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The study of biological soft tissue structures at the micron scale details the function of healthy and pathological tissues, which is vital in the diagnosis and treatment of diseases. Speckle based X-ray phase contrast tomographic scans at a nanometer scale have the potential to thoroughly analyse such tissues in a quantitative and qualitative manner. Diamond light source, the UKs national synchrotron facility developed and refined a 1-D X-ray speckle-based imaging technique, referred to as Fly scan mode. This novel image acquisition technique was used to perform a rapid structural composition scan of rodent lung histology samples. The rodent samples were taken from healthy and Staphylococcus aureus induced acute respiratory distress syndrome models. The analysis and cross comparison of the fly scan method, absorption-based tomography and conventional histopathology H&E staining microscopy are discussed in this paper. This analysis and cross comparison outline the ways the speckle-based technique can be of benefit. These advantages include improved soft tissue contrast, 3-D volumetric rendering, segmentation of specific gross tissue structures, quantitative analysis of gross tissue volume. A further advantage is the analysis of cellular distribution throughout the volumetric rendering of the tissue sample. The study also details the current limitations of this technique and points to ways in which future work on this imaging modality may progress., (© 2024. The Author(s).)

Two-directional beam-tracking (2DBT) is a method for phase-contrast imaging and tomography that uses an intensity modulator to structure the X-ray beam into an array of independent circular beamlets that are resolved by a high-resolution detector. It features isotropic spatial resolution, provides two-dimensional phase sensitivity, and enables the three-dimensional reconstructions of the refractive index decrement, δ, and the attenuation coefficient, μ. In this work, the angular sensitivity and the spatial resolution of 2DBT images in a synchrotron-based implementation is reported. In its best configuration, angular sensitivities of ∼20 nrad and spatial resolution of at least 6.25 µm in phase-contrast images were obtained. Exemplar application to the three-dimensional imaging of soft tissue samples, including a mouse liver and a decellularized porcine dermis, is also demonstrated., (open access.)

We apply X-ray ptycho-tomography to perform high-resolution, non-destructive, three-dimensional (3D) imaging of Fe-rich inclusions in paleomagnetically relevant materials (zircon single crystals from the Bishop Tuff ignimbrite). Correlative imaging using quantum diamond magnetic microscopy combined with X-ray fluorescence mapping was used to locate regions containing potential ferromagnetic remanence carriers. Ptycho-tomographic reconstructions with voxel sizes 85 nm and 21 nm were achievable across a field-of-view > 80 µm; voxel sizes as small as 5 nm were achievable over a limited field-of-view using local ptycho-tomography. Fe-rich inclusions 300 nm in size were clearly resolved. We estimate that particles as small as 100 nm-approaching single-domain threshold for magnetite-could be resolvable using this "dual-mode" methodology. Fe-rich inclusions (likely magnetite) are closely associated with apatite inclusions that have no visible connection to the exterior surface of the zircon (e.g., via intersecting cracks). There is no evidence of radiation damage, alteration, recrystallisation or deformation in the host zircon or apatite that could provide alternative pathways for Fe infiltration, indicating that magnetite and apatite grew separately as primary phases in the magma, that magnetite adhered to the surfaces of the apatite, and that the magnetite-coated apatite was then encapsulated as primary inclusions within the growing zircon. Rarer examples of Fe-rich inclusions entirely encapsulated by zircon are also observed. These observations support the presence of primary inclusions in relatively young and pristine zircon crystals. Combining magnetic and tomography results we deduce the presence of magnetic carriers that are in the optimal size range for carrying strong and stable paleomagnetic signals but that remain below the detection limits of even the highest-resolution X-ray tomography reconstructions. We recommend the use of focused ion beam nanotomography and/or correlative transmission electron microscopy to directly confirm the presence of primary magnetite in the sub 300 nm range as a necessary step in targeted paleomagnetic workflows., (© 2024. The Author(s).)

High-resolution spatial and temporal analysis and 3D visualization of time-dependent processes, such as human dental enamel acid demineralization, often present a challenging task. Overcoming this challenge often requires the development of special methods. Dental caries remains one of the most important oral diseases that involves the demineralization of hard dental tissues as a consequence of acid production by oral bacteria. Enamel has a hierarchically organized architecture that extends down to the nanostructural level and requires high resolution to study its evolution in detail. Enamel demineralization is a dynamic process that is best investigated with the help of in situ experiments. In previous studies, synchrotron tomography was applied to study the 3D enamel structure at certain time points (time-lapse tomography). Here, another distinct approach to time-evolving tomography studies is presented, whereby the sample image is reconstructed as it undergoes continuous rotation over a virtually unlimited angular range. The resulting (single) data set contains the data for multiple (potentially overlapping) intermediate tomograms that can be extracted and analyzed as desired using time-stepping selection of data subsets from the continuous fly-scan recording. One of the advantages of this approach is that it reduces the amount of time required to collect an equivalent number of single tomograms. Another advantage is that the nominal time step between successive reconstructions can be significantly reduced. We applied this approach to the study of acidic enamel demineralization and observed the progression of demineralization over time steps significantly smaller than the total acquisition time of a single tomogram, with a voxel size smaller than 0.5 μm. It is expected that the approach presented in this paper can be useful for high-resolution studies of other dynamic processes and for assessing small structural modifications in evolving hierarchical materials., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Co-published by Nanjing University and American Chemical Society.)

Caries, a major global disease associated with dental enamel demineralization, remains insufficiently understood to devise effective prevention or minimally invasive treatment. Understanding the ultrastructural changes in enamel is hampered by a lack of nanoscale characterization of the chemical spatial distributions within the dental tissue. This leads to the requirement to develop techniques based on various characterization methods. The purpose of the present study is to demonstrate the strength of analytic methods using a correlative technique on a single sample of human dental enamel as a specific case study to test the accuracy of techniques to compare regions in enamel. The science of the different techniques is integrated to genuinely study the enamel. The hierarchical structures within carious tissue were mapped using the combination of focused ion beam scanning electron microscopy with synchrotron X-ray tomography. The chemical changes were studied using scanning X-ray fluorescence (XRF) and X-ray wide-angle and small-angle scattering using a beam size below 80 nm for ångström and nanometer length scales. The analysis of XRF intensity gradients revealed subtle variations of Ca intensity in carious samples in comparison with those of normal mature enamel. In addition, the pathways for enamel rod demineralization were studied using X-ray ptychography. The results show the chemical and structural modification in carious enamel with differing locations. These results reinforce the need for multi-modal approaches to nanoscale analysis in complex hierarchically structured materials to interpret the changes of materials. The approach establishes a meticulous correlative characterization platform for the analysis of biomineralized tissues at the nanoscale, which adds confidence in the interpretation of the results and time-saving imaging techniques. The protocol demonstrated here using the dental tissue sample can be applied to other samples for statistical study and the investigation of nanoscale structural changes. The information gathered from the combination of methods could not be obtained with traditional individual techniques.

We present a flyscan compatible acquisition scheme for three-modal X-Ray Computed Tomography (CT) with two-dimensional phase sensitivity. Our approach is demonstrated using a "beam tracking" setup, through which a sample's attenuation, phase (refraction) and scattering properties can be measured from a single frame, providing three complementary contrast channels. Up to now, such setups required the sample to be stepped at each rotation angle to sample signals at an adequate rate, to prevent resolution losses, anisotropic resolution, and under-sampling artefacts. However, the need for stepping necessitated a step-and-shoot implementation, which is affected by motors' overheads and increases the total scan time. By contrast, our proposed scheme, by which continuous horizontal and vertical translations of the sample are integrated with its rotation (leading to a "cycloidal-spiral" trajectory), is fully compatible with continuous scanning (flyscans). This leads to greatly reduced scan times while largely preserving image quality and isotropic resolution., (© 2022. The Author(s).)

Digital volume correlation (DVC) in combination with high-resolution micro-computed tomography (microCT) imaging and in situ mechanical testing is gaining popularity for quantifying 3D full-field strains in bone and biomaterials. However, traditional in situ time-lapsed (i.e., interrupted) mechanical testing cannot fully capture the dynamic strain mechanisms in viscoelastic biological materials. The aim of this study was to investigate the time-resolved deformation of bone structures and analogues via continuous in situ synchrotron-radiation microCT (SR-microCT) compression and DVC to gain a better insight into their structure-function relationships. Fast SR-microCT imaging enabled the deformation behaviour to be captured with high temporal and spatial resolution. Time-resolved DVC highlighted the relationship between local strains and damage initiation and progression in the different biostructures undergoing plastic deformation, bending and/or buckling of their main microstructural elements. The results showed that SR-microCT continuous mechanical testing complemented and enhanced the information obtained from time-lapsed testing, which may underestimate the 3D strain magnitudes as a result of the stress relaxation occurring in between steps before image acquisition in porous biomaterials. Altogether, the findings of this study highlight the importance of time-resolved in situ experiments to fully characterise the time-dependent mechanical behaviour of biological tissues and biomaterials and to further explore their micromechanics under physiologically relevant conditions. STATEMENT OF SIGNIFICANCE: Time-resolved synchrotron X-ray tomography in combination with in situ mechanical testing provided the first four-dimensional analysis of the mechanical deformation of bone and bone analogues. To unravel the interplay of damage initiation and progression with local deformation, digital volume correlation was used to map the local strain field while microstructural changes were tracked with high temporal and spatial resolution. The results highlighted the importance of fast imaging and time-resolved in situ experiments to capture the real deformation of complex porous materials to fully characterize the local strain-damage relationship. The findings are notably improving the understanding of time-dependent mechanical behaviour of bone tissue, with the potential to be extend to highly viscoelastic biomaterials and soft tissues., Competing Interests: Declaration of Competing Interest F.W. is an employee of Biotrics Bioimplants AG which partially funded this study. The remaining authors declare that they have no known conflict of interests., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In this paper a practical solution for the reconstruction and segmentation of low-contrast X-ray tomographic data of protein crystals from the long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source is provided. The resulting segmented data will provide the path lengths through both diffracting and non-diffracting materials as basis for analytical absorption corrections for X-ray diffraction data taken in the same sample environment ahead of the tomography experiment. X-ray tomography data from protein crystals can be difficult to analyse due to very low or absent contrast between the different materials: the crystal, the sample holder and the surrounding mother liquor. The proposed data processing pipeline consists of two major sequential operations: model-based iterative reconstruction to improve contrast and minimize the influence of noise and artefacts, followed by segmentation. The segmentation aims to partition the reconstructed data into four phases: the crystal, mother liquor, loop and vacuum. In this study three different semi-automated segmentation methods are experimented with by using Gaussian mixture models, geodesic distance thresholding and a novel morphological method, RegionGrow, implemented specifically for the task. The complete reconstruction-segmentation pipeline is integrated into the MPI-based data analysis and reconstruction framework Savu, which is used to reduce computation time through parallelization across a computing cluster and makes the developed methods easily accessible., (open access.)

Extensive structural changes occur within the spinal cord following traumatic injury. Acute tissue debris and necrotic tissue are broken down, proliferating local glia and infiltrating leukocytes remodel tissue biochemical and biophysical properties, and a chronic cavity surrounded by a scar forms at the injury epicentre. Serial-section 2D histology has traditionally assessed these features in experimental models of spinal cord injury (SCI) to measure the extent of tissue pathology and evaluate efficacy of novel therapies. However, this 2D snapshot approach overlooks slice intervening features, with accurate representation of tissue compromised by mechanical processing artefacts. 3D imaging avoids these caveats and allows full exploration of the injured tissue volume to characterise whole tissue pathology. Amongst 3D imaging modalities, Synchrotron Radiation X-ray microtomography (SRμCT) is advantageous for its speed, ability to cover large tissue volumes at high resolution, and need for minimal sample processing. Here we demonstrate how extended lengths of formalin-fixed, paraffin-embedded (FFPE) rat spinal cord can be completely imaged by SRμCT with micron resolution. Label-free contrast derived from X-ray phase interactions with low-density soft tissues, reveals spinal cord white matter, gray matter, tissue damage and vasculature, with tissue still viable for targeted 2D-histology after 3D imaging. We used SRμCT to quantify tissue pathology after a midline, cervical level (C6), 225 kDyne contusion injury over acute-to-chronic (24 h to 5 weeks) post injury time points. Quantification revealed acute tissue swelling prior to chronic atrophy across the whole imaged region (spanning 2 spinal segments above and below injury), along with rostro-caudal asymmetries in white and gray matter volume loss. 3D volumes revealed satellite damage in tissue far removed from the epicentre, and extensive rostro-caudal spread of damage through the base of the dorsal columns at 24 h post injury. This damage overlapped regions of vasogenic oedema, confirmed with subsequent histology. Tissue damage at later time points in border regions was most prominent in the dorsal columns, where it overlapped sites of damaged venous vasculature. Elaborating rostro-caudal and spatiotemporal asymmetries in reduced traumatic injury models centred on these regions may inform future treatments that seek to limit the spread of tissue pathology to these 'at-risk' regions., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The mechanics of breathing is a fascinating and vital process. The lung has complexities and subtle heterogeneities in structure across length scales that influence mechanics and function. This study establishes an experimental pipeline for capturing alveolar deformations during a respiratory cycle using synchrotron radiation micro-computed tomography (SR-micro-CT). Rodent lungs were mechanically ventilated and imaged at various time points during the respiratory cycle. Pressure-Volume (P-V) characteristics were recorded to capture any changes in overall lung mechanical behaviour during the experiment. A sequence of tomograms was collected from the lungs within the intact thoracic cavity. Digital volume correlation (DVC) was used to compute the three-dimensional strain field at the alveolar level from the time sequence of reconstructed tomograms. Regional differences in ventilation were highlighted during the respiratory cycle, relating the local strains within the lung tissue to the global ventilation measurements. Strains locally reached approximately 150% compared to the averaged regional deformations of approximately 80-100%. Redistribution of air within the lungs was observed during cycling. Regions which were relatively poorly ventilated (low deformations compared to its neighbouring region) were deforming more uniformly at later stages of the experiment (consistent with its neighbouring region). Such heterogenous phenomena are common in everyday breathing. In pathological lungs, some of these non-uniformities in deformation behaviour can become exaggerated, leading to poor function or further damage. The technique presented can help characterize the multiscale biomechanical nature of a given pathology to improve patient management strategies, considering both the local and global lung mechanics.

Synchrotron radiation microtomography (SRμCT) is a nominally non-destructive 3D imaging technique which can visualise the internal structures of whole soft tissues. As a multi-stage technique, the cumulative benefits of optimising sample preparation, scanning parameters and signal processing can improve SRμCT imaging efficiency, image quality, accuracy and ultimately, data utility. By evaluating different sample preparations (embedding media, tissue stains), imaging (projection number, propagation distance) and reconstruction (artefact correction, phase retrieval) parameters, a novel methodology (combining reversible iodine stain, wax embedding and inline phase contrast) was optimised for fast (~12 minutes), high-resolution (3.2-4.8 μm diameter capillaries resolved) imaging of the full diameter of a 3.5 mm length of rat spinal cord. White-grey matter macro-features and micro-features such as motoneurons and capillary-level vasculature could then be completely segmented from the imaged volume for analysis through the shallow machine learning SuRVoS Workbench. Imaged spinal cord tissue was preserved for subsequent histology, establishing a complementary SRμCT methodology that can be applied to study spinal cord pathologies or other nervous system tissues such as ganglia, nerves and brain. Further, our 'single-scan iterative downsampling' approach and side-by-side comparisons of mounting options, sample stains and phase contrast parameters should inform efficient, effective future soft tissue SRμCT experiment design.

Background: Approximately 30-40% of children <1 year of age are Clostridium difficile colonized, and may represent a reservoir for adult C. difficile infections (CDI). Risk factors for colonization with toxigenic versus non-toxigenic C. difficile strains and longitudinal acquisition dynamics in infants remain incompletely characterized., Methods: Predominantly healthy infants (≤2 years) were recruited in Oxfordshire, UK, and provided ≥1 fecal samples. Independent risk factors for toxigenic/non-toxigenic C. difficile colonization and acquisition were identified using multivariable regression. Infant C. difficile isolates were whole-genome sequenced to assay genetic diversity and prevalence of toxin-associated genes, and compared with sequenced strains from Oxfordshire CDI cases., Results: 338/365 enrolled infants provided 1332 fecal samples, representing 158 C. difficile colonization or carriage episodes (107[68%] toxigenic). Initial colonization was associated with age, and reduced with breastfeeding but increased with pet dogs. Acquisition was associated with older age, Caesarean delivery, and diarrhea. Breastfeeding and pre-existing C. difficile colonization reduced acquisition risk. Overall 13% of CDI C. difficile strains were genetically related to infant strains. 29(18%) infant C. difficile sequences were consistent with recent direct/indirect transmission to/from Oxfordshire CDI cases (≤2 single nucleotide variants [SNVs]); 79(50%) shared a common origin with an Oxfordshire CDI case within the last ~5 years (0-10 SNVs). The hypervirulent, epidemic ST1/ribotype 027 remained notably absent in infants in this large study, as did other lineages such as STs 10/44 (ribotype 015); the most common strain in infants was ST2 (ribotype 020/014)(22%)., Conclusions: In predominantly healthy infants without significant healthcare exposure C. difficile colonization and acquisition reflect environmental exposures, with pet dogs identified as a novel risk factor. Genetic overlap between some infant strains and those isolated from CDI cases suggest common community reservoirs of these C. difficile lineages, contrasting with those lineages found only in CDI cases, and therefore more consistent with healthcare-associated spread.

I12 is the Joint Engineering, Environmental and Processing (JEEP) beamline, constructed during Phase II of the Diamond Light Source. I12 is located on a short (5 m) straight section of the Diamond storage ring and uses a 4.2 T superconducting wiggler to provide polychromatic and monochromatic X-rays in the energy range 50-150 keV. The beam energy enables good penetration through large or dense samples, combined with a large beam size (1 mrad horizontally × 0.3 mrad vertically). The beam characteristics permit the study of materials and processes inside environmental chambers without unacceptable attenuation of the beam and without the need to use sample sizes which are atypically small for the process under study. X-ray techniques available to users are radiography, tomography, energy-dispersive diffraction, monochromatic and white-beam two-dimensional diffraction/scattering and small-angle X-ray scattering. Since commencing operations in November 2009, I12 has established a broad user community in materials science and processing, chemical processing, biomedical engineering, civil engineering, environmental science, palaeontology and physics.