I18-Microfocus Spectroscopy
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Alexander P.
Morrell
,
Richard A.
Martin
,
Helen M
Roberts
,
Hiram
Castillo-Michel
,
J. Frederick W.
Mosselmans
,
Kalotina
Geraki
,
Adrian T.
Warfield
,
Paul
Lingor
,
Wasif
Qayyum
,
Daniel
Graf
,
Maria
Febbraio
,
Owen
Addison
Diamond Proposal Number(s):
[17638, 23569]
Open Access
Abstract: Exposures to exogenous particles is of increasing concern to human health. Characterising the concentrations, chemical species, distribution, and involvement of the stimulus with the tissue microanatomy is essential in understanding the associated biological response. However, no single imaging technique can interrogate all these features at once which confounds and limits correlative analyses. Developments of synchronous imaging strategies, allowing multiple features to be identified simultaneously, is essential to assess spatial relationships between these key features with greater confidence. Here we present data to first highlight complications of correlative analysis between the tissue microanatomy and elemental composition associated with imaging serial tissue sections. This is achieved by assessing both the cellular and elemental distribution in 3-dimensional space using optical microscopy on serial sections and confocal X-ray fluorescence spectroscopy on bulk samples respectively. We propose a new imaging strategy using lanthanide tagged antibodies with X-ray fluorescence spectroscopy. Using simulations, a series of lanthanide tags were identified as candidate labels for scenarios where tissue sections are imaged. The feasibility and value of the proposed approach is shown where an exposure of Ti was identified concurrently with CD45 positive cells at sub-cellular resolutions. Significant heterogeneity in the distribution of exogenous particles and cells can be present between immediately adjacent serial sections showing clear need of synchronous imaging methods. The proposed approach enables elemental compositions to be correlated with the tissue microanatomy in a highly multiplexed and non-destructive manner at high spatial resolutions with the opportunity for subsequent guided analysis.
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May 2023
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I08-Scanning X-ray Microscopy beamline (SXM)
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Matthew
Loxham
,
Jeongmin
Woo
,
Akul
Singhania
,
Natalie P.
Smithers
,
Alison
Yeomans
,
Graham
Packham
,
Alina M.
Crainic
,
Richard B.
Cook
,
Flemming R.
Cassee
,
Christopher H.
Woelk
,
Donna E.
Davies
Diamond Proposal Number(s):
[17801]
Open Access
Abstract: Airborne particulate matter (PM) is a leading cause of mortality and morbidity. However, understanding of the range and mechanisms of effects of PM components is poor. PM generated in underground railways is rich in metals, especially iron. In the ultrafine (UFPM; <0.1 μm diameter) fraction, the combination of small size and metal enrichment poses an unknown health risk. This study aimed to analyse transcriptomic responses to underground UFPM in primary bronchial epithelial cells (PBECs), a key site of PM deposition. The oxidation state of iron in UFPM from an underground station was determined by X-ray absorption near edge structure (XANES) spectroscopy. Antioxidant response was assayed using a reporter cell line transfected with an antioxidant response element (ARE)-luciferase construct. Differentiated PBECs were exposed to UFPM for 6 h or 24 h for RNA-Seq and RT-qPCR analysis. XANES showed predominance of redox-active Fe3O4, with ROS generation confirmed by induction of ARE-luciferase expression. 6 h exposure of PBECs to UFPM identified 52 differentially expressed genes (DEGs), especially associated with epithelial maintenance, whereas 24 h exposure yielded 23 DEGs, particularly involved with redox homeostasis and metal binding. At both timepoints, there was upregulation of members of the metallothionein family, low molecular weight proteins with antioxidant activity whose main function is binding and homeostasis of zinc and copper ions, but not iron ions. This upregulation was partially inhibited by metal chelation or ROS scavenging. These data suggest differential regulation of responses to metal-rich UFPM depending on exposure period, and highlight novel pathways and markers of PM exposure, with the role of metallothioneins warranting further investigation
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Apr 2020
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[8504]
Abstract: In this study, we measured the levels of elements in human brain microvascular endothelial cells (ECs) infected with T. gondii. ECs were infected with tachyzoites of the RH strain, and at 6, 24, and 48 hours post infection (hpi), the intracellular concentrations of elements were determined using a synchrotron–microfocus X-ray fluorescence microscopy (μ-XRF) system. This method enabled the quantification of the concentrations of Zn and Ca in infected and uninfected (control) ECs at sub-micron spatial resolution. T. gondii-hosting ECs contained less Zn than uninfected cells only at 48 hpi (p < 0.01). The level of Ca was not significantly different between infected and control cells (p > 0.05). Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis revealed infection-specific metallome profiles characterized by significant increases in the intracellular levels of Zn, Fe, Mn and Cu at 48 hpi (p < 0.01), and significant reductions in the extracellular concentrations of Co, Cu, Mo, V, and Ag at 24 hpi (p < 0.05) compared with control cells. Zn constituted the largest part (74%) of the total metal composition (metallome) of the parasite. Gene expression analysis showed infection-specific upregulation in the expression of five genes, MT1JP, MT1M, MT1E, MT1F, and MT1X, belonging to the metallothionein gene family. These results point to a possible correlation between T. gondii infection and increased expression of MT1 isoforms and altered intracellular levels of elements, especially Zn and Fe. Taken together, a combined μ-XRF and ICP-MS approach is promising for studies of the role of elements in mediating host–parasite interaction.
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Sep 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Abstract: Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P1-type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.
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Dec 2017
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B16-Test Beamline
I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[11372, 12446]
Open Access
Abstract: Aging leads to an increase in iron-loaded cellular structures in the choroid of the eye. This study was carried out to determine the distribution and content of iron, zinc and copper in the macular retina, choroid and retrobulbar optic nerve of young (4-5 years, n=3) and aged (15-16 years, n=5) male non-human primates, Macaca fascicularis, whose ocular anatomy is similar to humans. Thirty-µm-thick tissue sections were analysed with synchrotron X-ray fluorescence and stained histologically for iron deposition. Quantitative measurements showed high levels of iron, zinc and copper in the choroid and retinal pigment epithelium in the macular area and arachnoid layer in the retrobulbar optic nerve. In aged animals compared to young ones, there was an increase in iron in the choroid with larger deposits and and iron-loaded cellular structures. Iron-accumulation within these cellular structures may contribute to choroidal function impairment in aging and age-related macular degeneration.
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Aug 2016
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[9488, 8597]
Open Access
Abstract: Bone remodelling is a crucial biological process needed to maintain elemental homeostasis. It is important to understand the trace elemental inventories that govern these processes as malfunctions in bone remodelling can have devastating effects on an organism. In this study, we use a combination of X-ray techniques to map, quantify, and characterise the coordination chemistry of trace elements within the highly remodelled bone tissues of extant and extinct Sirenia (manatees and dugongs). The dense bone structure and unique body chemistry of sirenians represent ideal tissues for studying both high remodelling rates as well as unique fossilisation pathways. Here, elemental maps revealed uncorrelated patterning of Ca and Zn within secondary osteons in both extant and fossil sirenians, as well as elevated Sr within the connecting canals of fossil sirenians. Concentrations of these elements are comparable between extant and fossil material indicating geochemical processing of the fossil bone has been minimal. Zn was found to be bound in the same coordination within the apatite structure in both extant and fossil bone. Accurate quantification of trace elements in extant material was only possible when the organic constituents of the bone were included. The comparable distributions, concentrations, and chemical coordination of these physiologically important trace elements indicate the chemistry of bone remodelling has been preserved for 19 million years. This study signifies the powerful potential of merging histological and chemical techniques in the understanding of physiological processes in both extant and extinct vertebrates.
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Mar 2016
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[7454]
Abstract: Zinc levels are high in ocular tissues and the distribution is non-uniform. Zinc is particularly concentrated in the corneal epithelium and posterior stroma. Zinc is the most abundant trace metal in the retina. Bound-zinc is particularly located in the inner nuclear layer, (e.g. forming part of the structure of zinc finger transcription factors), while loosely-bound zinc is prominent in the retinal pigment epithelium and photoreceptor layers. Loosely-bound zinc ions in the photoreceptors might play a role in the phototransduction cascade and rhodopsin regeneration. Loosely-bound zinc is also found in presynaptic vesicles of photoreceptor cells in the outer plexiform and inner plexiform layers and can be synaptically released to affect both ionotropic and metabotropic receptors and also ion channels to modulate neurotransmission. The correct amount of loosely-bound zinc ions is maintained by regulating the function of zinc transporters, sensors and trafficking/storage proteins (i.e. metallothionein). The retinal homeostasis of zinc is dysregulated in systemic zinc depletion, aging and diseases such as age-related macular degeneration. Manipulation of retinal zinc metabolism in these situations might improve visual function.
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Jun 2014
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I18-Microfocus Spectroscopy
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N. P.
Edwards
,
P. L.
Manning
,
U.
Bergmann
,
P. L.
Larson
,
B. E.
Van Dongen
,
W. I.
Sellers
,
S. M.
Webb
,
D.
Sokaras
,
R.
Alonso-Mori
,
K.
Ignatyev
,
H. E.
Barden
,
A.
Van Veelen
,
J.
Anne
,
V. M.
Egerton
,
R. A.
Wogelius
Diamond Proposal Number(s):
[8597]
Open Access
Abstract: Large-scale Synchrotron Rapid Scanning X-ray Fluorescence (SRS-XRF) elemental mapping and X-ray absorption spectroscopy are applied here to fossil leaf material from the ∼50 Mya Green River Formation (USA) in order to improve our understanding of the chemistry of fossilized plant remains. SRS-XRF of fossilized animals has previously shown that bioaccumulated trace metals and sulfur compounds may be preserved in their original distributions and these elements can also act as biomarkers for specific biosynthetic pathways. Similar spatially resolved chemical data for fossilized plants is sparsely represented in the literature despite the multitude of other chemical studies performed. Here, synchrotron data from multiple specimens consistently show that fossil leaves possess chemical inventories consisting of organometallic and organosulfur compounds that: (1) map discretely within the fossils, (2) resolve fine scale biological structures, and (3) are distinct from embedding sedimentary matrices. Additionally, the chemical distributions in fossil leaves are directly comparable to those of extant leaves. This evidence strongly suggests that a significant fraction of the chemical inventory of the examined fossil leaf material is derived from the living organisms and that original bioaccumulated elements have been preserved in situ for 50 million years. Chemical information of this kind has so far been unknown for fossilized plants and could for the first time allow the metallome of extinct flora to be studied.
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Apr 2014
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[6303, 1640]
Abstract: X-ray absorption spectroscopy (XAS) and micro-synchrotron based X-ray fluorescence (micro-SXRF) are element specific spectroscopic techniques and have been proven to be valuable tools for the investigation of changes in the chemical environment of metal centres. XAS allows the determination of the oxidation state, the coordination motif of the probed element, the identity and the number of adjacent atoms and the absorber–ligand distances. It is further applicable to nearly all types of samples independent of their actual physical state (solid, liquid, gaseous) down to μM concentrations. Micro-SXRF can provide information on the distribution and concentration of multiple elements within a sample simultaneously, allowing for the chemical state of several elements within subcellular compartments to be probed. Modern third generation synchrotrons offer the possibility to investigate the majority of the biologically relevant elements. The biological mode of action of metal-based compounds often involves interactions with target and/or transport molecules. The presence of reducing agents may also give rise to changes in the coordination sphere and/or the oxidation state. XAS and micro-SXRF are ideal techniques for investigating these issues. This tutorial review introduces the use of XAS and micro-SXRF techniques into the field of inorganic medicinal chemistry. The results obtained for platinum, ruthenium, gallium, gold and cobalt compounds within the last few years are presented.
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Mar 2013
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I18-Microfocus Spectroscopy
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Marta
Ugarte
,
Geoffrey W.
Grime
,
Gillian
Lord
,
Tina
Geraki
,
Joanna F.
Collingwood
,
Mary E.
Finnegan
,
Hannah
Farnfield
,
Michael
Merchant
,
Melanie
Bailey
,
N. I.
Ward
,
Peter
Foster
,
Paul N.
Bishop
,
Neville N.
Osborne
Diamond Proposal Number(s):
[7454]
Abstract: Inductively coupled plasma mass spectrometry (ICP-MS) was used to quantify the total amount of trace elements in retina from adult male Sprague-Dawley rats (n = 6). Concentration of trace elements within individual retinal areas in frozen sections of the fellow eye was established with the use of two methodologies: (1) particle-induced X-ray emission (PIXE) in combination with 3D depth profiling with Rutherford backscattering spectrometry (RBS) and (2) synchrotron X-ray fluorescence (SXRF) microscopy. The most abundant metal in the retina was zinc, followed by iron and copper. Nickel, manganese, chromium, cobalt, selenium and cadmium were present in very small amounts. The PIXE and SXRF analysis yielded a non-homogenous pattern distribution of metals in the retina. Relatively high levels of zinc were found in the inner part of the photoreceptor inner segments (RIS)/outer limiting membrane (OLM), inner nuclear layer and plexiform layers. Iron was found to accumulate in the retinal pigment epithelium/choroid layer and RIS/OLM. Copper in turn, was localised primarily in the RIS/OLM and plexiform layers. The trace elements iron, copper, and zinc exist in different amounts and locations in the rat retina.
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Oct 2012
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