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.
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 (<250 μm) and inhalable (<10 μm) particle fractions. Pb IVBA was significantly reduced (p < 0.05) by phosphate amendments in the <250 μm fraction (solubility bioaccessibility research consortium assay) and by PA, MAP, and TSP in the <10 μm fraction (inhalation–ingestion bioaccessibility assay). A 21.1% reduction in Pb RBA (<250 μm fraction) and 56.4% reduction in blood Pb concentration (<10 μm fraction) were observed via the ingestion and inhalation pathways, respectively. XRF microscopy detected Pb in the stomach within 4 h, presumably via mucociliary clearance.
Metal biochemistry drives a diverse range of cellular processes associated with development, health and disease. Determining metal distribution, concentration and flux defines our understanding of ...these fundamental processes. A comprehensive analysis of biological systems requires a balance of analytical techniques that inform on metal quantity (sensitivity), chemical state (selectivity) and location (spatial resolution) with a high degree of certainty. A number of approaches are available for imaging metals from whole tissues down to subcellular organelles, as well as mapping metal turnover, protein association and redox state within these structures. Technological advances in micro- and nano-scale imaging are striving to achieve multi-dimensional and in vivo measures of metals while maintaining the native biochemical environment and physiological state. This Tutorial Review discusses state-of-the-art imaging technology as a guide to obtaining novel insight into the biology of metals, with sensitivity, selectivity and spatial resolution in focus.
Hard X-ray fluorescence microscopy is well-suited to in-situ investigations of trace metal distributions within whole, unstained, biological tissue, with sub-parts-per-million detection achievable in ...whole cells. The high penetration of X-rays indicates the use of X-ray fluorescence tomography for structural visualization, and recent measurements have realised sub-500-nm tomography on a 10-μm cell. Limitations of present approaches impact the duration of an experiment and imaging fidelity. Developments in X-ray resolution, detector speed, cryogenic environments, and the incorporation of auxiliary signals are being pursued within the synchrotron community. Several complementary approaches to X-ray fluorescence tomography will be routinely available to the biologist in the near future. We discuss these approaches and review applications of biological relevance.
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.
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.
Although zinc oxide (ZnO) nanoparticles (NPs) have been widely formulated in sunscreens, the relationship between reactive oxygen species (ROS) generation induced by these particles, zinc ions, and ...cytotoxicity is not clearly understood. This study explores whether these factors can be accurately quantified and related. The study demonstrates a strong correlation between ZnO NP-induced cytotoxicity and free intracellular zinc concentration (R (2) = .945) in human immune cells, indicating a requirement for NP dissolution to precede cytotoxicity. In addition, although direct exposure to ZnO NPs was found to induce cytotoxicity at relatively high concentrations, indirect exposure (via dialysis) was not cytotoxic, even at extremely high concentrations, highlighting a requirement for NP-to-cell contact. Elevated levels of ROS present in NP-exposed cells also correlated to both cytotoxicity and intracellular free zinc. Although the addition of antioxidant was able to reduce ROS, cytotoxicity to ZnO NPs was unaffected, suggesting ROS may be, in part, a result of cytotoxicity rather than a causal factor. This study highlights both the requirement and role of intracellular dissolution of zinc nanomaterials to elicit a cytotoxic response. This response is only partially ROS dependent, and therefore, modification of NP uptake and their intracellular solubility are key components in modulating the bioactivity of ZnO NPs.
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 h and 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.
Abstract
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.
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.
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