Kathryn Spiers

Maia X-ray fluorescence imaging: Capturing detail in complex natural samples

Motivated by the challenge of capturing complex hierarchical chemical detail in natural material from a wide range of applications, the Maia detector array and integrated realtime processor have been developed to acquire X-ray fluorescence images using X-ray Fluorescence Microscopy (XFM). Maia has been deployed initially at the XFM beamline at the Australian Synchrotron and more recently, demonstrating improvements in energy resolution, at the P06 beamline at Petra III in Germany. Maia captures fine detail in element images beyond 100 M pixels. It combines a large solid-angle annular energy-dispersive 384 detector array, stage encoder and flux counter inputs and dedicated FPGA-based real-time event processor with embedded spectral deconvolution. This enables high definition imaging and enhanced trace element sensitivity to capture complex trace element textures and place them in a detailed spatial context. Maia hardware and software methods provide per pixel correction for dwell, beam flux variation, dead-time and pileup, as well as off-line parallel processing for enhanced throughput. Methods have been developed for real-time display of deconvoluted SXRF element images, depth mapping of rare particles and the acquisition of 3D datasets for fluorescence tomography and XANES imaging using a spectral deconvolution method that tracks beam energy variation.

Laterally resolved speciation of arsenic in roots of wheat and rice using fluorescence-XANES imaging

• 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.

Visualising coordination chemistry: fluorescence X-ray absorption near edge structure tomography

Coordination chemistry underlies the structure/function of biological metal complexes. Contextualising this chemical information within an organisms physiology is critical for enhancing the understanding of bioinorganic chemistry but few high-fidelity probes are available. Here we develop fluorescence X-ray absorption near-edge structure tomography as a means for studying the spatial arrangement of biological coordination chemistry within intact organisms, and demonstrate the approach by mapping the distribution of cuprous and cupric complexes within Drosophila melanogaster.

The Maia detector array and x-ray fluorescence imaging system: locating rare precious metal phases in complex samples

X-ray fluorescence images acquired using the Maia large solid-angle detector array and integrated real-time processor on the X-ray Fluorescence Microscopy (XFM) beamline at the Australian Synchrotron capture fine detail in complex natural samples with images beyond 100M pixels. Quantitative methods permit real-time display of deconvoluted element images and for the acquisition of large area XFM images and 3D datasets for fluorescence tomography and chemical state (XANES) imaging. This paper outlines the Maia system and analytical methods and describes the use of the large detector array, with a wide range of X-ray take-off angles, to provide sensitivity to the depth of features, which is used to provide an imaging depth contrast and to determine the depth of rare precious metal particles in complex geological samples.

Localization of the Trace Elements Iron, Zinc and Selenium in Relation to Anatomical Structures in Bovine Ovaries by X-Ray Fluorescence Imaging.

X-ray fluorescence (XRF) was used to image 40 histological cross-sections of bovine ovaries (n=19), focusing on structures including: antral follicles at different stages of growth or atresia, corpora lutea at three stages of development (II-IV), and capillaries, arterioles, and other blood vessels. This method identified three key trace elements [iron (Fe), zinc (Zn), and selenium (Se)] within the ovarian tissue which appeared to be localized to specific structures. Owing to minimal preprocessing of the ovaries, important high-resolution information regarding the spatial distribution of these elements was obtained with elemental trends and colocalizations of Fe and Zn apparent, as well as the infrequent appearance of Se surrounding the antrum of large follicles, as previously reported. The ability to use synchrotron radiation to measure trace element distributions in bovine ovaries at such high resolution and over such large areas could have a significant impact on understanding the mechanisms of ovarian development. This research is intended to form a baseline study of healthy ovaries which can later be extended to disease states, thereby improving our current understanding of infertility and endocrine diseases involving the ovary.

An X-ray fluorescence microscopic analysis of the tissue surrounding the multi-channel cochlear implant electrode array

Objectives: The aim of this study was to analyse the tissue surrounding the University of Melbournes (UOMs) multi-channel cochlear implant electrode array and cochlear limited replacements, after long-term implantations. In particular, it aimed to identify the particulate material in the fibrous tissue capsule of the arrays implanted in 1978, 1983, and 1998, by using the Australian Synchrotron for X-ray fluorescence microscopy (XFM) to reveal the characteristic spectrum of metal, in particular platinum. This also helped to determine its format and chemical state. Tissue was retrieved following the recipients death in 2007. Methods: Tissue was fixed and sections taken across the UOM and Cochlear Corporation (CI-22 and CI-24) electrode tracks. These were stained with Massons trichrome. The Australian Synchrotron enabled XFM to accurately identify platinum from its characteristic fluorescence spectrum. Results: There was a fibrous tissue capsule (about 100-µm thick) and small regions of calcification around the UOM and CI-22 arrays, but a thinner capsule (40–60-µm thick) around CI-24, and a greater degree of calcification. Dark particulate matter was observed within macrophages and especially in fibrous tissue in proximity to the UOM and CI-22 arrays. This was identified as platinum using X-ray fluorescence. There was also diffusion of platinum into the tissue surrounding the UOM and CI-22 electrodes and fine particles had penetrated the spiral ligament. Discussion: The larger particulate matter in the tissue around the UOM and CI-22 arrays suggested that it had flaked off in the manufacturing of the UOM electrodes. The more diffuse spread of platinum in the tissue around the UOM and CI-22 electrodes was likely due to electrolysis, probably from charge imbalance with the bipolar pulses from the UOM implant. This did not occur with the Cochlear CI-24 device. Furthermore, the widespread fine particles of platinum could have also been due to corrosion, especially from the UOM electrodes.

The Topobiology of Chemical Elements in Seabird Feathers

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.

The impact of steeping, germination and hydrothermal processing of wheat (Triticum aestivum L.) grains on phytate hydrolysis and the distribution, speciation and bio-accessibility of iron and zinc elements

Chelation of iron and zinc in wheat as phytates lowers their bio-accessibility. Steeping and germination (15 °C, 120 h) lowered phytate content from 0.96% to only 0.81% of initial dry matter. A multifactorial experiment in which (steeped/germinated) wheat was subjected to different time (2-24 h), temperature (20-80 °C) and pH (2.0-8.0) conditions showed that hydrothermal processing of germinated (15 °C, 120 h) wheat at 50 °C and pH 3.8 for 24 h reduced phytate content by 95%. X-ray absorption near-edge structure imaging showed that it indeed abolished chelation of iron to phytate. It also proved that iron was oxidized during steeping, germination and hydrothermal processing. It was further shown that zinc and iron bio-accessibility were respectively 3 and 5% in wheat and 27 and 37% in hydrothermally processed wheat. Thus, hydrothermal processing of (germinated) wheat paves the way for increasing elemental bio-accessibility in whole grain-based products.

SLC30A10 mutation involved in parkinsonism results in manganese accumulation within nano-vesicles of the Golgi apparatus

Manganese (Mn) is an essential metal that can be neurotoxic when elevated exposition occurs leading to parkinsonian-like syndromes. Mutations in the Slc30a10 gene have been identified in new forms of familial parkinsonism. SLC30A10 is a cell surface protein involved in the efflux of Mn and protects the cell against Mn toxicity. Disease-causing mutations block the efflux activity of SLC30A10, resulting in Mn accumulation. Determining the intracellular localization of Mn when disease-causing SLC30A10 mutants are expressed is essential to elucidate the mechanisms of Mn neurotoxicity. Here, using organelle fluorescence microscopy and synchrotron X-ray fluorescence (SXRF) imaging, we found that Mn accumulates in the Golgi apparatus of human cells transfected with the disease-causing SLC30A10-Δ105-107 mutant under physiological conditions and after exposure to Mn. In cells expressing the wild-type SLC30A10 protein, cellular Mn content was low after all exposure conditions, confirming efficient Mn efflux. In nontransfected cells that do not express endogenous SLC30A10 and in mock transfected cells, Mn was located in the Golgi apparatus, similarly to its distribution in cells expressing the mutant protein, confirming deficient Mn efflux. The newly developed SXRF cryogenic nanoimaging (<50 nm resolution) indicated that Mn was trapped in single vesicles within the Golgi apparatus. Our results confirm the role of SLC30A10 in Mn efflux and the accumulation of Mn in cells expressing the disease-causing SLC30A10-Δ105-107 mutation. Moreover, we identified suborganelle Golgi nanovesicles as the main compartment of Mn accumulation in SLC30A10 mutants, suggesting interactions with the vesicular trafficking machinery as a cause of the disease.