I describe a method for accurately refining the geometrical parameters of segmented X-ray area detectors on the basis of serial crystallography data, using 'Millepede' - an algorithm created for a very similar problem in high-energy physics. The Millepede method for serial crystallography builds on the approach of Brewster et al. [Acta Cryst. (2018), D74, 877-894], in which the detector parameters are refined simultaneously with the parameters for each individual crystal. This accounts for the mutual dependency between the parameters and thereby avoids the bias and slow convergence problems that have afflicted older approaches in which the deviations between observed and calculated Bragg peak positions were taken directly as the updates for the detector panel positions. The Millepede method uses the special structure of the least-squares normal equations to reduce them to a much smaller form that can be solved very quickly, even compared with the sparse matrix methods used previously. This makes it practical to refine the detector geometry frequently and thereby maintain accurate calibration without specialized alignment campaigns. Tilts of detector panels out of the plane can be reliably refined, as can the overall distance of the detector in the beam direction. With a simulated test case, the new method produced panel shifts within 7% of the correct values with only one iteration, and produced almost exactly correct shifts after a second iteration. A simulated out-of-plane panel rotation was correctly determined to within 0.001°. Applied to experimental data from an X-ray free-electron laser, the method increased the indexable fraction of frames from 30% to 91% in a single iteration, and to 96% after two further iterations. Computing the geometry updates on the basis of 2060 crystals took only 0.819 s on desktop computing hardware, including the time taken to read the required data from disk. The scaling was found to be very close to linear for up to 100 980 sets of crystal parameters, which took only 78.2 s to process under the same conditions. The method has been applied as part of a real-time feedback system at a synchrotron radiation beamline, in which an out-of-plane detector tilt of 0.04° was detected and corrected. Possible further applications are also described here.
Bragg edge neutron transmission analysis is a non-destructive technique that can be used for the investigation of properties of crystalline solids, such as microstructure, texture, strain or defects. In this work, Bragg edge imaging is applied to characterize additively manufactured metal samples produced via powder bed fusion-laser-based, featuring an innovative star-shaped geometry. This process can induce microstructural inhomogeneities within the material, thereby compromising the mechanical integrity of the final component. For this reason, a comprehensive understanding of the manufacturing process is essential to identify optimal operational parameters. Because of the lack of non-invasive techniques allowing an in-depth study of the microstructure of these samples, Bragg edge imaging is applied for providing detailed quantitative information on the manufacturing process. In this context, the final aim of this work is to investigate how the production process influences the final manufactured components. To study these effects, three different additively manufactured samples made of different metal alloys have been characterized by Bragg edge analysis. Characterization of elastic lattice strain, density of crystallographic defects and texture reveals significant discrepancies between the samples and their respective starting powders. These findings elucidate the various effects induced by the manufacturing process, which alters the crystalline structure of the metal and introduces anisotropy, potentially leading to mechanical failure of the components.
Grazing-incidence small-angle X-ray scattering (GISAXS) is a technique of choice for providing information about the morphology of nano- and micro-structures at surfaces and interfaces, also in real time. The geometry of the sample, in particular its curvature, has an impact on the observed X-ray scattering signal. There are a multitude of systems with sophisticated geometries (including curvature), ranging from electronic devices on flexible substrates to biological membranes, for which GISAXS could provide valuable information. Therefore, in this work the effect of the sample geometry on the GISAXS signal is addressed. More specifically the influence of the substrate curvature and extent along the X-ray beam is considered. The analytical expressions accounting for the effects of those two geometrical parameters are provided, and the way to include them in the analysis of GISAXS patterns is described. The calculations reveal that no corrections are needed for small samples (length over distance to the detector ratio smaller than 1%) and radius of curvature |R| > 50 m. These results allow for a combination of GISAXS with substrate curvature measurements. The latter technique is a non-destructive in situ and real-time method providing information about the intrinsic stress in a thin film during its growth. Morphological information from GISAXS is supposed to complement this stress information. Herein this methodology is applied to the growth of Ag thin films deposited by magnetron sputtering with N2 plasma additive. The analysis of the GISAXS pattern obtained from the sample, which bends during the deposition, provided morphological parameters of the growing film. This methodology can be useful for understanding of the mechanisms at the nanoscale leading to the observed stress state. The ability to perform GISAXS on curved substrates enables its application to more complex systems.
Multipolar scattering models, such as the transferable aspherical atom model, account for atomic chemical interactions and provide a more accurate representation of experimental data. However, the simpler independent atom model (IAM), which assumes non-interacting atoms, is the only model available in the most widely used macromolecular refinement programs. This is primarily because IAM offers a hard-to-beat combination of computational efficiency and modelling power at typical macromolecular resolutions. By contrast, more accurate multipolar modelling has historically been limited due to its computational cost and the absence of an interface between software capable of calculating structure factors and gradients based on multipolar models and software designed for macromolecular refinement. This work introduces pyDiSCaMB, a Python software package designed to integrate between the computational crystallography toolbox (cctbx) and the quantum crystallography library DiSCaMB (Densities in Structural Chemistry and Molecular Biology), thus enabling multipolar scattering models in Phenix's toolkit. The implementation, features and capabilities of pyDiSCaMB are presented, the runtimes for the calculation of structure factor and target gradients with respect to atomic parameters are explored, and Fourier images of electrostatic potential, electron density and deformation maps are computed as illustrative examples. The pyDiSCaMB library will make multipolar modelling widely available to the structural biology community, potentially transforming refinement and model-building for both crystallography and cryogenic electron microscopy (cryoEM).
Understanding the interactions between microstructure, strain, phase and material behavior is crucial in scientific fields such as energy storage, carbon sequestration and biomedical engineering. However, quantifying these correlations is challenging, as it requires the use of multiple instruments and techniques, often separated by space and time. The Dual Imaging and Diffraction (DIAD) beamline at Diamond Light Source is designed to address this challenge. DIAD allows its users to visualize internal structures (in two and three dimensions), identify compositional/phase changes and measure strain. It enables in situ and operando experiments that require spatially correlated information. DIAD provides two independent beams combined at one sample position, allowing 'quasi-simultaneous' X-ray computed tomography and X-ray powder diffraction. A unique functionality of the DIAD configuration is the ability to perform 'image-guided diffraction', where the micrometre-sized diffraction beam is scanned over the complete area of the imaging field of view without moving the specimen. This moving-beam diffraction geometry enables the study of fast-evolving and motion-susceptible processes and samples. Here, we discuss the novel moving-beam diffraction geometry, presenting the latest findings on the reliability of both the geometry calibration and the data-reduction routines used. We provide a comprehensive quantitative assessment of the moving-beam diffraction geometry implemented at the DIAD beamline, which will serve as a reference for beamline users. Our measurements confirm that diffraction is most sensitive to the moving-beam geometry for the conventional transmission geometry of the detector. The observed data confirm that the motion of the Kirkpatrick-Baez mirror coupled with a fixed-aperture slit results in a rigid translation of the beam probe, without affecting the angle of the incident-beam path to the sample. Our measurements demonstrate that a nearest-neighbor calibration can achieve the same accuracy as a self-calibrated geometry when the distance between the calibrated and probed sample regions is smaller than or equal to the beam spot size. The absolute error of the moving-beam diffraction geometry at DIAD with typical calibration setup remains below 0.01%, which is the accuracy we observe for the beamline with stable beam operation.
We present LamelODF, a MATLAB-based software platform for automated extraction and mapping of axisymmetric orientation distribution functions (ODFs) from 2D X-ray diffraction patterns of lamellar minerals. Building on a maximum-entropy-method-derived ODF specifically for clay mineral systems, the software provides a streamlined workflow for texture analysis under the assumption of transverse isotropy. The program features dedicated file converters for both laboratory and synchrotron data formats, batch processing capabilities, and flexible background-correction algorithms. The analytical pipeline performs azimuthal intensity integration with background subtraction, followed by non-linear fitting to extract quantitative orientation parameters including the 〈P 2〉 order parameter, the deviation angle δ between the main orientation of lamellar particles and the detector reference, and integrated intensities, focusing specifically on basal 001 reflections to enable rapid processing of thousands of diffraction patterns for spatial mapping applications. Mapping of a laboratory-prepared porous clay medium and a natural soil sample demonstrates the software's ability to detect density stratification, sedimentation discontinuities and complex geological structures such as relict topsoil crusts. The software successfully discriminates between different clay mineral phases, providing a practical complementary tool for researchers investigating the organization of lamellar minerals in natural and engineered materials.
Ultrathin Fe3O4 films were grown on SrTiO3(001) substrates under systematically varied growth conditions (deposition rate and temperature, and film thickness) in order to determine the influence of these parameters on the formation of the coexisting (111) and (001) orientations of Fe3O4. Structural characterization was performed using grazing-incidence X-ray diffraction and high-energy X-ray diffraction, including mapping of the reciprocal space in plane and out of plane to obtain detailed information about the structure of the films in the lateral and vertical directions. Despite an expected cube-on-cube growth, an intermediate layer of (111)-oriented Fe3O4 with hexagonal surface symmetry formed in all samples examined. This intermediate phase occurs in the form of 3D islands during the initial growth phase and persists even after growth has transformed to preferential formation of the (001) orientation. The fraction of the (111) phase, on the other hand, depends heavily on the kinetic conditions during growth. Its formation is favoured by low deposition rates and, above all, high deposition temperatures. These results support earlier observations and provide new insights into the structural development of Fe3O4 films on SrTiO3(001) in the early stages.
Supported lipid bilayers (SLBs) are crucial model membrane platforms to study the structure and dynamics of cellular membranes. Vesicle fusion (VF) is one of the most widely used approaches to forming SLBs, though it suffers from compositional limitations and substrate compatibility constraints. The solvent-assisted lipid bilayer (SALB) technique enables the possibility of forming SLBs using a wider range of membrane compositions and substrate platforms through organic-solvent-mediated bilayer assembly, yet questions remain regarding structural equivalence and potential organic solvent incorporation effects. Using neutron reflectometry (NR), we systematically compare the structure and composition of phosphatidylcholine-based SLBs formed by either VF or SALB methodologies. SALB conditions were optimized for NR solid/liquid cells, and structural characterization revealed comparable bilayer architectures between the two formation methods, although some changes in the lipid acyl chain thickness were observed. SALBs showed up to 99.2 ± 0.9% surface coverage using ultrapure water for solvent exchange, but the reproducibility of the method was poor. Enhanced-contrast NR using either deuterated lipids or solvents allowed for the quantitative detection of residual organic solvent incorporation of the SALBs, which was up to 3.3 ± 0.9 vol.% in the tail regions. Making use of 1 mM CaCl2 during solvent exchange substantially improved SALB reproducibility, reducing coverage variability from 21-30 to 2  vol.%. Validation studies using the antimicrobial peptide melittin demonstrated that membrane-peptide interactions proceeded according to established mechanisms, with peptide incorporation of 18 vol.% for the low-coverage (69.7 ± 0.8%) SALB. The quantified solvent incorporation levels and small changes in acyl chain layer thickness in the SALBs must be considered when interpreting protein-membrane interaction studies, which suggests that validation of the SALB methodology for membrane research applications requires assessment on a case by case basis.
Mixed-halide perovskites (MHPs) offer good band-gap tunability via stoichiometry changes, and such tunability is an essential property for the creation of multijunction solar cells. However, under illumination halide ions in MHPs segregate and create I- and Br-rich regions, which decrease the efficiency of potential solar cells. In this work, a method for a detailed investigation of the distribution of halide ions within an MHP during and after illumination is introduced. Calculations of the strain field created by the halide segregation were performed, and the obtained local displacement of atoms was used to calculate the X-ray diffuse scattering. By fitting the experimental data measured on a thin polycrystalline layer of FA0.83Cs0.17Pb(I0.6Br0.4)3 (where FA stands for formamidinium), the distribution of Br- and I- ions within an illuminated MHP was determined and the subsequent relaxation process of the segregation in the dark was tracked. The creation of highly Br-rich regions within a slightly I-rich volume during illumination was observed.
This work reports the development and analytical validation of FACILE 2.0, a portable multifunctional potentiostat designed for electrochemical measurements under both static and microfluidic configurations. The platform integrates a compact electrochemical interface, embedded single-board computer, touchscreen control, and programmable flow handling, enabling autonomous execution of cyclic voltammetry, square wave voltammetry, chronoamperometry, and electrochemical impedance spectroscopy. Analytical performance was first validated using benchmark reversible redox systems and electroactive species exhibiting different electron-transfer kinetics. Under static conditions, calibration studies demonstrated high linearity (R2 up to 0.999), repeatability of 8% (RSD), and a limit of detection of 0.7 μM for representative redox probes, values comparable to commercial benchtop and portable potentiostats. Correlation analyses confirmed strong agreement in current response and extracted electrochemical parameters across different techniques. Measurements performed in fluidic mode further demonstrate stable and reproducible responses under controlled flow conditions. Following electrochemical validation, FACILE 2.0, was applied as a representative case study to a label-free immunosensor for the detection of Pseudomonas aeruginosa in untreated tap water. Using square wave voltammetry, the system achieved a limit of detection of 1000 CFU/mL (RSD <10%, n = 5), with high specificity against non-target microorganisms (<10% interference). With a compact footprint (W32 x D18 x H5 cm), low power consumption (<15W maximum), and complete portability (1.93 kg), FACILE 2.0 provides a robust, scalable and field-deployable solution for a broad range of electrochemical applications, including environmental monitoring, industrial quality control, materials research, teaching laboratories, and decentralized biosensing applications.
Quantum refinement (QR) is an approach in which the empirical restraints used in standard structural refinement to ensure that the details of the structure, e.g. bond lengths and angles, make chemical sense are replaced by more accurate quantum mechanical calculations for a small but interesting part of the structure. QR has previously been used for X-ray and neutron crystallography, cryogenic electron microscopy, nuclear magnetic resonance, and extended X-ray absorption fine structure. Here, QR is used for the first time for X-ray free-electron laser (XFEL) crystallography and microcrystal electron diffraction (MicroED). As a test case, we use six structures of the R2a protein of ribonucleotide reductase, concentrating on the binuclear Fe2 site in either the oxidized (Fe2 III) or reduced (Fe2 II) state, two each from single-crystal X-ray (SCX) crystallography, XFEL crystallography or MicroED. The results show that QR works well for data from all three radiation sources, even though scattering factors for neutral atoms had to be used for MicroED. QR corrects unrealistically short Fe-O distances in the reduced SCX structure and gives improved real-space Z scores for the reduced MicroED structure. The three methods give similar structures, apart from variation in the weak water ligands and in the binding of carboxylate groups (monodentate, bidentate or a mixture). By performing QR for three protonation states of the bridging solvent molecule, we could show that it is undoubtedly a water molecule in the reduced XFEL and MicroED structures (it is not present in the SCX structure) and that it is not water in the oxidized structures. The XFEL data indicate that it is O2- in the oxidized XFEL structure, in agreement with the spectroscopic results. However, for the SCX structure, O2- and OH- give comparable results, whereas OH- is slightly preferred in the MicroED structure. This indicates that the SCX and MicroED structures may be partly photoreduced during data collection.
Residual stress analysis in thin films and nanomultilayers (NMLs) is essential for understanding their mechanical, thermal and functional properties. However, accurate strain measurements in highly textured multilayers obtained by physical vapor deposition techniques can be experimentally challenging, particularly when strong epitaxial relationships and twinning are present. In this paper, texture and residual stresses were investigated for a representative NML system composed of immiscible face-centered cubic (fcc)/body-centered cubic (bcc) elemental layers, namely Cu/Mo NMLs grown on a sapphire (0001) substrate. The experimentally investigated Cu/Mo NML system exhibits a specific fcc Cu {111}||bcc Mo {110}||sapphire (0001) in-plane and out-of-plane texture with growth twinning of Cu and a Nishiyama-Wasserman orientation relationship between Cu and Mo. This combination results in an effective sixfold rotational symmetry in the pole figures. The presence of twinning significantly complicates residual stress determination, as the selection of inappropriate diffraction planes can lead to erroneous results and the failure of standard approaches like the crystallite group method. To address this issue, we have established a theoretical framework and practical methodology to select suitable diffraction planes for the reliable determination of residual stresses in fcc/bcc NMLs with strong in-plane and out-of-plane textures, including the presence of twinning, using X-ray diffraction. The experimental X-ray-diffraction-based analysis is complemented by high-resolution scanning transmission electron microscopy and orientation mapping, providing a comprehensive understanding of the microstructure and crystallographic texture. The presented method can be applied to other multilayer systems and extended to other orientation relationships.
Fixed-target platforms provide convenient support for microcrystals during serial X-ray crystallography studies using synchrotron radiation. Here, we describe a simple user-friendly 3D-printed support where the crystals are sandwiched between two layers of thin X-ray-transparent membrane resulting in very low scattering background. The platform is compatible with magnetic mounting onto the standard goniometer of macromolecular crystallography beamlines. Our design utilizes a 96-well frame that facilitates hanging-drop experiments directly on the membrane using conventional crystallization plates, thereby eliminating multiple pipetting and crystal handling steps. Crystals can be enclosed in a sandwich and packed into 'cassettes', preventing the risk of the sample drying out during room-temperature transportation to synchrotron sources. The versatility of the platform is demonstrated by five structures solved using different crystallization and data-collection strategies. Lysozyme single-crystal rotational crystallography at room temperature is shown, as well as microcrystal serial data collection under cryogenic conditions. On-chip microcrystallization is illustrated by use of a photosynthetic reaction center as an example. Finally, serial crystallography data collection at room temperature from microcrystals of the membrane protein cytochrome c oxidase crystallized in lipidic cubic phase is presented.
We have elucidated the polymer adsorption layer structure in filler-rubber systems by conducting spin-contrast-variation small-angle neutron scattering (SANS) on partially and fully swollen filler-rubber samples with and without a silane coupling agent. In spin-contrast-variation SANS, dynamic nuclear polarization (DNP) was used to polarize protons and change their scattering length with respect to polarized neutron beams significantly. SANS measurements were performed in dynamically polarized states using a DNP cryostat (1.2 K and 3.35 T). From SANS profiles obtained at various proton spin polarizations, partial scattering functions (PSFs) for each component were separated by regarding each sample as a three-component system composed of silica, polymer and deuterated toluene. To analyze the obtained PSFs in detail, we built a structure model for the silica aggregates and the surrounding polymer adsorption layer. Numerical calculation based on this model successfully reproduced the experimentally obtained PSFs, providing the structural parameters of the silica aggregates and polymer adsorption layer. The results showed a considerable difference in structural parameters between the partially and fully swollen states. For the sample with the silane coupling agent, the thickness of the polymer adsorption layer decreased as the solvent fraction increased. The difference in polymer volume fraction between the polymer adsorption layer and the outside matrix was very small in less swollen states but significant in the fully swollen state. Furthermore, the scattering contribution of the polymer chains in the solvent was accurately separated via contrast variation. In the swollen silica-filled rubber without the silane coupling agent, the size of the polymer-dense regions was almost constant, regardless of the swelling ratio. By contrast, in the swollen silica-filled rubber with the silane coupling agent, the size of the polymer-dense regions significantly increased by a factor of 2 with an increase in the swelling ratio.
The mean value of weighted residuals (〈ζ〉) was analysed for 8424 published single-crystal X-ray data sets of crystals containing only light elements (C, H, N, O). A striking asymmetry was observed: 71.5% of data sets exhibit positive 〈ζ〉 values, occurring 2.5 times more often than negative values. This imbalance suggests systematic errors, with evidence pointing to a slight overestimation of observed intensities (I obs). Simulations and theoretical analysis show that such overestimation artificially lowers common data-quality metrics, including the popular merging factor R merge, the redundancy independent factor R r.i.m., the precision indicating factor R p.i.m., the weighted agreement factor wR(F 2) and even atomic displacement parameters, creating a 'rewarding error' that may reinforce confirmation bias. Experimental data confirm these findings, as residual factors reach their minima for 〈ζ〉 > 0 rather than at zero. These results highlight the need for critical evaluation of data-processing strategies and caution against relying solely on conventional agreement factors as indicators of accuracy.
Neutron reflectometry (NR) at the ISIS Neutron and Muon Source has evolved into a mature, versatile technique for investigating the structure and dynamics of interfaces across a wide range of scientific disciplines, including soft matter, magnetism, quantum materials and environmental systems. This article provides a comprehensive overview of the current instrumentation available at ISIS, detailing the capabilities of the four operational reflectometers - OFFSPEC, INTER, POLREF and SURF - including their time-of-flight configurations, scattering geometries, polarization options and range of accessible sample environments. Special emphasis is placed on the integration of advanced sample environments, automated control software and data analysis tools, including IBEX, MANTID and the ISIS Data Analysis as a Service (IDAaaS) platform, which collectively facilitate efficient, reproducible and high-quality NR measurements. Representative scientific highlights demonstrate the unique potential of NR to resolve sub-nanometre structural and kinetic information in biological membranes, thin functional films, quantum fluids and environmental interfaces. By consolidating detailed technical information, operational characteristics and examples of cutting-edge research, this article serves as a practical guide for new and experienced users, helping them design experiments, select suitable instruments and sample environments, and fully exploit the capabilities of ISIS neutron reflectometers.
We have developed a compact tape drive (CoT) with on-demand sample delivery for time-resolved serial femtosecond crystallography (SFX) experiments, which can deliver sample droplets and/or initiate reactions with a drop-on-drop strategy. Two disposable piezoelectric injectors are positioned in tandem along the tape to produce a queue of nanolitre-scale droplets. X-ray free-electron laser pulses arrive perpendicular to and pass through the broad face of the tape. The pulse is synchronized and aligned to the droplets, thereby enabling highly efficient SFX data collection. The tape transport speed and the delivery distance can be varied to control the mixing time from approximately 130 ms to tens of seconds. We conducted time-resolved SFX experiments utilizing a basic enzymatic reaction model of hen egg white lysozyme (HEWL) and N-acetyl-D-glucosamine (GlcNAc) to demonstrate the drop-on-drop capabilities of the CoT, and the full binding process of GlcNAc to HEWL was observed at 1.3-9.7 s.
Contrast variation (CV) small-angle neutron scattering (SANS) enables the determination of structural parameters from individual components in multi-component biological complexes in solution. Researchers can selectively highlight different parts of a multi-component system by varying the contrast between the components, typically through adjusting the deuteration level of solvents or specific molecular constituents. While this approach provides unique insights into component shapes, spatial arrangements and interactions, SANS CV experiments require substantial resources, specialized facilities and, often, a steep learning curve. To address these challenges and improve the likelihood of a successful experiment, a new contrast software suite has been developed within the SASSIE-web framework, allowing researchers to plan and simulate experiments in silico, integrate structural modeling, and streamline both experiment design and CV data analysis into a unified platform.
The small-angle scattering form factors of two classes of composite particles with contrasting internal architectures have been studied: one consisting of inclusions of smaller spheres embedded within a larger sphere, and the other comprising a solid sphere with randomly distributed spherical voids. These systems serve as material- and void-based analogues, providing a model framework for examining how internal material distribution in porous particles influences scattering signatures. Monte Carlo simulations were used to generate scattering curves across a range of volume fractions and polydispersities, which were then employed to benchmark analytically derived form factor expressions. Steric repulsion between, respectively, spheres and voids was taken as hard-sphere interactions. The results reveal that internal structural asymmetries, especially in spatial correlations and contrast topology, significantly affect scattering patterns, despite the particles having similar overall structures and volume fractions. In particular, spheres-of-spheres structures exhibit features in the scattering signal from internal modulations, while void-based particles display smoother shell-like scattering features. The analytical models show excellent agreement with the simulated data, capturing both the global shape and fine structural characteristics. These findings demonstrate that relatively simple analytical approaches, validated against numerical simulations, can reliably describe complex heterogeneous particles. This methodology provides a robust basis for interpreting scattering data from porous and composite materials across a wide range of applications.
We present NuMagSANS, a GPU-accelerated software package for calculating nuclear and magnetic small-angle neutron scattering (SANS) cross sections and correlation functions. The program allows users to import position-dependent nuclear density and magnetization data, providing significant flexibility for analyzing the scattering signatures of complex systems, particularly magnetic materials. Full rotational control of the sample is supported, allowing a comprehensive exploration of angle-dependent scattering features. NuMagSANS includes a versatile library of approximately 100 response functions that encompass two-dimensional SANS cross sections, correlation functions and azimuthally averaged quantities. These capabilities allow users to gain detailed insight into the structural and magnetic characteristics of their samples. GPU acceleration ensures rapid computations, even for large data sets, making NuMagSANS a powerful and efficient tool for advanced SANS analysis.