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Type I collagen physiological scaffold for tissue regeneration is considered one of the widely used biomaterials for tissue engineering and medical applications. It is hierarchically organized: five laterally staggered molecules are packed within fibrils, arranged into fascicles and bundles. The structural organization is correlated to the direction and intensity of the forces which can be loaded onto the tissue. For a tissue-specific regeneration, the required macro- and microstructure of a suitable biomaterial has been largely investigated. Conversely, the function of multiscale structural integrity has been much less explored but is crucial for scaffold design and application. In this work, collagen was extracted from different animal sources with protocols that alter its structure. Collagen of tendon shreds excised from cattle, horse, sheep and pig was structurally investigated by wide- and small-angle X-ray scattering techniques, at both molecular and supramolecular scales, and thermo-mechanically with thermal and load-bearing tests. Tendons were selected because of their resistance to chemical degradation and mechanical stresses. The multiscale structural integrity of tendons’ collagen was studied in relation to the animal source, anatomic location and source for collagen extraction.

The supplementary data file contains detailed patient's characteristics including the number of MC detected for each sample, statistical significance calculations, classification and validation results, wide angle X-ray scattering (WAXS) diffractograms, WAXS and small angle X-ray scattering (SAXS) images, mean Raman spectra of different diagnostic categories, box-plot reporting phosphate peak position shift among diagnostic categories, loading of the first principal components emerging from PCA and correlation between Raman variables and a) age, b) BIRADS classification and c) MC size.

This chapter presents an investigation of V2 violations in the German contact variety Kiezdeutsch, making comparison with spoken and written Standard German (SG). We undertake a corpus study analyzing the distribution of V3-inducing resumption strategies otherwise unproblematic in SG: adverbial resumption, Left Dislocation, and Hanging Topic Left Dislocation. Unlike for SG, little is known about resumption strategies in Kiezdeutsch, yet we find similar behavior for spoken SG and Kiezdeutsch. We attempt to reconcile such V3 with a well-known noncanonical V3 pattern in Kiezdeutsch following the order Frame Setter > SubjectTOPIC> finite verb. We employ the framework proposed by Sam Wolfe in which strict-V2 systems have high locus of V2 in Force allowing V2 violations involving resumption and, for some languages, initial Frame Setters but not other violations. We suggest that microvariation in and between Kiezdeutsch and SG results from lexicalization of Frame Setters above ForceP in Kiezdeutsch and below it in SG.

Myelin insulates neuronal axons and enables fast signal transmission, constituting a key component of brain development, aging and disease. Yet, myelin-specific imaging of macroscopic samples remains a challenge. Here, we exploit myelin’s nanostructural periodicity, and use small-angle X-ray scattering tensor tomography (SAXS-TT) to simultaneously quantify myelin levels, nanostructural integrity and axon orientations in nervous tissue. Proof-of-principle is demonstrated in whole mouse brain, mouse spinal cord and human white and gray matter samples. Outcomes are validated by 2D/3D histology and compared to MRI measurements sensitive to myelin and axon orientations. Specificity to nanostructure is exemplified by concomitantly imaging different myelin types with distinct periodicities. Finally, we illustrate the method’s sensitivity towards myelin-related diseases by quantifying myelin alterations in dysmyelinated mouse brain. This non-destructive, stain-free molecular imaging approach enables quantitative studies of myelination within and across samples during development, aging, disease and treatment, and is applicable to other ordered biomolecules or nanostructures., Small-angle X-ray scattering (SAXS) combines the high tissue penetration of X-rays with specificity to periodic nanostructures. The authors use SAXS tensor tomography (SAXS-TT) on intact mouse and human brain tissue samples, to quantify myelin levels and determine myelin integrity, myelinated axon orientation, and fibre tracts non-destructively.

Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets., IUCrJ, 7 (6), ISSN:2052-2525

Across all branches of science, medicine and engineering, high-resolution microscopy is required to understand functionality. Although optical methods have been developed to `defeat' the diffraction limit and produce 3D images, and electrons have proven ever more useful in creating pictures of small objects or thin sections, so far there is no substitute for X-ray microscopy in providing multiscale 3D images of objects with a single instrument and minimal labeling and preparation. A powerful technique proven to continuously access length scales from 10 nm to 10 µm is ptychographic X-ray computed tomography, which, on account of the orthogonality of the tomographic rotation axis to the illuminating beam, still has the limitation of necessitating pillar-shaped samples of small (ca 10 µm) diameter. Large-area planar samples are common in science and engineering, and it is therefore highly desirable to create an X-ray microscope that can examine such samples without the extraction of pillars. Computed laminography, where the axis of rotation is not perpendicular to the illumination direction, solves this problem. This entailed the development of a new instrument, LamNI, dedicated to high-resolution 3D scanning X-ray microscopy via hard X-ray ptychographic laminography. Scanning precision is achieved by a dedicated interferometry scheme and the instrument covers a scan range of 12 mm × 12 mm with a position stability of 2 nm and positioning errors below 5 nm. A new feature of LamNI is a pair of counter-rotating stages carrying the sample and interferometric mirrors, respectively., Journal of Synchrotron Radiation, 27 (3), ISSN:0909-0495, ISSN:1600-5775

Simple Summary This study aims at using a multi-technique approach to detect and analyze the effects of high dose rate spatially fractionated radiation therapies and to compare them to seamless broad beam irradiation targeting healthy and glioblastoma-bearing rat brains and delivering three different doses per each irradiation geometry. Brains were analyzed post mortem by multi-scale X-ray phase contrast imaging–computed tomography, histology, immunohistochemistry, X-ray fluorescence, and small- and wide-angle X-ray scattering to achieve detailed visualization, characterization and classification in 3D of the radio-induced effects on brain tissues. The original results bring new insights to the understanding of the response of cerebral tissue and tumors treated with high dose rate spatially fractioned radiotherapies and put the basis for channeling studies of in-vivo applications for monitoring RT effects. Abstract The purpose of this study is to use a multi-technique approach to detect the effects of spatially fractionated X-ray Microbeam (MRT) and Minibeam Radiation Therapy (MB) and to compare them to seamless Broad Beam (BB) irradiation. Healthy- and Glioblastoma (GBM)-bearing male Fischer rats were irradiated in-vivo on the right brain hemisphere with MRT, MB and BB delivering three different doses for each irradiation geometry. Brains were analyzed post mortem by multi-scale X-ray Phase Contrast Imaging–Computed Tomography (XPCI-CT), histology, immunohistochemistry, X-ray Fluorescence (XRF), Small- and Wide-Angle X-ray Scattering (SAXS/WAXS). XPCI-CT discriminates with high sensitivity the effects of MRT, MB and BB irradiations on both healthy and GBM-bearing brains producing a first-time 3D visualization and morphological analysis of the radio-induced lesions, MRT and MB induced tissue ablations, the presence of hyperdense deposits within specific areas of the brain and tumor evolution or regression with respect to the evaluation made few days post-irradiation with an in-vivo magnetic resonance imaging session. Histology, immunohistochemistry, SAXS/WAXS and XRF allowed identification and classification of these deposits as hydroxyapatite crystals with the coexistence of Ca, P and Fe mineralization, and the multi-technique approach enabled the realization, for the first time, of the map of the differential radiosensitivity of the different brain areas treated with MRT and MB. 3D XPCI-CT datasets enabled also the quantification of tumor volumes and Ca/Fe deposits and their full-organ visualization. The multi-scale and multi-technique approach enabled a detailed visualization and classification in 3D of the radio-induced effects on brain tissues bringing new essential information towards the clinical implementation of the MRT and MB radiation therapy techniques.

Many pathological side-effects of diabetes and aging are directly linked to non-enzymatic glycation of collagen as an abundant, multi-purpose tissue in human beings. We describe a model that quantifies glycation and its structural effects as a function of sugar level (glucose and ribose) and time, based on SAXS and WAXS data., Glycosyl­ation is the process of combining one or more glucose molecules (or other monosaccharides) with molecules of a different nature (which are therefore glycosyl­ated). In biochemistry, glycosyl­ation is catalyzed by several specific enzymes, and assumes considerable importance since it occurs mainly at the expense of proteins and phospho­lipids which are thus transformed into glycoproteins and glycolipids. Conversely, in diabetes and aging, glycation of proteins is a phenomenon of non-enzymatic nature and thus not easily controlled. Glycation of collagen distorts its structure, renders the extracellular matrix stiff and brittle and at the same time lowers the degradation susceptibility thereby preventing renewal. Based on models detailed in this paper and with parameters determined from experimental data, we describe the glycation of type 1 collagen in bovine pericardium derived bio-tissues, upon incubation in glucose and ribose. With arginine and lysine/hy­droxy­lysine amino acids as the primary sites of glycation and assuming that the topological polar surface area of the sugar molecules determines the glycation rates, we modelled the glycation as a function of time and determined the glycation rate and thus the progression of glycation as well as the resulting volume increase.

The imaging of integrated circuits across different length scales is required for failure analysis, design validation and quality control. At present, such inspection is accomplished using a hierarchy of different probes, from optical microscopy on the millimetre length scale to electron microscopy on the nanometre scale. Here we show that ptychographic X-ray laminography can provide non-destructive, three-dimensional views of integrated circuits, yielding both images of an entire chip volume and high-resolution images of arbitrarily chosen subregions. We demonstrate the approach using chips produced with 16 nm fin field-effect transistor technology, achieving a reconstruction resolution of 18.9 nm, and compare our results with photolithographic mask layout files and more conventional imaging approaches such as scanning electron microscopy. The technique should also be applicable to other branches of science and engineering where three-dimensional X-ray images of planar samples are required. A technique that combines X-ray ptychography with laminography can provide three-dimensional views of integrated circuits, yielding both images of entire chip volumes and high-resolution images of arbitrarily chosen subregions, and is applicable to any imaging problem where the samples are planar.

Decellularized pericardium tissue exposed to solutions of sugars like glucose, galactose and ribose could potentially be a model system for diseases like diabetes and the effects of aging. Scanning X-ray diffraction and scattering reveals the different nano-structural changes of collagen induced by glucose, galactose and ribose., Diseases like widespread diabetes or rare galactosemia may lead to high sugar concentrations in the human body, thereby promoting the formation of glycoconjugates. Glycation of collagen, i.e. the formation of glucose bridges, is nonenzymatic and therefore cannot be prevented in any other way than keeping the sugar level low. It relates to secondary diseases, abundantly occurring in aging populations and diabetics. However, little is known about the effects of glycation of collagen on the molecular level. We studied in vitro the effect of glycation, with d-glucose and d-galactose as well as d-ribose, on the structure of type 1 collagen by preparing decellularized matrices of bovine pericardia soaked in different sugar solutions, at increasing concentrations (0, 2.5, 5, 10, 20 and 40 mg ml−1), and incubated at 37°C for 3, 14, 30 and 90 days. The tissue samples were analyzed with small- and wide-angle X-ray scattering in scanning mode. We found that glucose and galactose produce similar changes in collagen, i.e. they mainly affect the lateral packing between macromolecules. However, ribose is much faster in glycation, provoking a larger effect on the lateral packing, but also seems to cause qualitatively different effects on the collagen structure.

Diseases like widespread diabetes or rare galactosemia may lead to high sugar concentrations in the human body, thereby promoting the formation of glycoconjugates. Glycation of collagen, i.e. the formation of glucose bridges, is nonenzymatic and therefore cannot be prevented in any other way than keeping the sugar level low. It relates to secondary diseases, abundantly occurring in aging populations and diabetics. However, little is known about the effects of glycation of collagen on the molecular level. We studied in vitro the effect of glycation, with d-glucose and d-galactose as well as d-ribose, on the structure of type 1 collagen by preparing decellularized matrices of bovine pericardia soaked in different sugar solutions, at increasing concentrations (0, 2.5, 5, 10, 20 and 40 mg ml 1), and incubated at 37C for 3, 14, 30 and 90 days. The tissue samples were analyzed with small- and wide-angle X-ray scattering in scanning mode.We found that glucose and galactose produce similar changes in collagen, i.e. they mainly affect the lateral packing between macromolecules. However, ribose is much faster in glycation, provoking a larger effect on the lateral packing, but also seems to cause qualitatively different effects on the collagen structure

A microcrystal-prelocation method is demonstrated using low-dose 2D full-field propagation-based X-ray phase-contrast imaging on samples with an essentially flat geometry for automated serial crystallography data collection at a microfocus macromolecular crystallography beamline., Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets.

A growing number of X-ray sources based on the free-electron laser (XFEL) principle are presently under construction or have recently started operation. The intense, ultrashort pulses of these sources will enable new insights in many different fields of science. A key problem is to provide x-ray optical elements capable of collecting the largest possible fraction of the radiation and to focus into the smallest possible focus. As a key step towards this goal, we demonstrate here the first nanofocusing of hard XFEL pulses. We developed diamond based Fresnel zone plates capable of withstanding the full beam of the world's most powerful x-ray laser. Using an imprint technique, we measured the focal spot size, which was limited to 320 nm FWHM by the spectral band width of the source. A peak power density in the focal spot of 4×10(17)W/cm(2) was obtained at 70 fs pulse length.

In this paper, we present a data workflow developed to operate the adJUstiNg Gain detector FoR the Aramis User station (JUNGFRAU) adaptive gain charge integrating pixel-array detectors at macromolecular crystallography beamlines. We summarize current achievements for operating at 9 GB/s data-rate a JUNGFRAU with 4 Mpixel at 1.1 kHz frame-rate and preparations to operate at 46 GB/s data-rate a JUNGFRAU with 10 Mpixel at 2.2 kHz in the future. In this context, we highlight the challenges for computer architecture and how these challenges can be addressed with innovative hardware including IBM POWER9 servers and field-programmable gate arrays. We discuss also data science challenges, showing the effect of rounding and lossy compression schemes on the MX JUNGFRAU detector images., Structural Dynamics, 7 (1), ISSN:2329-7778

Breast microcalcifications are a common mammographic finding. Microcalcifications are considered suspicious signs of breast cancer and a breast biopsy is required, however, cancer is diagnosed in only a few patients. Reducing unnecessary biopsies and rapid characterization of breast microcalcifications are unmet clinical needs. In this study, 473 microcalcifications detected on breast biopsy specimens from 56 patients were characterized entirely by Raman mapping and confirmed by X-ray scattering. Microcalcifications from malignant samples were generally more homogeneous, more crystalline, and characterized by a less substituted crystal lattice compared with benign samples. There were significant differences in Raman features corresponding to the phosphate and carbonate bands between the benign and malignant groups. In addition to the heterogeneous composition, the presence of whitlockite specifically emerged as marker of benignity in benign microcalcifications. The whole Raman signature of each microcalcification was then used to build a classification model that distinguishes microcalcifications according to their overall biochemical composition. After validation, microcalcifications found in benign and malignant samples were correctly recognized with 93.5% sensitivity and 80.6% specificity. Finally, microcalcifications identified in malignant biopsies, but located outside the lesion, reported malignant features in 65% of in situ and 98% of invasive cancer cases, respectively, suggesting that the local microenvironment influences microcalcification features. This study confirms that the composition and structural features of microcalcifications correlate with breast pathology and indicates new diagnostic potentialities based on microcalcifications assessment. Significance: Raman spectroscopy could be a quick and accurate diagnostic tool to precisely characterize and distinguish benign from malignant breast microcalcifications detected on mammography.

The accuracy of X-ray diffraction data is directly related to how the X-ray detector records photons. Here we describe the application of a direct-detection charge-integrating pixel-array detector (JUNGFRAU) in macromolecular crystallography (MX). JUNGFRAU features a uniform response on the subpixel level, linear behavior toward high photon rates, and low-noise performance across the whole dynamic range. We demonstrate that these features allow accurate MX data to be recorded at unprecedented speed. We also demonstrate improvements over previous-generation detectors in terms of data quality, using native single-wavelength anomalous diffraction (SAD) phasing, for thaumatin, lysozyme, and aminopeptidase N. Our results suggest that the JUNGFRAU detector will substantially improve the performance of synchrotron MX beamlines and equip them for future synchrotron light sources. published

A recently developed computational imaging technique, X-ray ptychographic tomography, is used to study integrated circuits, and a 3D image of a processor chip with a resolution of 14.6 nm is obtained. As computer chips have become increasingly crammed with nanometre-scale devices and circuitry, new microscopy techniques that can resolve the smallest features are required to enable chip design and inspection. X-ray imaging is uniquely suited for non-destructive, high-resolution imaging and Mirko Holler et al. make use of a recently developed computational imaging technique, X-ray ptychography, to generate high-resolution three-dimensional images of integrated circuits. They test X-ray ptychography on a circuit with known features, and then apply it to an Intel processor chip manufactured in the 22-nanometre technology, obtaining detailed three-dimensional maps of the devices with a resolution down to 14.6 nanometres. This technique could be used to assist quality control during chip production. Modern nanoelectronics1,2 has advanced to a point at which it is impossible to image entire devices and their interconnections non-destructively because of their small feature sizes and the complex three-dimensional structures resulting from their integration on a chip. This metrology gap implies a lack of direct feedback between design and manufacturing processes, and hampers quality control during production, shipment and use. Here we demonstrate that X-ray ptychography3,4—a high-resolution coherent diffractive imaging technique—can create three-dimensional images of integrated circuits of known and unknown designs with a lateral resolution in all directions down to 14.6 nanometres. We obtained detailed device geometries and corresponding elemental maps, and show how the devices are integrated with each other to form the chip. Our experiments represent a major advance in chip inspection and reverse engineering over the traditional destructive electron microscopy and ion milling techniques5,6,7. Foreseeable developments in X-ray sources8, optics9 and detectors10, as well as adoption of an instrument geometry11 optimized for planar rather than cylindrical samples, could lead to a thousand-fold increase in efficiency, with concomitant reductions in scan times and voxel sizes.

Blood glucose supplies energy to cells and is critical for the human brain. Glycation of collagen, the nonenzymatic formation of glucose‐bridges, relates to diseases of aging populations and diabetics. This chemical reaction, together with its biomechanical effects, has been well studied employing animal models. However, the direct impact of glycation on collagen nano‐structure is largely overlooked, and there is a lack of ex vivo model systems. Here, we present the impact of glucose on collagen nanostructure in a model system based on abundantly available connective tissue of farm animals. By combining ex vivo small and wide‐angle X‐ray scattering (SAXS/WAXS) imaging, we characterize intra‐ and inter‐molecular parameters of collagen in decellularized bovine pericardium with picometer precision. We observe three distinct regimes according to glucose concentration. Such a study opens new avenues for inspecting the effects of diabetes mellitus on connective tissues and the influence of therapies on the resulting secondary disorders., The direct effects of nonenzymatic glycation on the structure of collagen have been quantified in a new model system for the study of diabetes and age‐related effects.

Advantages of using a wavelength of 2.7 Å over a wavelength of 1.9 Å for native single-wavelength anomalous dispersion phasing are presented, and potentials of using a wavelength of 3.3 Å are discussed., Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers (Z < 20). The anomalous signal of sulfur in particular, is enhanced at long wavelengths, however the absorption of diffracted X-rays owing to the crystal, the sample support and air affects the recorded intensities. Thereby, the optimal measurable anomalous signals primarily depend on the counterplay of the absorption and the anomalous scattering factor at a given X-ray wavelength. Here, the benefit of using a wavelength of 2.7 over 1.9 Å is demonstrated for native-SAD phasing on a 266 kDa multiprotein-ligand tubulin complex (T2R-TTL) and is applied in the structure determination of an 86 kDa helicase Sen1 protein at beamline BL-1A of the KEK Photon Factory, Japan. Furthermore, X-ray absorption at long wavelengths was controlled by shaping a lysozyme crystal into spheres of defined thicknesses using a deep-UV laser, and a systematic comparison between wavelengths of 2.7 and 3.3 Å is reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3 Å are discussed.

Microcalcifications are important pathological markers. Thus, it is relevant to study the occurring crystalline phases, their formation mechanisms, and the correlations between their structure and pathologic modifications of human tissue. Combining scanning X-ray microdiffraction and small-angle scattering reveals the crystalline phases of microcalcifications, and the abundance and orientation of collocated fibrous tissues (elastin, collagen, myofilament), spatially resolved over aneurysm tissue samples., Abdominal aortic and popliteal artery aneurysms are vascular diseases which show massive degeneration, weakening of the vascular wall and loss of the vascular tissue functionality. They are driven by inflammatory, hemodynamical factors and biological alterations that may lead, in the case of an abdominal aortic aneurysm, to sudden and dangerous ruptures of the arteries. Here, human aortic and popliteal aneurysm tissues were obtained during surgical repair, and studied by synchrotron radiation X-ray scanning microdiffraction and small-angle scattering, to investigate the microcalcifications present in the tissues. Data collected during the experiments were transformed into quantitative microscopy images through the combination of statistical approaches and crystallographic methods. As a result of this multi-step analysis, microcalcifications, which are markers of the pathology, were classified in terms of chemical and structural content. This analysis helped to identify the presence of nanocrystalline hy­droxy­apatite and microcrystalline cholesterol, embedded in myofilament, and elastin-containing tissue with low collagen content in predominantly nanocrystalline areas. The generality of the approach allows it to be transferred to other types of tissue and other pathologies affected by microcalcifications, such as thyroid carcinoma, breast cancer, testicular microli­thia­sis or glioblastoma.

Journal of Synchrotron Radiation, 26 (1), ISSN:0909-0495, ISSN:1600-5775

This article reports the incorporation of a fast continuous grid scan with both still and oscillation images into the Swiss Light Source macromolecular crystallography beamlines and its application in visualization of protein crystals with scanning transmission X-ray microscopy., A fast continuous grid scan protocol has been incorporated into the Swiss Light Source (SLS) data acquisition and analysis software suite on the macromolecular crystallography (MX) beamlines. Its combination with fast readout single-photon counting hybrid pixel array detectors (PILATUS and EIGER) allows for diffraction-based identification of crystal diffraction hotspots and the location and centering of membrane protein microcrystals in the lipid cubic phase (LCP) in in meso in situ serial crystallography plates and silicon nitride supports. Diffraction-based continuous grid scans with both still and oscillation images are supported. Examples that include a grid scan of a large (50 nl) LCP bolus and analysis of the resulting diffraction images are presented. Scanning transmission X-ray microscopy (STXM) complements and benefits from fast grid scanning. STXM has been demonstrated at the SLS beamline X06SA for near-zero-dose detection of protein crystals mounted on different types of sample supports at room and cryogenic temperatures. Flash-cooled crystals in nylon loops were successfully identified in differential and integrated phase images. Crystals of just 10 µm thickness were visible in integrated phase images using data collected with the EIGER detector. STXM offers a truly low-dose method for locating crystals on solid supports prior to diffraction data collection at both synchrotron microfocusing and free-electron laser X-ray facilities.

The mechanical properties of many materials are based on the macroscopic arrangement and orientation of their nanostructure. This nanostructure can be ordered over a range of length scales. In biology, the principle of hierarchical ordering is often used to maximize functionality, such as strength and robustness of the material, while minimizing weight and energy cost. Methods for nanoscale imaging provide direct visual access to the ultrastructure (nanoscale structure that is too small to be imaged using light microscopy), but the field of view is limited and does not easily allow a full correlative study of changes in the ultrastructure over a macroscopic sample. Other methods of probing ultrastructure ordering, such as small-angle scattering of X-rays or neutrons, can be applied to macroscopic samples; however, these scattering methods remain constrained to two-dimensional specimens or to isotropically oriented ultrastructures. These constraints limit the use of these methods for studying nanostructures with more complex orientation patterns, which are abundant in nature and materials science. Here, we introduce an imaging method that combines small-angle scattering with tensor tomography to probe nanoscale structures in three-dimensional macroscopic samples in a non-destructive way. We demonstrate the method by measuring the main orientation and the degree of orientation of nanoscale mineralized collagen fibrils in a human trabecula bone sample with a spatial resolution of 25 micrometres. Symmetries within the sample, such as the cylindrical symmetry commonly observed for mineralized collagen fibrils in bone, allow for tractable sampling requirements and numerical efficiency. Small-angle scattering tensor tomography is applicable to both biological and materials science specimens, and may be useful for understanding and characterizing smart or bio-inspired materials. Moreover, because the method is non-destructive, it is appropriate for in situ measurements and allows, for example, the role of ultrastructure in the mechanical response of a biological tissue or manufactured material to be studied.

Small-angle X-ray scattering tensor tomography, which allows reconstruction of the local three-dimensional reciprocal-space map within a three-dimensional sample as introduced by Liebi et al. [Nature (2015), 527, 349–352], is described in more detail with regard to the mathematical framework and the optimization algorithm. For the case of trabecular bone samples from vertebrae it is shown that the model of the three-dimensional reciprocal-space map using spherical harmonics can adequately describe the measured data. The method enables the determination of nanostructure orientation and degree of orientation as demonstrated previously in a single momentum transfer q range. This article presents a reconstruction of the complete reciprocal-space map for the case of bone over extended ranges of q. In addition, it is shown that uniform angular sampling and advanced regularization strategies help to reduce the amount of data required., Acta Crystallographica Section A: Foundations and Advances, 74 (1), ISSN:2053-2733

X-ray ptychographic tomography has been recently developed for quantitative imaging of specimens on the nanometer scale. Here we present its application for the characterization of carbon fibers by mapping in three dimensions the mass density of entire fibers with diameters of several tens of micrometers with a resolution of about 100 nm. We characterized two fibers produced from two different precursors, revealing the spatial distribution of porosity and highly graphitized regions within the fibers. We further discuss the potential of ptychographic tomography as a new complementary technique for the characterization of carbon materials.

Carious lesions exhibit a complex structural organization composed of zones of higher and lower mineralization, formed by successive periods of cyclic de- and re-mineralization. A thorough understanding of the lesion morphology is necessary for the development of suitable treatments aiming to repair rather than replace the damaged tissue. This detailed understanding includes the entire lesion down to individual crystallites and nanopores within the natural organization of the crown. A moderate lesion, with surface loss and reaching dentin, and a very early lesion were studied. Scanning small-angle X-ray scattering (SAXS) with a pixel size of 20 × 20 μm(2) was used to characterize these lesions, allowing for the identification of distinct zones with varied absorption and scattering behavior, indicative of varied porosity and pore morphology. Despite these differences, the overall orientation and anisotropy of the SAXS signal was unaltered throughout both lesions, indicating that an anisotropic scaffold is still present in the lesion. The finding that crystallite orientation is preserved throughout the lesions facilitates the identification of preventive re-mineralizing strategies with the potential to recreate the original nanostructure.

Osteoarthritis (OA), among other bone pathologies, is expected to determine supramolecular changes at the level of the mineralized collagen fiber. In a proof-of-principle study, bone biopsies were collected from six coxarthritis-affected patients, aged 62–87 years, during hip prosthesis implant surgery, sliced down to 100 µm-thick tissues, and investigated using scanning small-angle and wide-angle X-ray scattering (SAXS and WAXS) transmission microscopy. A multi-modal imaging evaluation of the SAXS and WAXS data, combined with principal component and canonical correlation analyses, allowed the transformation of the raw data into microscopy images and inspection of the nanoscale structure of the mineralized collagen fibers across mm2tissue areas. The combined scanning SAXS and WAXS microscopy is shown to be a suitable choice for characterizing and quantifying the nanostructural properties of collagen over extended areas. The results suggest the existence of a correlation between age and cross-linking-induced rigidity of collagen fibers.

The nanoscale structural order of air-dried rat-tail tendon is investigated using small-angle X-ray scattering (SAXS). SAXS fiber diffraction patterns were collected with a superbright laboratory microsource at XMI-LAB [Altamura, Lassandro, Vittoria, De Caro, Siliqi, Ladisa & Giannini (2012).J. Appl. Cryst.45, 869–873] for increasing integration times (up to 10 h) and a novel algorithm was used to estimate and subtract background, and to deconvolve the beam-divergence effects. Once the algorithm is applied, the peak visibility improves considerably and reciprocal space information up to the 22nd diffraction order is retrieved (q= 0.21 Å−1,d= 29 Å) for an 8–10 h integration time. The gain in the visibility is already significant for patterns collected for 0.5 h, at least on the more intense peaks. This demonstrates the viability of detecting structural changes on a molecular/nanoscale level in tissues with state-of-the-art laboratory sources and also the technical feasibility to adopt SAXS fiber diffraction as a future potential clinical indicator for disease.

Ptychographic X-ray computed tomography (PXCT) allows for a non-destructive, three-dimensional mapping of the electron density. Its quantitativeness combined with a resolution in the 100 nm range makes it a suitable tool for the assessment of densities of the individual phases in complex materials, such as hardened cement pastes. Here we present results of an experiment performed on a cylindrical sample of epoxy-impregnated hardened cement paste of about 30 μm in diameter. Two-dimensional cross sections of the three-dimensional electron density map show a microstructure that bears distinct similarity to that observed by back-scattered scanning electron microscopy. Domains of various residues of cement grains, calcium hydroxide, calcium carbonate, epoxy-resin impregnated calcium silicate hydrates, epoxy-resin impregnated porosity, and unimpregnated porosity are revealed and are manifested as distinguishable peaks in the histogram of the three-dimensional electron density map. On assumptions of (i) a priori knowledge of the chemical composition and (ii) the purity of the analysed regions, the mass densities of the above mentioned individual material phases are estimated. The potential of PXCT for the science of cement and concrete is discussed.