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Dementia has no cure and is an international health crisis. In addition to the immeasurable loss of QOL caused by dementia, the global economic cost is predicted to reach $2 trillion (USD) by 2030. Although much remains unknown about the biochemical pathways driving cognitive decline and memory loss during dementia, metals have been implicated in neurodegenerative disease. For example, total levels of Fe and Cu increase, which has been proposed to drive oxidative stress; and Fe, Cu, and Zn can bind amyloid-β, catalysing aggregation and formation of amyloid plaques. Unfortunately, despite these known facets through which metal ions may induce pathology, studies in greater detail have been hampered by a lack of microscopy methods to directly visualise metal ions, and their chemical form, within brain cells. Herein we report the use of synchrotron X-ray fluorescence microscopy to simultaneously image Fe, Cu, and Zn within neurons in ex vivo brain tissue sections. Using animal models of dementia, we now demonstrate for the first time that despite global increases in brain metal content and metal ion accumulation within amyloid plaques, key brain regions may also become metal ion deficient. Such deficiency could contribute to cognitive decline because of the essential roles metal ions play in neurotransmitter synthesis and energy metabolism. These recent findings are discussed in the context of memory loss, and the impact that metal ion dis-homeostasis may have on diagnostic and therapeutic development.

Zinc is a prominent trace metal required for normal memory function. Memory loss and cognitive decline during natural ageing and neurodegenerative disease have been associated with altered brain-Zn homeostasis. Yet, the exact chemical pathways through which Zn influences memory function during health, natural ageing, or neurodegenerative disease remain unknown. The gap in the literature may in part be due to the difficulty to simultaneously image, and therefore, study the different chemical forms of Zn within the brain (or biological samples in general). To this extent, we have begun developing and optimising protocols that incorporate X-ray absorption near-edge structure (XANES) spectroscopic analysis of tissue at the Zn K-edge as an analytical tool to study Zn speciation in the brain. XANES is ideally suited for this task as all chemical forms of Zn are detected, the technique requires minimal sample preparation that may otherwise redistribute or alter the chemical form of Zn, and the Zn K-edge has known sensitivity to coordination geometry and ligand type. Herein, we report our initial results where we fit K-edge spectra collected from micro-dissected flash-frozen brain tissue, to a spectral library prepared from standard solutions, to demonstrate differences in the chemical form of Zn that exist between two brain regions, the hippocampus and cerebellum. Lastly, we have used an X-ray microprobe to demonstrate differences in Zn speciation within sub-regions of thin air-dried sections of the murine hippocampus; but, the corresponding results highlight that the chemical form of Zn is easily perturbed by sample preparation such as tissue sectioning or air-drying, which must be a critical consideration for future work.

Aims: There are major knowledge gaps in understanding the translocation leading from nickel uptake in the root to accumulation in other tissues in tropical nickel hyperaccumulator plant species. This study focuses on two species, Rinorea cf. bengalensis and Rinorea cf. javanica and aims to elucidate the similarities and differences in the distribution of nickel and physiologically relevant elements (potassium, calcium, manganese and zinc) in various organs and tissues. Methods: High-resolution X-ray fluorescence microscopy (XFM) of frozen-hydrated and fresh-hydrated tissue samples and nuclear microprobe (micro-PIXE) analysis of freeze-dried samples were used to provide insights into the in situ elemental distribution in these plant species. Results: This study has shown that the distribution pattern of nickel hyperaccumulation is typified by very high levels of accumulation in the phloem bundles of roots and stems. In the leaves, nickel is preferentially located in epidermal cell region, whereas manganese is located mainly in the lower epidermis and zinc in the upper epidermis and palisade mesophyll. The abundant formation of calcium-oxalate crystals, lining the collenchyma, is a prominent feature of both Rinorea cf. bengalensis and Rinorea cf. javanica. Conclusions: Future investigations on Rinorea cf. bengalensis and Rinorea cf. javanica should focus on unravelling the mechanisms of nickel uptake in the root, specifically targeting the identification of nickelspecific membrane transporters.

Sediments that are rich in organic matter (OM) can trap and accumulate metals, including uranium (U), and provide favourable conditions for the formation of minerals such as pyrite. In OM-sediments, pyrite may play an important role in sorption and reduction of U and other metals that are sensitive to redox change. Mulga Rock, located in Western Australia, is an Eocene polymetallic deposit hosting economic uranium within its OM-rich sediments. Our previous work on the Mulga Rock deposits showed that OM may host U(VI) for long periods and suggested that reduction to U(IV) may occur through different pathways. Framboidal-like pyrite, embedded in lignite, displaying U(IV)-rich rims, >100 µm, were investigated using synchrotron-X-ray fluorescence microscopy-micro-X-ray absorption near edge structure (XFM-µXANES), laboratory micro-X-ray diffraction (µXRD) and electron microscopy. Micro-XRD analyses indicated that the U(IV) phase was mostly coffinite [USiO4] mixed with uraninite [UO2]. Analyses of transects across U-pyrite rims using XFM and XFM-µXANES revealed higher concentrations of U(IV) near the pyrite, and proportionally more U(VI) away from the edge of the pyrite. We infer that within OM environments, pyrite has a local influence on the oxidation state of U, which may be influential when considering U mobility.

The first comprehensive structural study of the Ni–U–O system is reported. Single crystals of α-NiUO4, β-NiUO4, and NiU3O10 were synthesized, and their structures were refined—using synchrotron single-crystal X-ray diffraction data supported by X-ray absorption spectroscopic measurements. α-NiUO4 adopts an orthorhombic structure in space group Pbcn and is isostructural to CrUO4 containing corrugated two-dimensional (2D) layers of corner-sharing UO6 polyhedra and edge-sharing one-dimensional (1D) zigzag α-PbO2 rutile-like chains of NiO6 polyhedra in the [001] direction. β-NiUO4 is isostructural to MgUO4 and has an orthorhombic structure in space group Ibmm, which contains alternating 1D chains of edge-sharing UO6 and NiO6 polyhedra in the [001] direction as in regular TiO2 rutile. NiU3O10 forms a triclinic structure in space group P1 and is isostructural with CuU3O10, where it forms a three-dimensional (3D) framework structure built through a mixture of UO6 and UO7 polyhedra in which the NiO6 polyhedra si...

Intravenous administration of the last-line polymyxins results in poor drug exposure in the lungs and potential nephrotoxicity, whereas inhalation therapy offers better pharmacokinetics/pharmacodynamics for pulmonary infections by delivering the antibiotic directly to the infection site. However, polymyxin inhalation therapy has not been optimized, and adverse effects can occur. This study aimed to quantitatively determine the intracellular accumulation and distribution of polymyxins in single human alveolar epithelial A549 cells. Cells were treated with the iodine-labeled polymyxin probe FADDI-096 (5.0 and 10.0 μM) for 1, 4, and 24 h. Concentrations of FADDI-096 in single A549 cells were determined by synchrotron-based X-ray fluorescence microscopy. Concentration- and time-dependent accumulation of FADDI-096 within A549 cells was observed. The intracellular concentrations (mean ± standard error of the mean [SEM], n ≥ 189) of FADDI-096 were 1.58 ± 0.11, 2.25 ± 0.10, and 2.46 ± 0.07 mM following 1, 4, and 24 h of treatment at 10 μM, respectively. The corresponding intracellular concentrations following the treatment at 5 μM were 0.05 ± 0.01, 0.24 ± 0.04, and 0.25 ± 0.02 mM (n ≥ 189). Over 24 h, FADDI-096 was mainly localized throughout the cytoplasm and nuclear region. The intracellular zinc concentration increased in a concentration- and time-dependent manner. This is the first study to quantitatively map the accumulation of polymyxins in human alveolar epithelial cells, and it provides crucial insights for deciphering the mechanisms of their pulmonary toxicity. Importantly, our results may shed light on the optimization of inhaled polymyxins in patients and the development of new-generation, safer polymyxins.

High-accuracy X-ray mass attenuation coefficients were measured from the first X-ray Extended Range Technique (XERT)-like experiment at the Australian Synchrotron. Experimentally measured mass attenuation coefficients deviate by ∼50% from the theoretical values near the zinc absorption edge, suggesting that improvements in theoretical tabulations of mass attenuation coefficients are required to bring them into better agreement with experiment. Using these values the imaginary component of the atomic form factor of zinc was determined for all the measured photon energies. The zinc K-edge jump ratio and jump factor are determined and results raise significant questions regarding the definitions of quantities used and best practice for background subtraction prior to X-ray absorption fine-structure (XAFS) analysis. The XAFS analysis shows excellent agreement between the measured and tabulated values and yields bond lengths and nanostructure of zinc with uncertainties of from 0.1% to 0.3% or 0.003 Å to 0.008 Å. Significant variation from the reported crystal structure was observed, suggesting local dynamic motion of the standard crystal lattice. XAFS is sensitive to dynamic correlated motion and in principle is capable of observing local dynamic motion beyond the reach of conventional crystallography. These results for the zinc absorption coefficient, XAFS and structure are the most accurate structural refinements of zinc at room temperature.

Hyperaccumulation is generally highly specific for a single element, for example nickel (Ni). The recently-discovered hyperaccumulator Glochidion cf. sericeum (Phyllanthaceae) from Malaysia is unusual in that it simultaneously accumulates nickel and cobalt (Co) with up to 1500 μg g−1 foliar of both elements. We set out to determine whether distribution and associated ligands for Ni and Co complexation differ in this species. We postulated that Co hyperaccumulation coincides with Ni hyperaccumulation operating on similar physiological pathways. However, the ostensibly lower tolerance for Co at the cellular level results in the exudation of Co on the leaf surface in the form of lesions. The formation of such lesions is akin to phytotoxicity responses described for manganese (Mn). Hence, in contrast to Ni, which is stored principally inside the foliar epidermal cells, the accumulation response to Co consists of an extracellular mechanism. The chemical speciation of Ni and Co, in terms of the coordinating ligands involved and principal oxidation state, is similar and associated with carboxylic acids (citrate for Ni and tartrate or malate for Co) and the hydrated metal ion. Some oxidation to Co3+, presumably on the surface of leaves after exudation, was observed.

Coordination chemistry enables a variety of vital functions in biological systems; however, characterising the chemical form of metal ions in cells and tissue is notoriously difficult. One technique that is gaining substantial momentum in this research area is X-ray absorption near-edge structure (XANES) spectroscopy. The XANES spectrum can be a rich source of information with respect to the coordination environment of metal ions. Further, XANES spectroscopy is compatible with microscopy mapping protocols as the spectra are recorded across a relatively narrow range of data points (typically 50–100). Although the potential of XANES spectroscopy to study metal ion coordination chemistry has long been known, data collection speed has only relatively recently reached the state in which maps can be collected with a full spectrum per pixel. The realisation of this capability now places XANES spectroscopic mapping among a suite of other spectroscopic imaging techniques, such as Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy, which are available to characterise biochemical composition, in situ within cells and tissue. Herein, we report a proof-of-concept application of XANES spectroscopic mapping to begin exploration of Fe speciation in brain tissue, which demonstrates the potential of this method for the biomedical sciences, and identifies important areas for consideration with respect to future protocol developments.

Analytical approaches that preserve the endogenous state of the examined system are essential for the in vivo study of bioinorganics. X-ray fluorescence microscopy of biological samples can map elements in vivo at subcellular resolutions in tissue samples and multicellular organisms. However, X-ray irradiation induces modifications that accumulate with dose. Consequently, the utility of X-ray fluorescence microscopy is intrinsically limited by the radiation damage it causes and the degree to which it alters the target features of interest. Identification of the dose threshold, below which the integrity of the specimen and its elemental distribution is preserved, is required to ensure valid interpretation of concentrations. Here we use the nematode, Caenorhabditis elegans, to explore these issues using three chemical-free specimen preparations: lyophilization, cryofixation, and live. We develop quantitative methods for investigating damage and present dose limits for each preparation pertaining to the micrometer-scale spatial distribution of specific analytes (potassium, calcium, manganese, iron, and zinc), and discuss dose-appropriate guidelines for X-ray fluorescence microscopy of microscale biological samples.

Lactobacillus spp. are known to accumulate large amounts of inorganic manganese, which protects against oxidative damage by scavenging free radicals. The ability of probiotic L. paracasei ATCC 55544 to maintain viability during long-term ambient storage may be enhanced by this microorganism's ability to accumulate manganese, which may act as a free radical scavenger. To investigate this hypothesis, X-ray fluorescence microscopy (XFM) was employed to determine the changes in the elemental composition of L. paracasei during growth in the MRS medium with or without added manganese. Moreover, manganese uptake by cells as a function of physiological growth state, early log vs. stationary phase was evaluated. The semiquantitative X-ray fluorescence microscopy results revealed that lower levels of manganese accumulation occurred during the early log phase of bacterial growth of L. paracasei cells (0.0064 µg/cm2) compared with the stationary phase cells (0.1355 µg/cm2). L. paracasei cells grown in manganese deficient MRS medium resulted in lower manganese uptake by cells (0.0027 µg/cm2). The L. paracasei cells were further embedded in milk powder matrix using a fluidized-bed drying technique and stored at a water activity (aw) of 0.33 at 25 °C for 15 days. The viability counts of L. paracasei cells grown in MRS medium harvested after 18 h growth and embedded in milk powder matrix retained viability of (9.19 ± 0.12 log CFU/g). No viable L. paracasei cells were observed in the case of embedded L. paracasei cells grown in manganese-deficient MRS medium harvested after 18 h growth or in the case of L. paracasei cells harvested after 4 h when grown in MRS medium. The lower level of manganese accumulation was found to be related to the loss of bacterial viability during storage.

This study compared lead (Pb) immobilization efficacies in mining/smelting impacted soil using phosphate and iron amendments via ingestion and inhalation pathways using in vitro and in vivo assays, in conjunction with investigating the dynamics of dust particles in the lungs and gastro-intestinal tract via X-ray fluorescence (XRF) microscopy. Phosphate amendments [phosphoric acid (PA), hydroxyapatite, monoammonium phosphate (MAP), triple super phosphate (TSP), and bone meal biochar] and hematite were applied at a molar ratio of Pb:Fe/P = 1:5. Pb phosphate formation was investigated in the soil/post-in vitro bioaccessibility (IVBA) residuals and in mouse lung via extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge structures (XANES) spectroscopy, respectively. EXAFS analysis revealed that anglesite was the dominant phase in the ingestible (

Fingermarks are an important form of crime-scene trace evidence; however, their usefulness may be hampered by a variation in response or a lack of robustness in detection methods. Understanding the chemical composition and distribution within fingermarks may help explain variation in latent fingermark detection with existing methods and identify new strategies to increase detection capabilities. The majority of research in the literature describes investigation of organic components of fingermark residue, leaving the elemental distribution less well understood. The relative scarcity of information regarding the elemental distribution within fingermarks is in part due to previous unavailability of direct, micron resolution elemental mapping techniques. This capability is now provided at third generation synchrotron light sources, where X-ray Fluorescence Microscopy (XFM) provides micron or sub-micron spatial resolution and direct detection with sub-μM detection limits. XFM has been applied in this study to reveal the distribution of inorganic components within fingermark residue, including endogenous trace metals (Fe, Cu, Zn), diffusible ions (Cl-, K+, Ca2+), and exogeneous metals (Ni, Ti, Bi). This study incorporated a multi-modal approach using XFM and Infrared Microspectroscopy (IRM) analyses to demonstrate co-localisation of endogenous metals within the hydrophilic organic components of fingermark residue. Additional experiments were then undertaken to investigate how sources of exogenous metals (e.g. coins and cosmetics) may be transferred to, and distributed within latent fingermarks. Lastly, this study reports a preliminary assessment of how environmental factors such as exposure to aqueous environments may effect elemental distribution within fingermarks. Taken together, the results of this study advance our current understanding of fingermark composition and its spatial distribution of chemical components, and may help explain detection variation observed during detection of fingermarks using standard forensic protocols.

It is well established that an array of avian species sense the Earth's magnetic field and use this information for orientation and navigation. While the existence of a magnetic sense can no longer be disputed, the underlying cellular and biophysical basis remains unknown. It has been proposed that pigeons exploit a magnetoreceptor based on magnetite crystals (Fe3O4) that are located within the lagena [1], a sensory epithelium of the inner ear. It has been hypothesised that these magnetic crystals form a bed of otoconia that stimulate hair cells transducing magnetic information into a neuronal impulse. We performed a systematic high-sensitivity screen for iron in the pigeon lagena using synchrotron X-ray fluorescence microscopy coupled with the analysis of serial sections by transmission electron microscopy. We find no evidence for extracellular magnetic otoconia or intracellular magnetite crystals, suggesting that if an inner ear magnetic sensor does exist it relies on a different biophysical mechanism.

Lead (Pb) exposure from household dust is a major childhood health concern because of its adverse impact on cognitive development. This study investigated the absorption kinetics of Pb from indoor dust following a single dose instillation into C57BL/6 mice. Blood Pb concentration (PbB) was assessed over 24 h, and the dynamics of particles in the lung and gastro-intestinal (GI)tract were visualized using X-ray fluorescence (XRF) microscopy. The influence of mineralogy on Pb absorption and particle retention was investigated using X-ray absorption near-edge structure spectroscopy. A rapid rise in PbB was observed between 0.25 and 4 h after instillation, peaking at 8 hand slowly declining during a period of 24 h. Following clearance from the lungs, Pb particles were detected in the stomach and small intestine at 4 and 8 h, respectively. Analysis of Pb mineralogy in the residual particles in tissues at 8 h showed that mineral-sorbed Pb and Pb-phosphates dominated the lung, while organic-bound Pb and galena were the main phases in the small intestines. This is the first study to visualize Pb dynamics in the lung and GI tract using XRF microscopy and link the inhalation and ingestion pathways for metal exposure assessment from dust. Refereed/Peer-reviewed

The fern Pteris vittata has been the subject of numerous studies because of its extreme arsenic hyperaccumulation characteristics. However, information on the arsenic chemical speciation and distribution across cell types within intact frozen-hydrated Pteris vittata fronds is necessary to better understand the arsenic biotransformation pathways in this unusual fern. While 2D X-ray absorption spectroscopy imaging studies show that different chemical forms of arsenic, As(III) and As(V), occur across the plant organs, depth-resolved information on arsenic distribution and chemical speciation in different cell types within tissues of Pteris vittata have not been reported. By using a combination of planar and confocal μ-X-ray fluorescence imaging and fluorescence computed μ-tomography, we reveal, in this study, the localization of arsenic in the endodermis and pericycle surrounding the vascular bundles in the rachis and the pinnules of the fern. Arsenic is also accumulated in the vascular bundles connecting into each sporangium, and in some mature sori. The use of 2D X-ray absorption near edge structure imaging allows for deciphering arsenic speciation across the tissues, revealing arsenate in the vascular bundles and arsenite in the endodermis and pericycle. This study demonstrates how different advanced synchrotron X-ray microscopy techniques can be complementary in revealing, at tissue and cellular levels, elemental distribution and chemical speciation in hyperaccumulator plants.

The biologically important metals such as zinc, copper and iron play key roles in retinal function, yet no study has mapped the spatio-temporal distribution of retinal biometals in healthy or diseased retina. We investigated a natural mouse model of retinal degeneration, the Cln6nclf mouse. As dysfunctional metabolism of biometals is observed in the brains of these animals and deregulated metal homeostasis has been linked to retinal degeneration, we focused on mapping the elemental distribution in the healthy and Cln6nclf mouse retina with age. Retinal and RPE elemental homeostasis was mapped in Cln6nclf and C57BL6/J mice from 1 to 8 months of age using X-ray Fluorescence Microscopy at the Australian Synchrotron. In the healthy retina, we detected a progressive loss of phosphorus in the outer nuclear layer and significant reduction in iron in the inner segments of the photoreceptors. Further investigation revealed a unique elemental signature for each retinal layer, with high areal concentrations of iron and sulfur in the photoreceptor segments and calcium, phosphorus, zinc and potassium enrichment predominantly in the nuclear layers. The analysis of retinae from Cln6nclf mice did not show significant temporal changes in elemental distributions compared to age matched controls, despite significant photoreceptor cell loss. Our data therefore demonstrates that retinal layers have unique elemental composition. Elemental distribution is, with few exceptions, stably maintained over time in healthy and Cln6nclf mouse retina, suggesting conservation of elemental distribution is critical for basic retinal function with age and is not modulated by processes underlying retinal degeneration.

Gold nanoparticles (AuNPs) have been widely investigated in drug delivery and imaging. However, for such biomedical applications, the modification of AuNPs is necessary to improve their aqueous dispersion stability and biocompatibility, especially in a salt environment. Here, we report a simple and highly efficient method to create a library of polymer-modified gold nanoparticles (PAuNPs) and screen their dispersion stability utilizing high-throughput facilities (in total 1000 experiments). Three types of water soluble polymers with different charge and chain length were prepared using Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization technology. The polymers were then converted into thiol-capped polymers by aminolysis and coated on AuNPs via thiol–gold binding to improve aqueous dispersion stability as well as the biocompatibility of AuNPs. Remarkably, we present the first report of imaging zebrafish embryos injected with a PAuNPs sample (selected from our PAuNPs library) using synchrotron X-ray fluorescence microscopy (XFM) beamline. The selected PAuNPs sample, which has been evaluated to be non-cytotoxic to L929 cells and biocompatible to zebrafish larvae in a wide range of concentrations, was injected into zebrafish larvae via the cardinal vein and could be clearly visualized in the whole circulatory system including both peripheral blood vessels and the head region by XFM. The result indicates that zebrafish larvae could be a potential animal model for probing the ability of AuNPs to cross the blood–brain barrier (BBB) and therefore hold promise for investigating AuNPs in biomedical applications such as detecting cancer and Alzheimer's disease (AD).

Heavy metal pollution is a concern in many coastal locations where it is frequently deleterious to the survival of young shellfish. Consequently, a great number of commercial shellfish hatcheries around the world rely on the addition of ethylenediaminetetraacetic acid (EDTA) to seawater to ensure successful larval production. Despite the importance of this practice to global shellfish production the mode of action of EDTA in larval production remains undetermined. It is assumed EDTA chelates heavy metals in seawater preventing interference in larval development. Larval mussels (Perna canaliculus) raised in seawater with 3 μM EDTA had a 15 fold higher yield than those without EDTA. The concentration and spatial arrangement of heavy metals in larvae as determined by Inductively Coupled Plasma Mass Spectrometry (ICPMS) and X-ray Fluorescence Microscopy (XFM) was consistent with reduced bioavailability of several metals, especially copper and zinc. This is the first study to confirm the effectiveness of EDTA for managing metal pollution commonly encountered in coastal shellfish hatcheries.

Background and Aims: Understanding the spatial distribution of inorganic nutrients within edible parts of plant products helps biofortification efforts to identify and focus on specific uptake pathways and storage mechanisms. Methods: Kernels of sweetcorn (Zea mays) variety 'High zeaxanthin 103146' and maize inbred line 'Thai Floury 2' were harvested at two different maturity stages, and the distributions of K, P, S, Ca, Zn, Fe and Mn were examined in situ using synchrotron-based X-ray fluorescence microscopy. Key Results: The distribution of inorganic nutrients was largely similar between maize and sweetcorn, but differed markedly depending upon the maturity stage after further embryonic development. The micronutrients Zn, Fe and Mn accumulated primarily in the scutellum of the embryo during early kernel development, while trace amounts of these were found in the aleurone layer at the mature stage. Although P accumulated in the scutellum, there was no direct relationship between the concentrations of P and those of the micronutrients, compared with the linear trend between Zn and Fe concentrations. Conclusions: This study highlights the important role of the embryo as a micronutrient reserve for sweetcorn and maize kernels, and the need to understand how biofortification efforts can further increase the inorganic nutrient concentration of the embryo for human consumption.

Background and Aims The pathways whereby foliar-applied nutrients move across the leaf surface remain unclear. The aim of the present study was to examine the pathways by which foliar-applied Zn moves across the sunflower (Helianthus annuus) leaf surface, considering the potential importance of the cuticle, stomata and trichomes.Methods Using synchrotron-based X-ray florescence microscopy and nanoscale secondary ion mass spectrometry (NanoSIMS), the absorption of foliar-applied ZnSO4 and nano-ZnO were studied in sunflower. The speciation of Zn was also examined using synchrotron-based X-ray absorption spectroscopy.Key Results Non-glandular trichomes (NGTs) were particularly important for foliar Zn absorption, with Zn preferentially accumulating within trichomes in 15 min. The cuticle was also found to have a role, with Zn appearing to move across the cuticle before accumulating in the walls of the epidermal cells. After 6 h, the total Zn that accumulated in the NGTs was approx. 1.9 times higher than in the cuticular tissues. No marked accumulation of Zn was found within the stomatal cavity, probably indicating a limited contribution of the stomatal pathway. Once absorbed, the Zn accumulated in the walls of the epidermal and the vascular cells, and trichome bases of both leaf sides, with the bundle sheath extensions that connected to the trichomes seemingly facilitating this translocation. Finally, the absorption of nano-ZnO was substantially lower than for ZnSO4, with Zn probably moving across the leaf surface as soluble Zn rather than nanoparticles.Conclusions In sunflower, both the trichomes and cuticle appear to be important for foliar Zn absorption. Refereed/Peer-reviewed

Contents 'Summary' I. 'Introduction' II. 'Preparation of plant samples for X-ray micro-analysis' III. 'X-ray elemental mapping techniques' IV. 'X-ray data analysis' V. 'Case studies' VI. 'Conclusions' 'Acknowledgements' 'Author contributions' References Summary Hyperaccumulators are attractive models for studying metal(loid) homeostasis, and probing the spatial distribution and coordination chemistry of metal(loid)s in their tissues is important for advancing our understanding of their ecophysiology. X-ray elemental mapping techniques are unique in providing in situ information, and with appropriate sample preparation offer results true to biological conditions of the living plant. The common platform of these techniques is a reliance on characteristic X-rays of elements present in a sample, excited either by electrons (scanning/transmission electron microscopy), protons (proton-induced X-ray emission) or X-rays (X-ray fluorescence microscopy). Elucidating the cellular and tissue-level distribution of metal(loid)s is inherently challenging and accurate X-ray analysis places strict demands on sample collection, preparation and analytical conditions, to avoid elemental redistribution, chemical modification or ultrastructural alterations. We compare the merits and limitations of the individual techniques, and focus on the optimal field of applications for inferring ecophysiological processes in hyperaccumulator plants. X-ray elemental mapping techniques can play a key role in answering questions at every level of metal(loid) homeostasis in plants, from the rhizosphere interface, to uptake pathways in the roots and shoots. Further improvements in technological capabilities offer exciting perspectives for the study of hyperaccumulator plants into the future.

WOS:000355152300030 Biofortification of wheat with zinc (Zn) through foliar Zn application has been proposed as an agronomic strategy to increase grain Zn concentration, which could serve as a nutritional intervention in regions with dietary Zn deficiency. Bread wheat (Triticum aestivum L.) was biofortified through foliar Zn applications at different growth stages. The concentration of Zn and associated micronutrient in harvested whole grains was determined by ICP-OES. Synchrotron-based X-ray fluorescence microscopy (XFM) was then used to investigate the localization of Zn and associated micronutrients in cross sections of these grains. The concentration of Zn and other micronutrients (Mn, Fe, and Cu) was higher in grains treated with foliar Zn during grain-filling (early milk/dough) than those treated at stem elongation. The increase in Zn concentration of wheat grain with foliar application during grain-filling can be attributed to the intense localization of Zn in the aleurone layer, modified aleurone, crease tissue, vascular bundle, and endosperm cavity, and to a modest localization in endosperm, which is the most dominant grain tissue. These tissues and the Zn they contain are presumed to remain after milling and can potentially increase the Zn concentration in wheat flour. By using XFM, it was shown that foliar Zn spray represents an important agronomic tool for a substantial Zn enrichment of different fractions of wheat grain, especially the endosperm. Further investigation of the chemical speciation of Zn in the endosperm is recommended to assess Zn bioavailability in harvested whole grain of wheat that has been biofortified through different timing of foliar Zn application. HarvestPlus program; Mosaic Co This research was undertaken on the X-ray Fluorescence Microscopy (XFM) beamline at the Australian Synchrotron, Victoria Australia. The sectioning of the grains with the vibratome was carried out at the Australian Centre for Plant Functional Genomics (ACPFG), which was facilitated by Dr Gwen Mayo. Ms Casey Doolette helped in preparing the transverse sections. This study was partially supported by the HarvestPlus program (www.harvestplus.org). Financial support by The Mosaic Co to the Fertilizer Technology Research Centre is duly acknowledged. The authors will like to thank Enzo Lombi for his useful comments during the draft manuscript review.

Iron is essential for eukaryotic biochemistry. Systematic trafficking and storage is required to maintain supply of iron while preventing it from catalysing unwanted reactions, particularly the generation of oxidising reactive species. Iron dyshomeostasis has been implicated in major age-associated diseases including cancers, neurodegeneration and heart disease. Here, we employ population-level X-ray fluorescence imaging and native-metalloproteomic analysis to determine that altered iron coordination and distribution is a pathological imperative of ageing in the nematode, Caenorhabditis elegans. Our approach provides a method to simultaneously study iron metabolism across different scales of biological organisation, from populations to cells. Here we report how and where iron homeostasis is lost during C. elegans ageing, and its relationship to the age-related elevation of damaging reactive oxygen species. We find that wild types utilise ferritin to sustain longevity, buffering against exogenous iron and showing rapid ageing if ferritin is ablated. After reproduction, escape of iron from safe-storage in ferritin raised cellular Fe2+ load in the ageing C. elegans, and increased generation of reactive species. These findings support the hypothesis that iron-mediated processes drive senescence. We propose that loss of iron homeostasis may be a fundamental and inescapable consequence of ageing that could represent a critical target for therapeutic strategies to improve health outcomes in ageing.

Gold(I) complexes are an important tool in the arsenal of established approaches for treating rheumatoid arthritis (RA), while some recent studies have suggested that gold nanoparticles (Au NPs) may also be therapeutically efficacious. These observations prompted the current biological studies involving gold(I) anti-RA agents and Au NPs, which are aimed towards improving our knowledge of how they work. The cytotoxicity of auranofin, aurothiomalate, aurothiosulfate and Au NPs towards RAW264.7 macrophages was evaluated using the MTT assay, with the former compound proving to be the most toxic. The extent of cellular uptake of the various gold agents was determined using graphite furnace atomic absorption spectrometry, while their distribution within macrophages was examined using microprobe synchrotron radiation X-ray fluorescence spectroscopy. The latter technique showed accumulation of gold in discrete regions of the cell, and co-localisation with sulfur in the case of cells treated with aurothiomalate or auranofin. Electrospray ionization mass spectrometry was used to characterize thioredoxin reductase (TrxR) in which the penultimate selenocysteine residue was replaced by cysteine. Mass spectra of solutions of TrxR and aurothiomalate, aurothiosulfate or auranofin showed complexes containing bare gold atoms bound to the protein, or protein adducts containing gold atoms retaining some of their initial ligands. These results support TrxR being an important target of gold(I) drugs used to treat RA, while the finding that Au NPs are incorporated into macrophages, but elicit little toxicity, indicates further exploration of their potential for treatment of RA is warranted.

A structure–activity relationship study of a library of novel bifunctional GdIII complexes covalently linked to arylphosphonium cations is reported. Such complexes have been designed for potential application in binary cancer therapies such as neutron capture therapy and photon activation therapy. A positive correlation was found between lipophilicity and cytotoxicity of the complexes. Mitochondria uptake was determined by means of inductively coupled plasma mass spectrometry (ICP‐MS), and Gd uptake was determined by means of quantification using synchrotron X‐ray fluorescence (XRF) imaging. A negative correlation between lipophilicity and tumour selectivity of the GdIII complexes was demonstrated. This study highlights the delicate balance required to minimise in vitro cytotoxicity and optimise in vitro tumour selectivity and mitochondrial localisation for this new class of mitochondrially‐targeted binary therapy agents. We also report the highest in vitro tumour selectivity for any Gd agent reported to date, with a T/N (tumour/normal cell) ratio of up to 23.5±6.6. Australian Research Council (ARC)

Adequate amounts of copper in milk are critical for normal neonatal development, however the mechanisms regulating copper supply to milk have not been clearly defined. PMC42-LA cell cultures representative of resting, lactating and suckled mammary epithelia were used to investigate the regulation of the copper uptake protein, CTR1. Both the degree of mammary epithelial differentiation (functionality) and extracellular copper concentration greatly impacted upon CTR1 expression and its plasma membrane association. In all three models (resting, lactating and suckling) there was an inverse correlation between extracellular copper concentration and the level of CTR1. Cell surface biotinylation studies demonstrated that as extracellular copper concentration increased membrane associated CTR1 was reduced. There was a significant increase in CTR1 expression (total and membrane associated) in the suckled gland model in comparison to the resting gland model, across all copper concentrations investigated (0-50 μM). Regulation of CTR1 expression was entirely post-translational, as quantitative real-time PCR analyses showed no change to CTR1 mRNA between all models and culture conditions. X-ray fluorescence microscopy on the differentiated PMC42-LA models revealed that organoid structures distinctively accumulated copper. Furthermore, as PMC42-LA cell cultures became progressively more specialised, successively more copper accumulated in organoids (resting

The metal ions of iron, copper, and zinc have long been associated with the aggregation of β-amyloid (Aβ) plaques in Alzheimer's disease; an interaction that has been suggested to promote increased oxidative stress and neuronal dysfunction. We examined plaque metal load in the hippocampus of APP/PS1 mice using X-ray fluorescence microscopy to assess how the anatomical location of Aβ plaques was influenced by the metal content of surrounding tissue. Immunohistochemical staining of Aβ plaques colocalized with areas of increased X-ray scattering power in unstained tissue sections, allowing direct X-ray based-assessment of plaque metal levels in sections subjected to minimal chemical fixation. We identified and mapped 48 individual plaques in four subregions of the hippocampus from four biological replicates. Iron, Cu, and Zn areal concentrations (ng cm-2) were increased in plaques compared to the surrounding neuropil. However, this elevation in metal load reflected the local metal makeup of the surrounding neuropil, where different brain regions are enriched for different metal ions. After correcting for tissue density, only Zn levels remained elevated in plaques. This study suggests that the in vivo binding of Zn to plaques is not simply due to increased protein deposition.

Asbestos bodies are the histological hallmarks of asbestos exposure. Both conventional and advanced techniques are used to evaluate abundance and composition in histological samples. We previously reported the possibility of using synchrotron X-ray fluorescence microscopy (XFM) for analyzing the chemical composition of asbestos bodies directly in lung tissue samples. Here we applied a high-performance synchrotron X-ray fluorescence (XRF) set-up that could allow new protocols for fast monitoring of the occurrence of asbestos bodies in large histological sections, improving investigation of the related chemical changes. A combination of synchrotron X-ray transmission and fluorescence microscopy techniques at different energies at three distinct synchrotrons was used to characterize asbestos in paraffinated lung tissues. The fast chemical imaging of the XFM beamline (Australian Synchrotron) demonstrates that asbestos bodies can be rapidly and efficiently identified as co-localization of high calcium and iron, the most abundant elements of these formations inside tissues (Fe up to 10% w/w; Ca up to 1%). By following iron presence, we were also able to hint at small asbestos fibers in pleural spaces. XRF at lower energy and at higher spatial resolution was afterwards performed to better define small fibers. These analyses may predispose for future protocols to be set with laboratory instruments.

• Accumulation of arsenic (As) within plant tissues represents a human health risk, but there remains much to learn regarding the speciation of As within plants. • We developed synchrotron-based fluorescence-X-ray absorption near-edge spectroscopy (fluorescence-XANES) imaging in hydrated and fresh plant tissues to provide laterally resolved data on the in situ speciation of As in roots of wheat (Triticum aestivum) and rice (Oryza sativa) exposed to 2 μM As(V) or As(III). • When exposed to As(V), the As was rapidly reduced to As(III) within the root, with As(V) calculated to be present only in the rhizodermis. However, no uncomplexed As(III) was detected in any root tissues, because of the efficient formation of the As(III)-thiol complex - this As species was calculated to account for all of the As in the cortex and stele. The observation that uncomplexed As(III) was below the detection limit in all root tissues explains why the transport of As to the shoots is low, given that uncomplexed As(III) is the major As species transported within the xylem and phloem. • Using fluorescence-XANES imaging, we have provided in situ data showing the accumulation and transformation of As within hydrated and fresh root tissues.

Many metals and metalloids, jointly termed metal(loid)s, are toxic to plants even at low levels. This has limited the study of their uptake, distribution, and modes of action in plant roots grown at physiologically relevant concentrations. Synchrotron-based X-ray fluorescence microscopy was used to examine metal(loid)s in hydrated cowpea (Vigna unguiculata L.) roots exposed to Zn(II), Ni(II), Mn(II), Cu(II), Hg(II), Se(IV), Se(VI), As(III), or As(V). Development of a mathematical model enabled in situ quantitative determination of their distribution in root tissues. The binding strength of metals influenced the extent of their movement through the root cylinder, which influenced the toxic effects exerted-metals (e.g. Cu, Hg) that bind more strongly to hard ligands had high concentrations in the rhizodermis and caused this tissue to rupture, while other metals (e.g. Ni, Zn) moved further into the root cylinder and did not cause ruptures.When longitudinal distributionswere examined, the highest Se concentration in roots exposed to Se(VI) was in the more proximal root tissues, suggesting that Se(VI) is readily loaded into the stele. This contrasted with other metal(loid)s (e.g. Mn, As), which accumulated in the apex. These differences inmetal(loid) spatial distribution provide valuable quantitative data on metal(loid) physiology, including uptake, transport, and toxicity in plant roots. Refereed/Peer-reviewed

The speciation and spatial distribution of selenium (Se) in hydrated plant tissues is not well understood. Using synchrotron-based x-ray absorption spectroscopy and x-ray fluorescence microscopy (two-dimensional scanning [and associated mathematical model] and computed tomography), the speciation and distribution of toxic Se were examined within hydrated roots of cowpea (Vigna unguiculata) exposed to either 20 µm selenite or selenate. Based upon bulk solution concentrations, selenate was 9-fold more toxic to the roots than selenite, most likely due to increased accumulation of organoselenium (e.g. selenomethionine) in selenate-treated roots. Specifically, uptake of selenate (probably by sulfate transporters) occurred at a much higher rate than for selenite (apparently by both passive diffusion and phosphate transporters), with bulk root tissue Se concentrations approximately 18-fold higher in the selenate treatment. Although the proportion of Se converted to organic forms was higher for selenite (100%) than for selenate (26%), the absolute concentration of organoselenium was actually approximately 5-fold higher for selenate-treated roots. In addition, the longitudinal and radial distribution of Se in roots differed markedly: the highest tissue concentrations were in the endodermis and cortex approximately 4 mm or more behind the apex when exposed to selenate but in the meristem (approximately 1 mm from the apex) when exposed to selenite. The examination of the distribution and speciation of Se in hydrated roots provides valuable data in understanding Se uptake, transport, and toxicity. Refereed/Peer-reviewed

In spite of the similar chemical properties of gold (Au) and platinum (Pt), Au is highly mobile in surface environments, whereas Pt appears to be far less so. In this study we assess if geomicrobial processes are likely to cause these differences, as the mobility of Pt and Au should differ little based on thermodynamic solubility alone. To achieve an accurate comparison it is important that both metals occur in the same environment. Mineral- and groundwater samples were obtained from the Fifield Pt–Au field in New South Wales, Australia, where Pt and Au nuggets occur in a series of Tertiary eluvial and alluvial paleo-placers. In particular, we studied the μm-scale dispersion of Au and Pt within an extraordinary 10 mm-sized fragment of ferruginous paleochannel material, which contained abundant native Au- and isoferroplatinum grains. Gold grains displayed complex secondary morphologies indicative of biogeochemical transformations, whereas isoferroplatinum grains appeared smooth and well-rounded and showed no signs of supergene transformation. Gold grains were surrounded by a dusting of highly pure metallic Au particles ( 10 μm in diameter), whereas no metallic Pt particles were detected. A search for ionic Pt was also unsuccessful. A series of biotic and abiotic incubation experiments was conducted to investigate the hypothesis that these differences in mobility are driven by interactions with microbiota. Biofilms consisting of metallophilic bacteria formed on ultraflat Au surfaces and caused significant surface transformations. In contrast, only subtle changes were observed on Pt surfaces incubated under similar conditions. Minimal inhibitory concentrations for Au complexes are more than an order of magnitude lower than those measured for Pt complexes in Cupriavidus metallidurans cells. This higher cell-toxicity of mobile Au- compared to Pt-complexes can lead to toxic levels of mobile Au in the vicinity of Au grain surfaces. The elevated toxicity drives the formation of Au-detoxifying biofilms that catalyze the biomineralization of spheroidal nano-particulate and bacteriomorphic Au. In contrast, this does not occur on isoferroplatinum grains due to the lower toxicity of Pt. In conclusion, these results are consistent with microbial adaptation to element toxicity driving the cycling of precious metals in surface environments.

Determining the age of sharks using vertebral banding is a vital component of management, but the causes of banding are not fully understood. Traditional shark ageing is based on fish otolith ageing methods where growth bands are assumed to result from varied seasonal calcification rates. Here we investigate these assumptions by mapping elemental distribution within the growth bands of vertebrae from six species of sharks representing four different taxonomic orders using scanning x-ray fluorescence microscopy. Traditional visual growth bands, determined with light microscopy, were more closely correlated to strontium than calcium in all species tested. Elemental distributions suggest that vertebral strontium bands may be related to environmental variations in salinity. These results highlight the requirement for a better understanding of shark movements, and their influence on vertebral development, if confidence in age estimates is to be improved. Analysis of shark vertebrae using similar strontium-focused elemental techniques, once validated for a given species, may allow more successful estimations of age on individuals with few or no visible vertebral bands.

Arsenic (As) is commonly associated with Cu ore minerals, with the resultant risk that As can be released offsite from mine tailings. We used synchrotron-based fluorescence X-ray absorption near-edge spectroscopy (XANES) imaging to provide in situ , laterally-resolved speciation of As within tailings which differed in magnetite content (5–12%) and organic matter content (0–5%). Although the total As content was lower in tailings with low magnetite (LM), the soluble (pore water) As was actually 7-times higher in LM tailings than in high magnetite (HM) tailings. Additionally, amendment with 5% sugarcane mulch residues (SMR) (for revegetation) further increased soluble As due to the dissolution and oxidation of arsenopyrite or orpiment. Indeed, in HM tailings, arsenopyrite and orpiment initially accounted for 88% of the total As, which decreased to 48% upon the addition of SMR – this being associated with an increase in As V -ferrihydrite from 12% to 52%. In LM tailings, the pattern of As distribution and speciation was similar, with As as As V -ferrihydrite increasing from 57% to 75% upon the addition of SMR. These findings indicate that changes in ore processing technology, such as the recovery of magnetite could have significant environmental consequences regarding the As mobilisation and transformation in mine tailings.

We have developed an X-ray absorption near edge structure spectroscopy method using fluorescence detection for visualizing in vivo coordination environments of metals in biological specimens. This approach, which we term fluorescence imaging XANES (φXANES), allows us to spatially depict metal-protein associations in a native, hydrated state whilst avoiding intrinsic chemical damage from radiation. This method was validated using iron-challenged Caenorhabditis elegans to observe marked alterations in redox environment.

The effect of different polishing techniques on loss of mineral elements from rice grains was quantified using a panel of indica and tropical japonica genotypes, previously classified as differing in ease of polishing. Gradients in mineral elements across the bran-endosperm interface were quantified using micro-scaled precision abrasive polishing in combination with inductively coupled plasma mass spectrometry and synchrotron X-ray fluorescence microscopy. Frictional polishing, similar to that of commercial mills, i.e. 8–10% loss of grain weight, reduced the concentration of Fe, Mg, P, K and Mn by 60–80% in all genotypes. Following gentler polishing (3–5% weight loss), genotypes classified as difficult to polish showed smaller decreases in Fe, Mg, P, K and Mn compared to genotypes classified as easy to polish. The concentration of other elements, e.g. Zn, S, Ca, Cu, Mo and Cd, showed comparable reductions (

Determining the chemical and biological compositions of the tumour models used in pharmacological studies is crucial for understanding the interactions between the drug molecules and the tumour micro-environment. Conventional techniques for spheroid characterisation require intensive chemical pre-treatments that result in the removal of unbound metabolites. In this study, the spectroscopic techniques, scanning transmission ion microscopy (STIM), proton-induced X-ray emission (PIXE) mapping, scanning X-ray fluorescence microscopy (SXFM), and Fourier transform infrared (FT-IR) imaging were employed to gain complementary information on the compositions of untreated DLD-l cancer cell spheroids. When used together, these techniques exhibited great potential for providing a comprehensive over-view of the density, biochemistry and elemental compositions within the different regions of the spheroids. STIM density and elemental maps correlated well with cellular density across the spheroid, and showed the accumulation of S, Cu and various lighter elements in the necrotic region. High levels of oxidative stress were evident in the hypoxic region, and different degrees of cellular necrosis as well as high levels of lactate and collagen within the necrotic region were suggested by FT-IR markers. FT-IR imaging was further employed to study the pharmacodynamics of known the cytotoxins, cisplatin and Pt1C3. Cisplatin was observed to induce minimal biochemical changes to the spheroids following 24 hour incubations, whereas Pt1C3 caused severe cellular damage to the spheroid periphery; consistent with their different modes of action.

The oxidation of linear low density polyethylene (LLDPE) films containing the combination of pro-oxidants titanium (IV) dioxide (TiO2) with either cobalt (II) stearate (CoSt) or iron (II) stearate (FeSt) have been evaluated under accelerated photo- and thermo-oxidative conditions as well as on outdoor weathering. LLDPE containing only surface-compatibilised nano-TiO2 rapidly photo-whitens and embrittles at a low apparent extent of oxidation (as measured by carbonyl index) due to formation of microscopic voids of similar to 150 nm. When CoSt was also included in the film, antagonism occurred shown by embrittlement times longer by similar to 90%, higher carbonyl index and absence of film whitening. In contrast, films containing TiO2/FeSt whitened during photo-oxidation and exhibited lower antagonism with only 44% longer times to embrittlement and lower carbonyl index. Antagonism between pro-oxidants was not observed under dark thermo-oxidative conditions. X-ray Fluorescence Microspectroscopy elemental maps revealed that the TiO2 nanoparticles were spatially correlated with iron and cobalt metal ions allowing scavenging of electrons and holes through cycling of the redox states of the metal without producing radical species to initiate polymer oxidation. It is suggested that the antagonism differences between TiO2/CoSt and TiO2/FeSt pro-oxidants is related to the respective reduction potentials of Co3+/2+ and Fe3+/2+ and their effect on the UV conduction and valence band edges of the TiO2 particle. In these ways the photochemistry of TiO2 is suppressed and the photo-oxidative lifetime is governed by the chemistry of the transition metal pro-oxidant. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

Arsenic (As) is considered to be the environmental contaminant of greatest concern due to its potential accumulation in the food chain and in humans. Using novel synchrotron-based x-ray fluorescence techniques (including sequential computed tomography), short-term solution culture studies were used to examine the spatial distribution of As in hydrated and fresh roots of cowpea (Vigna unguiculata ‘Red Caloona’) seedlings exposed to 4 or 20 µm arsenate [As(V)] or 4 or 20 µm arsenite. For plants exposed to As(V), the highest concentrations were observed internally at the root apex (meristem), with As also accumulating in the root border cells and at the endodermis. When exposed to arsenite, the endodermis was again a site of accumulation, although no As was observed in border cells. For As(V), subsequent transfer of seedlings to an As-free solution resulted in a decrease in tissue As concentrations, but growth did not improve. These data suggest that, under our experimental conditions, the accumulation of As causes permanent damage to the meristem. In addition, we suggest that root border cells possibly contribute to the plant’s ability to tolerate excess As(V) by accumulating high levels of As and limiting its movement into the root.

In situ spatially resolved X-ray absorption near edge structure (μ-XANES) spectra are obtained for natural Cu-In-bearing sphalerite. Copper K -edge spectral data show that, in Cu-In-bearing sphalerite, in which an excellent correlation between the Cu and In contents is noted, Cu is present in the Cu + state. This offers indirect proof for the coupled substitution 2 Zn 2+ ↔ Cu + + In 3+ , which allows indium to enter the sphalerite structure. The study clearly demonstrates the utility of synchrotron radiation to accurately determine oxidation state in small volumes of mineral in which the concentration of the element of interest is low or very low. The study also demonstrates that good quality μ-XANES spectra can be collected on TEM foils prepared in situ at a chosen position on the surface of a polished sample using the focused ion beam–scanning electron microscope method.

Here, we present a sub-μm multimodal approach to image essential elements in Caenorhabditis elegans. A combination of chemical imaging technologies reveals total metal concentration, chemical state and the protein to which an element is associated. This application of distinct yet complementary chemical imaging techniques provided unique insight into essential and trace elements at the subcellular level.

Selenite is an inorganic form of selenium that has a cytotoxic effect against several human cancer cell lines: one or more selenite metabolites are considered to be responsible for its toxicity. X-ray absorption spectroscopy was used to monitor Se speciation in A549 human lung cancer cells incubated with selenite over 72 h. As anticipated, selenodiglutathione and elemental Se both comprised a large proportion of Se in the cells between 4 and 72 h after treatment, which is in accordance with the reductive metabolism of selenite in the presence of glutathione and glutathione reductase/NADPH system. Selenocystine was also present in the cells, but was only detected as a significant component between 24 h and 48 h concomitant with a decrease in the proportion of selenocysteine and the viability of the cells. The change in speciation from the selenol, selenocysteine, to the diselenide, selenocystine, is indicative of a change in the redox status of the cells to a more oxidizing environment, likely brought about by metabolites of selenite. X-ray fluorescence microscopy of single cells treated with selenite for 24 h revealed a punctate distribution of Se in the cytoplasm. The accumulation of Se was associated with a greater than two-fold increase in Cu, which was colocalized with Se. Selenium K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy revealed Se-Se and Se-S bonding, but not Se-Cu bonding, despite the spatial association of Se and Cu. Microprobe X-ray absorption near-edge structure spectroscopy (μ-XANES) showed that the highly localized Se species was mostly elemental Se.

Synchrotron-based X-ray fluorescence microscopy (XFM) can localise chemical elements at a subcellular level. 99mTechnetium stannous (TcSn) colloid is taken up by phagocytes via a Complement Receptor 3 mediated phagocytic process. In the current study, XFM was used to examine the intracellular trafficking of TcSn colloid in neutrophils. XFM was performed on TcSn colloid, and neutrophils labelled with TcSn colloid, in whole blood. We developed a set of pixel by pixel analysis and mapping techniques incorporating cluster analysis that allowed us to differentiate neutrophils and artefactual contaminants, and we examined the changes in element distribution that accompany neutrophil phagocytosis of TcSn colloid. Sn became associated with half the neutrophils. Within cells, Sn colocalised with iron (Fe) and sulphur (S), and was negatively associated with calcium (Ca). Despite the high sensitivity of XFM, Tc was not detected. XFM can help clarify the intracellular processes that accompany neutrophil phagocytosis. The subcellular colocalisation of Sn with Fe is consistent with fusion of the colloid-containing phagosome with neutrophil granules. The association of Sn with S suggests that proteins rich in S-containing amino acids are present in the phagosome. The negative colocalisation with Ca indicates that ongoing maturation of the TcSn colloid phagosome is no longer calcium dependent one hour after phagocytosis.The online version of this article (doi:10.1007/s10867-011-9233-9) contains supplementary material, which is available to authorized users.

Despite its pivotal role in determining the risks and time frames associated with contaminant release, metal speciation remains a poorly understood aspect of biosolids chemistry. The work reported here used synchrotron-based spectroscopy techniques to investigate the speciation of copper and zinc in a range of Australian biosolids. High resolution element mapping of biosolids samples using micro X-ray fluorescence spectroscopy revealed considerable heterogeneity in key element associations, and a combination of both organic and inorganic copper and zinc binding environments. Linear combination fitting of K-edge X-ray absorption spectra indicated consistent differences in metal speciation between freshly produced and stockpiled biosolids. While sulfide minerals play a dominant role in metal binding in freshly dewatered biosolids, they are of lesser importance in dried biosolids that have been stockpiled. A degree of metal binding with iron oxide minerals was apparent but the results did not support the hypothesis that biosolids metals are chiefly associated with iron minerals. This work has potential implications for the long-term stability of metals in biosolids and their eventual fate following land application. Refereed/Peer-reviewed

X-ray fluorescence microscopy (XFM) facilitates high-sensitivity quantitative imaging of trace metals at high spatial resolution over large sample areas and can be applied to a diverse range of biological samples. Accurate determination of elemental content from recorded spectra requires proper calibration of the XFM instrument under the relevant operating conditions. Here, we describe the manufacture, characterization, and utilization of multi-element thin-film reference foils for use in calibration of XFM measurements of biological and other specimens. We have used these internal standards to assess the two-dimensional distribution of trace metals in a thin tissue section of a rat hippocampus. The data used in this study was acquired at the XFM beamline of the Australian Synchrotron using a new 384-element array detector (Maia) and at beamline 2-ID-E at the Advanced Photon Source. Post-processing of samples by different fixation techniques was investigated, with the conclusion that differences in solvent type and sample handling can significantly alter elemental content. The present study highlights the quantitative capability, high statistical power, and versatility of the XFM technique for mapping trace metals in biological samples, e.g., brain tissue samples in order to help understand neurological processes, especially when implemented in conjunction with a high-performance detector such as Maia.

The phytotoxicity of trace metals is of global concern due to contamination of the landscape by human activities. Using synchrotron-based x-ray fluorescence microscopy and x-ray absorption spectroscopy, the distribution and speciation of copper (Cu), nickel (Ni), and zinc (Zn) was examined in situ using hydrated roots of cowpea (Vigna unguiculata) exposed to 1.5 μm Cu, 5 μm Ni, or 40 μm Zn for 1 to 24 h. After 24 h of exposure, most Cu was bound to polygalacturonic acid of the rhizodermis and outer cortex, suggesting that binding of Cu to walls of cells in the rhizodermis possibly contributes to the toxic effects of Cu. When exposed to Zn, cortical concentrations remained comparatively low with much of the Zn accumulating in the meristematic region and moving into the stele; approximately 60% to 85% of the total Zn stored as Zn phytate within 3 h of exposure. While Ni concentrations were high in both the cortex and meristem, concentrations in the stele were comparatively low. To our knowledge, this is the first report of the in situ distribution and speciation of Cu, Ni, and Zn in hydrated (and fresh) plant tissues, providing valuable information on the potential mechanisms by which they are toxic.

A rapid scanning synchrofron-based X-ray fluorescence microprobe technique is applied to relatively impure crystalline silicon feedstock for solar cells. The results reveal the distributions of metallic impurities in the material over regions several millimetres in size, allowing scans across several grains. Relatively high concentrations of Fe, Cu and Zn were observed, with traces of Mn and Ni. The metals were mostly present as discrete particles up to 60 μm in size, while Cu was more uniformly distributed. More than 50% of the detected Fe was present as large particles at the grain boundaries, probably due to diffusion and precipitation during cooling. In contrast, less than 5% of the Cu resided in such large particles. The particles contained multiple metallic elements, with strongly varying proportions of their metal constituents.

The development of shark vertebrae and the possible drivers of inter- and intra-specific differences in vertebral structure are poorly understood. Shark vertebrae are used to examine life-history traits related to trophic ecology, movement patterns, and the management of fisheries; a better understanding of their development would be beneficial to many fields of research that rely on these calcified structures. This study used Scanning X-ray Fluorescence Microscopy to observe zinc distribution within vertebrae of ten shark species from five different orders. Zinc was mostly localised within the intermedialis and was generally detected at levels an order of magnitude lower in the corpus calcareum. In most species, zinc concentrations were higher pre-birth mark, indicating a high rate of pre-natal zinc deposition. These results suggest there are inter-specific differences in elemental deposition within vertebrae. Since the deposition of zinc is physiologically-driven, these differences suggest that the processes of growth and deposition are potentially different in the intermedialis and corpus calcareum, and that caution should be taken when extrapolating information such as annual growth bands from one structure to the other. Together these results suggest that the high inter-specific variation in vertebral zinc deposition and associated physiologies may explain the varying effectiveness of ageing methodologies applied to elasmobranch vertebrae.

For a toposequence with increasing groundwater influence (Cambisol, Stagnosol, Histosol) and with different groundwater regimes (Histosols 1 and 2) in a forested watershed in the Fichtelgebirge (Germany), the speciation of sulfur (S) and iron (Fe) in the soils was assessed by X-ray absorption near-edge spectroscopy (XANES) after anoxic and conventional oxic sample pretreatments. For samples with anoxic pretreatment, the contribution of reduced inorganic S compounds (monosulfide, pyrite) to total S increased with soil depth for the Cambisol and the Stagnosol, but decreased for the Histosols; the opposite trend was noticed for the contribution of reduced organic S (organic mono- and disulfides, thiols). The contribution of reduced S to the soil S pool increased and the contribution of oxidized S compounds decreased in the sequence Cambisol–Stagnosol–Histosol 1 (permanently anoxic). Histosol 2 (seasonally oxic) showed a markedly larger contribution of oxidized and intermediate S compounds to total S than Histosol 1. The dominating Fe-bearing phases in the Cambisol were Fe(III) oxyhydroxides; the contribution of sulfide-bound Fe was 2 during the entire period between sampling and analysis is crucial for a correct S and Fe speciation. Only after appropriate sample preparation, clear relationships between the mean oxidation states of S and Fe (MOFe) on one hand and soil hydrological conditions on the other become evident: a concomitant systematic decrease of MOS and MOFe from the well-aerated Cambisol to the permanently anoxic Histosol 1, and larger MOS and MOFe in the seasonally oxic Histosol 2 than in Histosol 1 indicate a close coupling of S and Fe cycling in the soils. Finally, the results of our study suggest that in organic horizons of wetland soils inorganic sulfide S is overestimated and reduced organic S is underestimated by S K -edge XANES, if a significant portion of the thiol groups in reduced organic S is complex-bound to Fe 2+ or other chalcophilic metal cations. This is supported by the observation that synthetic organic compounds (cysteine; 1,3,5-trimer-captotriazine [TMT]; ferredoxin) after addition of Fe show spectra with pre-edge peaks at energies