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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.

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.

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.

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.

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.

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

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.

The highly organized morphogenesis of bird feathers holds important phylo- and ontogenetic information on the evolution of birds, organogenesis, tissue regeneration, and the health status of individual animals. Altered topobiological patterns are regularly used as retrospective evidence for disturbed developmental trajectories due to the past exposure to environmental stressors. Using the most advanced high-resolution (5–70 µm) X-ray fluorescence microscopy (XFM), we describe in the feathers from three species of Procellariiformes hitherto unknown, depositions of elements (Zn, Ca, Br, Cu, Fe) that are independent of pigmentation or any underlying variation in density or polymer structure. In the case of Zn, the pattern across several species of Procellariiformes, but not other species, consisted of highly regular bands of Zn numbering 30–32, which may reflect the estimated number of days of active feather growth or the duration of the moult period. Thus, speculatively, the highly consistent Zn pattern might be the result of a so far unknown diurnal systemic regulation rather than local heterogeneity amongst the follicular stem cells.

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

Palaeomagnetic analysis indicates that Haasgat, a fossil-bearing palaeocave in the Gauteng Province of South Africa, is dominated by reversed magnetic polarity in its oldest, deepest layers and normal polarity in the younger layers. The presence of in-situ Equus specimens suggests an age of less than ~2.3 Ma, while morphological analysis of faunal specimens from the ex-situ assemblage suggests an age greater than 1.8 Ma. Given this faunal age constraint, the older reversed polarity sections most likely date to the beginning of the Matuyama Chron (2.58–1.95 Ma), while the younger normal polarity deposits likely date to the very beginning of the Olduvai Sub-Chron (1.95–1.78 Ma). The occurrence of a magnetic reversal from reversed to normal polarity recorded in the sequence indicates the deposits of the Bridge Section date to ~1.95 Ma. All the in-situ fossil deposits that have been noted are older than the 1.95 Ma reversal, but younger than 2.3 Ma. Haasgat therefore dates to an interesting time period in South African human evolution that saw the last occurrence of two australopith species at ~2.05–2.02 Ma (Sts5 Australopithecus africanus from Sterkfontein Member 4) to ~1.98 Ma ( Australopithecus sediba from Malapa) and the first occurrence of early Homo (Sk847), Paranthropus and the Oldowan within Swartkrans Member 1 between ~2.0 Ma and ~1.8 Ma.

• 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

Melita plumulosa is an epibenthic, detritivorous amphipod found in estuaries along the eastern coast of Australia. It has been utilized as a test organism in rapid ten to thirteen days reproduction toxicity tests for sediment quality assessment. The fecundity of females in the toxicity test has been found to be inhibited by exposure of the amphipods to contaminated sediments enriched with zinc and other metals. This study investigated the proposal that interference in vitellogenesis is the cause of reproductive toxicity of metals in crustaceans. Inspection of the ovaries from amphipods on day 6 of the test either from control or Zn/Pb/Cd/Cu-spiked sediment, that were nearing completion of vitellogenesis, showed that the females in all treatments were producing similar numbers of oocytes undergoing secondary vitellogenesis. The distribution of the Zn, Cu and Pb in the oocytes and ventral caeca of females was examined by X-ray fluorescence microscopy. Elemental mapping revealed a dense accumulation of Zn in primary oocytes and a uniform distribution of Zn and Cu in the secondary oocytes in all treatments. Zn and Cu were also observed to be uniformly distributed in the ventral caeca. Pb was not detected in either of these tissues. The apparent normal morphology and the typical number of oocytes undergoing secondary vitellogenesis suggest that vitellogenesis was not being disrupted by Pb displacing Zn in the metal-binding domain of vitellogenin in amphipods exposed to the contaminated sediment during the test. Alternative mechanisms for the reproductive toxicity of amphipods exposed for six days to metal-contaminated sediment are discussed. © 2013.

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.

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 (

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.

Multimodal spectroscopic imaging resolved controversies on biochemical changes associated with cerebral malaria pathology., Using a multimodal biospectroscopic approach, we settle several long-standing controversies over the molecular mechanisms that lead to brain damage in cerebral malaria, which is a major health concern in developing countries because of high levels of mortality and permanent brain damage. Our results provide the first conclusive evidence that important components of the pathology of cerebral malaria include peroxidative stress and protein oxidation within cerebellar gray matter, which are colocalized with elevated nonheme iron at the site of microhemorrhage. Such information could not be obtained previously from routine imaging methods, such as electron microscopy, fluorescence, and optical microscopy in combination with immunocytochemistry, or from bulk assays, where the level of spatial information is restricted to the minimum size of tissue that can be dissected. We describe the novel combination of chemical probe–free, multimodal imaging to quantify molecular markers of disturbed energy metabolism and peroxidative stress, which were used to provide new insights into understanding the pathogenesis of cerebral malaria. In addition to these mechanistic insights, the approach described acts as a template for the future use of multimodal biospectroscopy for understanding the molecular processes involved in a range of clinically important acute and chronic (neurodegenerative) brain diseases to improve treatment strategies.

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.

Understanding the accumulation and distribution of essential nutrients in cereals is of primary importance for improving the nutritional quality of this staple food. While recent studies have improved the understanding of micronutrient loading into the barley grain, a detailed characterization of the distribution of micronutrients within the grain is still lacking. High-definition synchrotron X-ray fluorescence was used to investigate the distribution and association of essential elements in barley grain at the micro scale. Micronutrient distribution within the scutellum and the embryo was shown to be highly variable between elements in relation to various morphological features. In the rest of the grain, the distribution of some elements such as Cu and Zn was not limited to the aleurone layer but extended into the endosperm. This pattern of distribution was less marked in the case of Fe and, in particular, Mn. A significant difference in element distribution was also found between the ventral and dorsal part of the grains. The correlation between the elements was not consistent between and within tissues, indicating that the transport and storage of elements is highly regulated. The complexity of the spatial distribution and associations has important implications for improving the nutritional content of cereal crops such as barley. Refereed/Peer-reviewed

X-ray fluorescence tomography promises to map elemental distributions in unstained and unfixed biological specimens in three dimensions at high resolution and sensitivity, offering unparalleled insight in medical, biological, and environmental sciences. X-ray fluorescence tomography of biological specimens has been viewed as impractical—and perhaps even impossible for routine application—due to the large time required for scanning tomography and significant radiation dose delivered to the specimen during the imaging process. Here, we demonstrate submicron resolution X-ray fluorescence tomography of a whole unstained biological specimen, quantifying three-dimensional distributions of the elements Si, P, S, Cl, K, Ca, Mn, Fe, Cu, and Zn in the freshwater diatom Cyclotella meneghiniana with 400-nm resolution, improving the spatial resolution by over an order of magnitude. The resulting maps faithfully reproduce cellular structure revealing unexpected patterns that may elucidate the role of metals in diatom biology and of diatoms in global element cycles. With anticipated improvements in data acquisition and detector sensitivity, such measurements could become routine in the near future.

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.

Methods for imaging cellular architecture and ultimately macromolecular complexes and individual proteins, within a cellular environment, are an important goal for cell and molecular biology. Coherent diffractive imaging (CDI) is a method of lensless imaging that can be applied to any individual finite object. A diffraction pattern from a single biological structure is recorded and an iterative Fourier transform between real space and reciprocal space is used to reconstruct information about the architecture of the sample to high resolution. As a test system for cellular imaging, we have applied CDI to an important human pathogen, the malaria parasite, Plasmodium falciparum. We have employed a novel CDI approach, known as Fresnel CDI, which uses illumination with a curved incident wavefront, to image red blood cells infected with malaria parasites. We have examined the intrinsic X-ray absorption contrast of these cells and compared them with cells contrasted with heavy metal stains or immunogold labeling. We compare CDI images with data obtained from the same cells using scanning electron microscopy, light microscopy, and scanning X-ray fluorescence microscopy. We show that CDI can offer new information both within and at the surface of complex biological specimens at a spatial resolution of better than 40 nm. and we demonstrate an imaging modality that conveniently combines scanning X-ray fluorescence microscopy with CDI. The data provide independent confirmation of the validity of the coherent diffractive image and demonstrate that CDI offers the potential to become an important and reliable new high-resolution imaging modality for cell biology. CDI can detect features at high resolution within unsectioned cells. ' 2008 International Society for Advancement of Cytometry

The biogeochemical cycles of many elements in the ocean are linked by their simultaneous incorporation into protists. In order to understand these elemental interactions and their implications for global biogeochemical cycles, accurate measures of cellular element stoichiometries are needed. Bulk analysis of size-fractionated particulate material obscures the unique biogeochemical roles of different functional groups such as diatoms, calcifying protists, and diazotrophs. Elemental analysis of individual protist cells can be performed using electron, proton, and synchrotron X-ray microprobes. Here we review the capabilities and limitations of each approach and the application of these advanced techniques to cells collected from natural communities. Particular attention is paid to recent studies of plankton biogeochemistry in low-iron waters of the Southern Ocean. Single-cell analyses have revealed significant inter-taxa differences in phosphorus, iron, and nickel quotas. Differences in the response of autotrophs and heterotrophs to iron fertilization were also observed. Two-dimensional sub-cellular mapping indicates the importance of iron to photosynthetic machinery and of zinc to nuclear organelles. Observed changes in diatom silicification and cytoplasm content following iron fertilization modify our understanding of the relationship between iron availability and silicification. These examples demonstrate the advantages of studying ocean biogeochemistry at the level of individual cells.

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.

The amyloid precursor protein (APP) gene family includes APP and the amyloid precursor-like proteins, APLP1 and APLP2. These proteins contain metal binding sites for copper, zinc and iron and are known to have physiological roles in modulating the metal homeostasis in brain cells. Here we report the application of X-ray fluorescence microscopy (XFM) to investigate the subcellular distribution patterns of the metal ions Cu, Zn, Fe, and Ca in individual neurons derived from APP and APLP2 knockout mice brains to further define their role in metal homeostasis. These studies add to the growing body of data that the APP family of proteins are metalloproteins that have shared as well as distinct effects on metals. As we continue to delineate the cellular effects of the APP family of proteins it is important to consider how metals are involved in their actions.

Cereal crops accumulate low levels of iron (Fe) of which only a small fraction (5e10%) is bioavailable in human diets. Extensive co-localization of Fe in outer grain tissues with phytic acid, a strong chelator of metal ions, results in the formation of insoluble complexes that cannot be digested by humans. Here we describe the use of synchrotron X-ray fluorescence microscopy (XFM) and high resolution secondary ion mass spectrometry (NanoSIMS) to map the distribution of Fe, zinc (Zn), phosphorus (P) and other elements in the aleurone and subaleurone layers of mature grain from wild-type and an Fe-enriched line of rice (Oryza sativa L.). The results obtained from both XFM and NanoSIMS indicated that most Fe was colocalized with P (indicative of phytic acid) in the aleurone layer but that a small amount of Fe, often present as “hotspots”, extended further into the subaleurone and outer endosperm in a pattern that was not co-localized with P. We hypothesize that Fe in subaleurone and outer endosperm layers of rice grain could be bound to low molecular weight chelators such as nicotianamine and/or deoxymugineic acid. 2014 Published by Elsevier Ltd.

Metals are essential in biochemistry, with highly regulated localisation and concentration. To continue elucidating the impact of inorganic physiology on cellular process in health and disease requires development of suitable eukaryotic models and experimental approaches. The multicellular organism Caenorhabditis elegans has been widely adopted as a model system and proved to be highly valuable in biomedical research, and here we record the distribution of physiologically important 4th row elements throughout anesthetised, hydrated adult C. elegans without the need for chemical fixation or mechanical sectioning. Probing the partitioning of elements between intact anatomical structures at subcellular length scales allowed the impact of preparation techniques commonly in use for analytical microanalysis to be assessed. In addition, taking advantage of C. elegans as a research tool, these studies were carried out across a cohort of genetically homogenous individuals with specific expression of green fluorescent protein within intestinal cells. These studies allowed direct visualisation of elemental co-localisation with anatomical structures and facilitated analysis of inter-animal variability in biometal abundance and localisation. These data reflect stochastic metabolic activity and confirm C. elegans as a sensitive system in which to explore the effects of altered metal homeostasis.

SummaryThe heavy metal zinc is an essential component of the human diet and is incorporated as a structural component in up to 10% of all mammalian proteins. The physiological importance of zinc homeostasis at the cellular level and the molecular mechanisms involved in this process have become topics of increasing interest in recent years. We have performed a systematic functional characterization of the majority of the predicted Drosophila Zip (Zinc/iron regulated transporter-related protein) and ZnT genes, using the Gal4-UAS system to carry out both ubiquitous and targeted over expression and suppression studies for thirteen of the seventeen putative zinc transport genes identified to date. We find that six of these thirteen genes may be essential for fly viability and that three of the remaining seven demonstrate over expression phenotypes. Our findings reaffirm the previously proposed function of dZnT63C (CG17723: FBgn005432) as an important zinc efflux protein and indicate that the fly homolog of hZip1, dZip42C.1 (CG9428: FBgn0033096), is a strong zinc importer in Drosophila. By combining over expression of dZip42C.1 with suppression of dZnT63C we were able to produce easily identifiable zinc toxicosis phenotypes which can be rescued or worsened by modifying dietary zinc content. Our findings show that a genetically based zinc toxicosis situation can be therapeutically treated or exacerbated by modifications to the diet, providing a sensitized background for future, more detailed studies of Zip / ZNT function.

The phytotoxicity of Mn is important globally due to its increased solubility in acid or waterlogged soils. Short-term (≤24 h) solution culture studies with 150 µM Mn were conducted to investigate the in situ distribution and speciation of Mn in apical tissues of hydrated roots of cowpea [Vigna unguiculata (L.) Walp. cv. Red Caloona] using synchrotron-based techniques. Accumulation of Mn was rapid; exposure to 150 µM Mn for only 5 min resulting in substantial Mn accumulation in the root cap and associated mucigel. The highest tissue concentrations of Mn were in the root cap, with linear combination fitting of the data suggesting that ≥80% of this Mn(II) was associated with citrate. Interestingly, although the primary site of Mn toxicity is typically the shoots, concentrations of Mn in the stele of the root were not noticeably higher than in the surrounding cortical tissues in the short-term (≤24 h). The data provided here from the in situ analyses of hydrated roots exposed to excess Mn are, to our knowledge, the first of this type to be reported for Mn and provide important information regarding plant responses to high Mn in the rooting environment. Refereed/Peer-reviewed

Bioinorganic chemistry is critical to cellular function. Homeostasis of manganese (Mn), for example, is essential for life. A lack of methods for direct in situ visualization of Mn and other biological metals within intact multicellular eukaryotes limits our understanding of management of these metals. We provide the first quantitative subcellular visualization of endogenous Mn concentrations (spanning two orders of magnitude) associated with individual cells of the nematode, Caenorhabditis elegans.

Tissue-specific manipulation of known copper transport genes in Drosophila tissues results in phenotypes that are presumably due to an alteration in copper levels in the targeted cells. However direct confirmation of this has to date been technically challenging. Measures of cellular copper content such as expression levels of copper-responsive genes or cuproenzyme activity levels, while useful, are indirect. First-generation copper-sensitive fluorophores show promise but currently lack the sensitivity required to detect subtle changes in copper levels. Moreover such techniques do not provide information regarding other relevant biometals such as zinc or iron. Traditional techniques for measuring elemental composition such as inductively coupled plasma mass spectroscopy are not sensitive enough for use with the small tissue amounts available in Drosophila research. Here we present synchrotron x-ray fluorescence microscopy analysis of two different Drosophila tissues, the larval wing imaginal disc, and sectioned adult fly heads and show that this technique can be used to detect changes in tissue copper levels caused by targeted manipulation of known copper homeostasis genes.

Metals and metalloids play a key role in plant and other biological systems as some of them are essential to living organisms and all can be toxic at high concentrations. It is therefore important to understand how they are accumulated, complexed and transported within plants. In situ imaging of metal distribution at physiological relevant concentrations in highly hydrated biological systems is technically challenging. In the case of roots, this is mainly due to the possibility of artifacts arising during sample preparation such as cross sectioning. Synchrotron x-ray fluorescence microtomography has been used to obtain virtual cross sections of elemental distributions. However, traditionally this technique requires long data acquisition times. This has prohibited its application to highly hydrated biological samples which suffer both radiation damage and dehydration during extended analysis. However, recent advances in fast detectors coupled with powerful data acquisition approaches and suitable sample preparation methods can circumvent this problem. We demonstrate the heightened potential of this technique by imaging the distribution of nickel and zinc in hydrated plant roots. Although 3D tomography was still impeded by radiation damage, we successfully collected 2D tomograms of hydrated plant roots exposed to environmentally relevant metal concentrations for short periods of time. To our knowledge, this is the first published example of the possibilities offered by a new generation of fast fluorescence detectors to investigate metal and metalloid distribution in radiation-sensitive, biological samples. Refereed/Peer-reviewed

Cystic fibrosis, an autosomal recessive disorder caused by a mutation in a gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), remains a leading cause of childhood respiratory morbidity and mortality. The respiratory consequences of cystic fibrosis include the generation of thick, tenacious mucus that impairs lung clearance, predisposing the individual to repeated and persistent infections, progressive lung damage and shortened lifespan. Currently there is no cure for cystic fibrosis. With this in mind, we investigated the ability of human amnion epithelial cells (hAECs) to express functional CFTR. We found that hAECs formed 3-dimensional structures and expressed the CFTR gene and protein after culture in Small Airway Growth Medium (SAGM). We also observed a polarized CFTR distribution on the membrane of hAECs cultured in SAGM, similar to that observed in polarized airway cells in vivo. Further, hAECs induced to express CFTR possessed functional iodide/chloride (I(-/)Cl(-)) ion channels that were inhibited by the CFTR-inhibitor CFTR-172, indicating the presence of functional CFTR ion channels. These data suggest that hAECs may be a promising source for the development of a cellular therapy for cystic fibrosis.

The aim of this study was to determine specific distribution of metals in the termite Tumulitermes tumuli (Froggatt) and identify specific organs within the termite that host elevated metals and therefore play an important role in the regulation and transfer of these back into the environment. Like other insects, termites bio-accumulate essential metals to reinforce cuticular structures and utilize storage detoxification for other metals including Ca, P, Mg and K. Previously, Mn and Zn have been found concentrated in mandible tips and are associated with increased hardness whereas Ca, P, Mg and K are accumulated in Malpighian tubules. Using high resolution Particle Induced X-Ray Emission (PIXE) mapping of whole termites and Scanning Electron Microscope (SEM) Energy Dispersive X-ray (EDX) spot analysis, localised accumulations of metals in the termite T. tumuli were identified. Tumulitermes tumuli was found to have proportionally high Mn concentrations in mandible tips. Malpighian tubules had significant enrichment of Zn (1.6%), Mg (4.9%), P (6.8%), Ca (2.7%) and K (2.4%). Synchrotron scanning X-ray Fluorescence Microprobe (XFM) mapping demonstrated two different concretion types defined by the mutually exclusive presence of Ca and Zn. In-situ SEM EDX realisation of these concretions is problematic due to the excitation volume caused by operating conditions required to detect minor amounts of Zn in the presence of significant amounts of Na. For this reason, previous researchers have not demonstrated this surprising finding.