We present the development of a piezo-actuated shutter system designed for high-power broadband X-ray beams at fourth-generation synchrotron sources. The device combines a flexure-based mechanical design with efficient water cooling, achieving full open–close transitions in 2 ms while reliably withstanding continuous thermal loads exceeding 20 W. This performance enables safe operation with multilayer monochromators, which introduce higher heat loads than conventional optics such as Si(111) monochromators. The shutter provides an open aperture of 1.9 mm, is fully ultrahigh vacuum compatible, and was validated through mechanical and thermal characterization under realistic operating conditions. Its millisecond-scale actuation allows precise blocking of the beam during idle phases such as sample alignment or repositioning, thereby minimizing radiation damage and maximizing the effective use of the delivered photon flux. This makes the system particularly well suited for high-throughput scanning and imaging techniques, including ptychography, tomography, scanning small-angle X-ray scattering and protein crystallography, on modern synchrotron beamlines.

Dynamic processes such as crystallization, sintering and phase separation play pivotal roles in defining the structure and performance of engineered and natural materials. Yet, these phenomena are often challenging to study because they are transient, spatially heterogeneous, and span multiple length and time scales. Visualizing them in three dimensions under realistic conditions therefore requires imaging techniques capable of probing representative sample volumes with nanoscale resolution and minute-scale temporal resolution, sustained over extended observation times and across a wide range of environmental conditions, capabilities that current in situ methods rarely combine. Here, we present an integrated platform for in situ time- and temperature-resolved ptychographic X-ray nanotomography that meets these demands, and demonstrate its capability by tracking the crystallization of amorphous calcium carbonate from room temperature to 500°C. Quantitative tomograms are acquired at five-minute intervals, producing a 4D dataset that reveals multiple simultaneous crystallization pathways, and rare and transient events. Among these is the formation and recrystallization of a metastable polymorph, calcium carbonate hemihydrate, which has previously only been observed in additive-stabilized systems. We also demonstrate how volume defect evolution and structural rearrangements within individual crystals contribute to the mechanisms underlying Ostwald ripening. This platform offers a general method for in situ visualization of material transformations, providing insights into the processes that govern material structure and functionality.

Nanoparticles supported on the surface of porous carrier materials are the dominant form of heterogeneous catalysts today. Yet, they suffer from a common deactivation mechanism: the loss of active surface area under industrial use conditions. Deactivation often stems from the sintering of nanoparticles, a mass transport process whose mechanism and operating length scale are a topic of controversy. Investigating this process is challenging, requiring not only a behavioural characterisation of thousands of individual particles within the spatial confines of a hierarchically structured support but also a characterisation of their ensemble behaviour and local support interactions. Here, we introduce in-situ ptychographic X-ray computed nanotomography as a tool to facilitate this characterisation, allowing a local examination of catalysts in their use-geometry under operational-relevant conditions. Applied to methane oxidation over a palladium-on-silica supported catalyst, we reveal two concurrently operating deactivation drivers, short-range ripening and long-range particle migration, each with different temperature and atmosphere dependencies. The latter enables particles to traverse hundreds of nanometres through the support. These observations expand the current understanding of sintering behaviour in supported catalysts and demonstrate PXCT’s capability to resolve restructuring processes within complex porous materials.

Phase retrieval is an inverse problem that, on one hand, is crucial in many applications across imaging and physics, and, on the other hand, leads to deep research questions in theoretical signal processing and applied harmonic analysis. This survey paper is an outcome of the recent workshop Phase Retrieval in Mathematics and Applications (PRiMA) (held on August 5–9 2024 at the Lorentz Center in Leiden, The Netherlands) that brought together experts working on theoretical and practical aspects of the phase retrieval problem with the purpose to formulate and explore essential open problems in the field.

The link between the structural organization of the fibrillar components of lung extracellular matrix (ECM), local tissue stiffness and global viscoelastic behaviour is not known. Here we investigated the effect of injurious mechanical ventilation on the local lung tissue stiffness using 4D synchrotron phase-contrast micro-CT, in normal lung and 7 days after intratracheal bleomycin induced lung injury in anesthetized rats. Quantitative maps of local lung strain (ε) were computed within aerated lung acini, using a stepwise image registration method. Fibrillar organization of collagen and elastin at the nanoscale was measured using synchrotron small-angle x-ray scattering (SAXS). Local microscopic tissue ε was reduced in the aerated acini of normal lungs post injurious ventilation and in bleomycin-injured lungs and was associated with an increase in dynamic elastance (H). The scattering peak angle (q) which is inversely related to fibril D-spacing, was decreased by injurious ventilation indicating an elongation of the collagen fibril spacing in both normal and bleomycin-injured lung. There was a positive relationship between collagen periodicity and global tissue elastance, while an inverse relation was observed with tissue hysteresis. Our data demonstrate the effect of both bleomycin-induced lung injury and high-strain mechanical ventilation on the nanoscale fibrillar organization of collagen and for the first time, a link between collagen D-spacing and global lung tissue stiffening and viscoelastic behaviour.

Maps of biological tissues at subcellular detail are key for understanding how organs function. X-ray nanotomography is a promising alternative to volume electron microscopy: it has the potential to nondestructively image millimeter-sized samples at ultrastructural resolution within a few days. A fundamental barrier is that the intense X-rays required for imaging also deform and disintegrate the tissue samples. Here we show a combination of solutions that overcome this barrier: We used a cryogenic and stable sample stage, tailored nonrigid tomographic reconstruction algorithms and an epoxy resin developed for the nuclear and aerospace industry. Tissue samples were resistant to radiation doses exceeding 1.15 × 1010 Gy, and sub-40 nm isotropic resolution allowed identifying axon bundles, dendrites and synapses in mouse brain tissue without physical sectioning. Using volume electron microscopy, we demonstrate that tissue ultrastructure remains intact after X-ray imaging. Together, this unlocks the potential of X-ray tomography for high-resolution tissue imaging. Optimizations of X-ray nanotomography including the choice of resin allows high-resolution imaging of mouse brain tissue, approaching the resolution of volume electron microscopy. Since it does not require slicing the tissue, this technology may become an attractive alternative to current standard methods for connectomics.

Hard X-ray nanotomography is a promising technology for nondestructive imaging of biological tissues with three-dimensional isotropic resolution. The implementation of fourth-generation synchrotron sources brings coherent X-ray microscopy to the central stage and fosters further development of this class of techniques. Here, we present an experimental comparison of X-ray near-field ptychography and X-ray holography, two high-resolution X-ray microscopy techniques applied under cryogenic conditions to the exact same sample at two different synchrotron sources. Using a heavy-metal-stained, resin-embedded brain tissue sample, we obtain similar contrast and spatial resolutions at equivalent radiation doses with these two approaches. We discuss the current benefits and limitations of the two methods. These results provide a basis for developments in X-ray microscopy of biological samples at present and future beamlines of fourth-generation synchrotron sources.

Microtubule plus-end tracking proteins (+TIPs) participate in nearly all microtubule-based cellular processes and have recently been proposed to function as liquid condensates. However, their formation and internal organization remain poorly understood. Here, we have study the phase separation of Bik1, a CLIP-170 family member and key +TIP involved in budding yeast cell division. Bik1 is a dimer with a rod-shaped conformation primarily defined by its central coiled-coil domain. Its liquid condensation likely involves the formation of higher-order oligomers that phase separate in a manner dependent on the protein's N-terminal CAP-Gly domain and C-terminal EEY/F-like motif. This process is accompanied by conformational rearrangements in Bik1, leading to at least a two-fold increase in multivalent interactions between its folded and disordered domains. Unlike classical liquids, Bik1 condensates exhibit a heterogeneous, fractal supramolecular structure with protein- and solvent-rich regions. This structural evidence supports recent percolation-based models of biomolecular condensates. Together, our findings offer insights into the structure, dynamic rearrangement, and organization of a complex, oligomeric, and multidomain protein in both dilute and condensed states. Our experimental framework can be applied to other biomolecular condensates, including more complex +TIP networks.

The coacervation and structural rearrangement of the protein alpha-synuclein (αSyn) into cytotoxic oligomers and amyloid fibrils are considered pathological hallmarks of Parkinson's disease. While aggregation is recognized as the key element of amyloid diseases, liquid-liquid phase separation (LLPS) and its interplay with aggregation have gained increasing interest. Previous work showed that factors promoting or inhibiting amyloid formation have similar effects on phase separation. Here, we provide a detailed scanning of a wide range of parameters including protein, salt and crowding concentrations at multiple pH values, revealing different salt dependencies of aggregation and phase separation. The influence of salt on aggregation under crowded conditions follows a non-monotonic pattern, showing increased effects at medium salt concentrations. This behavior can be elucidated through a combination of electrostatic screening and salting-out effects on the intramolecular interactions between the N-terminal and C-terminal regions of αSyn. By contrast, we find a monotonic salt dependence of phase separation due to the intermolecular interaction. Furthermore, we observe the time evolution of the two distinct assembly states, with macroscopic fibrillar-like bundles initially forming at medium salt concentration but subsequently converting into droplets after prolonged incubation. The droplet state is therefore capable of inhibiting aggregation or even dissolving the aggregates through a variety of heterotypic interactions, thus preventing αSyn from its dynamically arrested state.

Small-angle X-ray tensor tomography and the related wide-angle X-ray tensor tomography are X-ray imaging techniques that tomographically reconstruct the anisotropic scattering density of extended samples. In previous studies, these methods have been used to image samples where the scattering density depends slowly on the direction of scattering, typically modeling the directionality, i.e. the texture, with a spherical harmonics expansion up until order ℓ = 8 or lower. This study investigates the performance of several established algorithms from small-angle X-ray tensor tomography on samples with a faster variation as a function of scattering direction and compares their expected and achieved performance. The various algorithms are tested using wide-angle scattering data from an as-drawn steel wire with known texture to establish the viability of the tensor tomography approach for such samples and to compare the performance of existing algorithms.

A correlative, multiscale imaging methodology for visualising and quantifying the morphology of solid dosage forms by combining ptychographic X-ray computed nanotomography (PXCT) and scanning small- and wide-angle X-ray scattering (S/WAXS) is presented. The methodology presents a workflow for multiscale analysis, where structures are characterised from the nanometre to millimetre regime. Here, the method is demonstrated by characterising a hot-melt extruded, partly crystalline, solid dispersion of carbamazepine in ethyl cellulose. Characterisation of the morphology and solid-state phase of the drug in solid dosage forms is central as this affects the performance of the final formulation. The 3D morphology was visualised at a resolution of 80 nm over an extended volume through PXCT, revealing an oriented structure of crystalline drug domains aligned in the direction of extrusion. Scanning S/WAXS showed that the nanostructure is similar over the cross section of the extruded filament, with minor radial changes in domain sizes and degree of orientation. The polymorphic forms of carbamazepine were qualified with WAXS, showing a heterogeneous distribution of the metastable forms I and II. This demonstrates the methodology for multiscale structural characterization and imaging to enable a better understanding of the relationships between morphology, performance, and processing conditions of solid dosage forms.

Wiring diagrams of neural circuits are of central importance in delineating mechanisms of computation in the brain ([Lichtman and Sanes, 2008][1]; [Litwin-Kumar and Turaga, 2019][2]). To generate these diagrams, the individual parts of neurons - axons, dendrites and synapses - must be densely identified in 3-dimensional volumes of neuronal tissue. This is typically achieved by electron microscopy ([Kornfeld and Denk, 2018][3]), necessitating physical sectioning of the specimen either before or during the image acquisition process using ultrathin sectioning techniques or gallium or gas cluster ion beams ([Denk and Horstmann, 2004][4]; [Hayworth et al., 2020][5]; [Kasthuri et al., 2015][6]; [Xu et al., 2017][7]). Here, we demonstrate that X-ray ptychography ([Pfeiffer, 2018][8]), a coherent diffractive X-ray imaging technique, can faithfully acquire 3-dimensional images of metal-stained mouse neuronal tissue. Achieving high imaging quality requires minimization of the radiation damage to the sample, which we achieve by imaging at cryogenic temperatures and using specialised tomographic reconstruction algorithms ([Odstrcil et al., 2019b][9]). Using a newly identified tri-functional epoxy resin we demonstrate radiation resistance to X-ray doses exceeding 1011 Gy. Sub-40 nm resolution makes it possible to densely resolve axon bundles, boutons, dendrites, and synapses without physical sectioning. Moreover, the tissue volumes can subsequently be imaged in 3D using high-resolution focused ion beam scanning electron microscopy (FIB-SEM) ([Heymann et al., 2006][10]; [Knott et al., 2008][11]) showing intact ultrastructure, suggesting that metal-stained neuronal tissue can be highly radiation-stable. Ongoing improvements in synchrotron, X-ray and detector physics ([Yabashi and Tanaka, 2017][12]), as well as further optimization of sample preparation and staining procedures ([Hua et al., 2015][13]; [Karlupia et al., 2023][14]; [Lu et al., 2023][15]; [Mikula and Denk, 2015][16]; [Pallotto et al., 2015][17]; [Song et al., 2022][18]), could lead to substantial improvements in acquisition speed ([Du et al., 2021][19]), whilst widening the volumes that can be imaged with X-ray techniques using laminography ([Helfen et al., 2005][20]; [Helfen et al., 2013][21]; [Holler et al., 2020b][22]; [Holler et al., 2019][23]) and nano-holotomography ([Cloetens et al., 1999][24]; [Kuan et al., 2020][25]) could allow for non-destructive X-ray imaging of synapses and neural circuits contained in volumes of increasing size.### Competing Interest StatementThe authors have declared no competing interest. [1]: #ref-59 [2]: #ref-60 [3]: #ref-55 [4]: #ref-13 [5]: #ref-25 [6]: #ref-50 [7]: #ref-92 [8]: #ref-73 [9]: #ref-69 [10]: #ref-31 [11]: #ref-54 [12]: #ref-93 [13]: #ref-42 [14]: #ref-49 [15]: #ref-61 [16]: #ref-63 [17]: #ref-71 [18]: #ref-82 [19]: #ref-15 [20]: #ref-27 [21]: #ref-28 [22]: #ref-34 [23]: #ref-35 [24]: #ref-12 [25]: #ref-56

The performance of functional materials is either driven or limited by nanoscopic heterogeneities distributed throughout the material's volume. To better our understanding of these materials, we need characterization tools that allow us to determine the nature and distribution of these heterogeneities in their native geometry in 3D. Here, we introduce a method based on x-ray near-edge spectroscopy, ptychographic x-ray computed nanotomography, and sparsity techniques. The method allows the acquisition of quantitative multimodal tomograms of representative sample volumes at sub-30 nm half-period spatial resolution within practical acquisition times, which enables local structure refinements in complex geometries. To demonstrate the method's capabilities, we investigated the transformation of vanadium phosphorus oxide catalysts with industrial use. We observe changes from the micrometer to the atomic level and the formation of a location-specific defect so far only theorized. These results led to a reevaluation of these catalysts used in the production of plastics.

Strain and defects in crystalline materials are responsible for the distinct mechanical, electric and magnetic properties of a desired material, making their study an essential task in material characterization, fabrication and design. Existing techniques for the visualization of strain fields, such as transmission electron microscopy and diffraction, are destructive and limited to thin slices of the materials. On the other hand, non-destructive X-ray imaging methods either have a reduced resolution or are not robust enough for a broad range of applications. Here we present X-ray ptychographic topography, a new method for strain imaging, and demonstrate its use on an InSb micro-pillar after micro-compression, where the strained region is visualized with a spatial resolution of 30 nm. Thereby, X-ray ptychographic topography proves itself as a robust non-destructive approach for the imaging of strain fields within bulk crystalline specimens with a spatial resolution of a few tens of nanometers.

Ptychographic hard X-ray computed tomography (PXCT) is a recent method allowing imaging with quantitative electron-density contrast. Here, we imaged, at cryogenic temperature and without sectioning, cellular and subcellular structures of a chemically fixed and stained wild-type mouse retina, including axons and synapses, with complete isotropic 3D information over tens of microns. Comparison with tomograms of degenerative retina from a mouse model of retinitis pigmentosa illustrates the potential of this method for analyzing disease processes like neurodegeneration at sub-200 nm resolution. As a non-destructive imaging method, PXCT is very suitable for correlative imaging. Within the outer plexiform layer containing the photoreceptor synapses, we identified somatic synapses. We used a small region inside the X-ray-imaged sample for further high-resolution focused ion beam/scanning electron microscope tomography. The subcellular structures of synapses obtained with the X-ray technique matched the electron microscopy data, demonstrating that PXCT is a powerful scanning method for tissue volumes of more than 60 cells and sensitive enough for identification of regions as small as 200 nm, which remain available for further structural and biochemical investigations.

We introduced a novel X-ray measurement and reconstruction method that allows the acquisition of high-resolution hyperspectral tomograms. We applied it for compositional analysis of VPO catalysts which lead to a reevaluation of their working mechanisms.

Over the past decade, ptychography has been proven to be a robust tool for non-destructive high-resolution quantitative electron, X-ray and optical microscopy. It allows for quantitative reconstruction of the specimen's transmissivity, as well as recovery of the illuminating wavefront. Additionally, various algorithms have been developed to account for systematic errors and improved convergence. With fast ptychographic microscopes and more advanced algorithms, both the complexity of the reconstruction task and the data volume increase significantly. PtychoShelves is a software package which combines high-level modularity for easy and fast changes to the data-processing pipeline, and high-performance computing on CPUs and GPUs.

Pulsed laser ablation in liquids can be used to produce nanoparticles for a wide range of materials without the use of harmful additives. In the ablation process, however, the size distribution is not well controlled. Time-resolved X-ray scattering reveals how the larger nanoparticles in the size distribution are formed.