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Abstract: Correlative microscopy linking synchrotron X-ray fluorescence (SXRF) with optical imaging is valuable for contextualizing chemical element distributions in biology. The spatial correlation necessary to achieve this presents fundamental challenges and can be a significant constraint on accuracy and data interpretation. We present a technical solution based on a finder grid concept, optimized for SXRF correlative studies of metals in biological tissues, with scope for wider adaptation and application. A hierarchically patterned fiducial system was directly etched onto spectroscopically clean quartz substrates via femtosecond laser ablation. This design enables improved correlation among SXRF, optical imaging, and histological staining over a greater range of length scales than conventional registration methods such as the use of tissue architecture from serial sections and the use of electron-microscopy-resolution finder grids and applied fiduciary markers that can introduce XRF-signal-dominating levels of elements such as copper, nickel, gold, and titanium. We present two quartz finder grid formats: a microgrid and a nanogrid design. We demonstrate their utility for rapid ROI relocalization and same-section correlative workflows using human brain tissue. The etched quartz finder grid approach facilitates rapid and reproducible ROI relocalization and alignment across instruments, particularly where integral fiducial markers are sparse or ambiguous.

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Abstract: Metals play an essential role in cellular homeostasis and are key components of several formulations currently used in the clinic. Synchrotron-based X-ray microscopy at submicron resolution is a powerful approach to map intracellular elemental distributions and to monitor how these patterns change upon genetic or pharmacological perturbations. However, existing sample-preparation protocols often rely on costly and highly specialized equipment for vitrification and dehydration, limiting their widespread adoption. Here, we present an adapted plunge-freezing and freeze-drying workflow that enables the preparation of mammalian cell samples for X-ray fluorescence (XRF) and X-ray absorption spectroscopy (XAS) studies with submicron resolution in a cost-effective and versatile manner. Furthermore, we define acquisition parameters optimized for the reliable detection of low-abundance metals, such as endogenous iron. We anticipate that this accessible protocol will facilitate the broader implementation of synchrotron-based inner-shell spectromicroscopy in cell biology.

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Abstract: Nickel-based single crystal superalloys exploit a γ/γʹ microstructure, providing exceptional high- temperature strength and environmental stability, outperforming other known alloy systems. These properties make them useful for gas turbine applications. However, enhancing gas turbine efficiency requires higher operating temperatures, which place greater temperature and stress demands on these materials. One critical factor influencing their mechanical properties is the lattice misfit between the γ and γʹ phases. Consequently, extensive research has been dedicated to optimising lattice misfit values to achieve the desired mechanical performance. Accurate determination of lattice misfit is challenging due to the similar cubic crystal structures of the two phases. Conventional diffraction analyses are complicated by peak overlap of these phases, making precise peak position measurements difficult. To address this, a series of synchrotron X-ray, and neutron diffraction studies were conducted to explore the possibility of more reliable lattice misfit determination and how it varies across the microstructure as well as during deformation. Firstly, a monochromatic synchrotron X-ray source was used to investigate a high misfit Ni-based single crystal superalloy, which involved rocking the sample to ensure the Bragg condition was maintained during in situ loading at elevated temperature. Whilst this technique highlighted difficulties in fitting diffraction peaks where the Ewald sphere was not perfectly intersecting the reciprocal lattice point, it also revealed substantial differences in the lattice parameter when sampling regions across the γ/γʹ interfacial region and the bulk material. This suggested that further exploration of the interfacial region and the effect it has on the overall mechanical properties of the material is required. For this reason, a novel X-ray diffraction (XRD) approach utilising a synchrotron X-ray nanoprobe was developed to analyse local lattice parameters in the γʹ and γ phases. As a result of current technological constraints, and challenges in aligning single crystal superalloys to satisfy the Bragg condition, there were complications in accessing the diffraction signal. However, the method yielded reasonable results and demonstrated capabilities beyond those of conventional techniques that only probe larger volumes of material. Local misorientations and significant lattice parameter variations were observed, demonstrating the heterogeneity of the microstructure at smaller length scales. As such, future in situ studies using this technique appear promising. Whilst in situ heating experiments were unsuccessful in yielding diffraction data, the X-ray fluorescence (XRF) collected in conjunction demonstrated the potential for compositional analysis at elevated temperatures, particularly for polycrystalline or thermally unstable alloys. Additionally, pulsed source neutron diffraction was used to track lattice rotations during tensile deformation, employing a multi-peak 2D Gaussian fitting approach. While the measurements from this experiment do not directly indicate how slip progressed the extent of misorientation occurring during slip was successfully quantified. Furthermore, conventional fitting techniques were used to assess the evolution of lattice misfit during tensile deformation, corroborating the measurements obtained in previous studies. This research demonstrates both the challenges and potential of diffraction techniques for enhanced characterisation. While the deconvolution of γʹ and γ phase peak positions remains a significant challenge, valuable insights have been gained into stress gradients at interfacial regions and local misorientations within the microstructure. Furthermore, a foundation for methods to probe local microstructural changes and track lattice rotation during deformation have been established.

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Abstract: The deposition of calcium phosphate mineral within a collagen matrix plays a pivotal role in the formation of bone and teeth, yet understanding its precise mechanism and time-dependence remains a significant challenge. Conventional approaches are often constrained to ex situ studies and as a result the intrinsic dynamics governing collagen mineralization remains unclear. To address this knowledge gap, we developed a custom thermal flow cell to enable in situ characterization using Raman spectroscopy and small/wide-angle X-ray scattering for comparable sample settings. This approach allowed us to monitor the intricate process of collagen matrix mineralization from the initial infiltration of precursor phases to the formation of intermediate phosphate phases, and ultimately to the predominant growth of hydroxyapatite. Our findings reveal a striking expansion of the collagen matrix during initial infiltration, followed by compression in the early stages of mineralization, likely driven by water expulsion, which suggests the development of pre-stress similar to that observed in bone. As mineralization progressed, the matrix expanded once again, correlated with crystal growth. Post-mortem analyses confirmed the presence of intrafibrillar mineralization, with remarkable agreement to bone formation at up to 9 h of mineralization before over-mineralization occurred. Our study further identified a tessellated mineralization pattern within the collagen matrix, a feature also seen in bone, pointing to a highly regulated physico-chemical control of the mineralization dynamics. These insights deepen our understanding of the fundamental processes governing bone mineralization with broad implications for designing advanced biomaterials.

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Abstract: Understanding the role of metal ions in normal and abnormal cell function continues to emerge as a critical research area in the biological and biochemical sciences. This is especially true in the context of brain health and neurodegenerative diseases, as the brain is especially enriched in metal ions. A range of microscopy and bioanalytical techniques are available to assist in characterizing and observing changes to the brain metallome. As is the case in many other scientific fields, the integration of multiple analytical methods often yields a more complete chemical picture and deeper biological understanding. Herein, we present a case study applying 4 different analytical methods to provide spatially resolved characterization of chemical and biochemical parameters relating to the iron (Fe) metallome within a specific brain region, cornu ammonis sector 1 (CA1) of the hippocampus. The CA1 hippocampal sector was chosen for investigation due to its known endogenous enrichment in Fe and its selective vulnerability to neurodegeneration. The 4 analytical techniques applied were X-ray fluorescence microscopy (to quantify Fe distribution); X-ray absorption near-edge structure (XANES) spectroscopy to reveal information on Fe oxidation state and coordination environment; immuno-fluorescence to reveal relative abundance of Fe storage proteins (heavy chain ferritin and mitochondrial ferritin); and spatial transcriptomics to reveal gene expression pathways relevant to Fe homeostasis. Collectively, the results highlight that although pyramidal neurons in lateral and medial regions of the hippocampal CA1 sector are morphologically similar, key differences in the Fe metallome are evident. The observed differences within the hippocampal CA1 sector potentially indicate a higher oxidative environment and higher metabolic turnover in medial CA1 neurons relative to lateral CA1 neurons, which may account for the heightened vulnerability to neurodegeneration that is observed in the medial CA1 sector.