protocols. Developments on the next-generation automounter with improvements in robustness, automated alignment and sample tracking are under way, with an end-to-end data-flow process being developed to allow remote data collection and monitoring.

X‐ray imaging in advanced studies of ophthalmic diseases

Microscopic characterization of pathological tissues has one major intrinsic limitation, the small sampling areas with respect to the extension of the tissues. Mapping possible changes on vast tissues and correlating them with large ensembles of clinical cases is not a feasible procedure for studying most diseases, as for instance vision loss related diseases and, in particular, the cataract. Although intraocular lens implants are successful treatments, cataract still is a leading public-health issue that grows in importance as the population increases and life expectancy is extended worldwide. In this work we have exploited the radiation-tissue interaction properties of hard x-rays--very low absorption and scattering--to map distinct lesions on entire eye lenses. At the used synchrotron x-ray photon energy of 20 keV (wavelength lambda=0.062 nm), scattering and refraction are angular resolved effects. It allows the employed x-ray image technique to efficiently characterize two types of lesions in eye lenses under cataractogenesis: distributions of tiny scattering centers and extended areas of fiber cell compaction. The data collection procedure is relatively fast; allowing dozens of samples to be totally imaged (scattering, refraction, and mass absorption images) in a single day of synchrotron beam time. More than 60 cases of canine cataract, not correlated to specific causes, were investigated in this first application of x-rays to image entire lenses. Cortical opacity cases, or partial opacity, could be related to the presence of calcificated tissues at the cortical areas, clearly visible in the images, whose elemental contents were verified by micro x-ray fluorescence as very rich in calcium. Calcificated tissues were also observed at nuclear areas in some cases of hypermature cataract. Total opacity cases without distinguishable amount of scattering centers consist in 70% of the analyzed cases, where remarkable fissure marks owing to extended areas of fiber cell compaction are diagnosed.