I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[15854, 19779]
Open Access
Abstract: Transition metals have essential roles in brain structure and function, and are associated with pathological processes in neurodegenerative disorders classed as proteinopathies. Synchrotron x-ray techniques, coupled with ultrahigh-resolution mass spectrometry, have been applied to study iron and copper interactions with amyloid β (1–42) or α-synuclein. Ex vivo tissue and in vitro systems were investigated, showing the capability to identify metal oxidation states, probe local chemical environments, and localize metal-peptide binding sites. Synchrotron experiments showed that the chemical reduction of ferric (Fe3+) iron and cupric (Cu2+) copper can occur in vitro after incubating each metal in the presence of Aβ for one week, and to a lesser extent for ferric iron incubated with α-syn. Nanoscale chemical speciation mapping of Aβ-Fe complexes revealed a spatial heterogeneity in chemical reduction of iron within individual aggregates. Mass spectrometry allowed the determination of the highest-affinity binding region in all four metal-biomolecule complexes. Iron and copper were coordinated by the same N-terminal region of Aβ, likely through histidine residues. Fe3+ bound to a C-terminal region of α-syn, rich in aspartic and glutamic acid residues, and Cu2+ to the N-terminal region of α-syn. Elucidating the biochemistry of these metal-biomolecule complexes and identifying drivers of chemical reduction processes for which there is evidence ex-vivo, are critical to the advanced understanding of disease aetiology.
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Oct 2019
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[12879, 24642]
Open Access
Abstract: Biometals such as iron, copper, potassium, and zinc are essential regulatory elements of several biological processes. The homeostasis of biometals is often affected in age-related pathologies. Notably, impaired iron metabolism has been linked to several neurodegenerative disorders. Autophagy, an intracellular degradative process dependent on the lysosomes, is involved in the regulation of ferritin and iron levels. Impaired autophagy has been associated with normal pathological aging, and neurodegeneration. Non-mammalian model organisms such as Drosophila have proven to be appropriate for the investigation of age-related pathologies. Here, we show that ferritin is expressed in adult Drosophila brain and that iron and holoferritin accumulate with aging. At whole-brain level we found no direct relationship between the accumulation of holoferritin and a deficit in autophagy in aged Drosophila brain. However, synchrotron X-ray spectromicroscopy revealed an additional spectral feature in the iron-richest region of autophagy-deficient fly brains, consistent with iron–sulfur. This potentially arises from iron–sulfur clusters associated with altered mitochondrial iron homeostasis.
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Jul 2019
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[1125, 7453]
Open Access
Abstract: Background: Chemical imaging of the human brain has great potential for diagnostic and monitoring purposes. The heterogeneity of human brain iron distribution, and alterations to this distribution in Alzheimer’s disease, indicate iron as a potential endogenous marker. The influence of iron on certain magnetic resonance imaging (MRI) parameters increases with magnetic field, but is under-explored in human brain tissues above 7 T. New Method: Magnetic resonance microscopy at 9.4 T is used to calculate parametric images of chemically-unfixed post-mortem tissue from Alzheimer’s cases (n = 3) and healthy controls (n = 2). Iron-rich regions including caudate nucleus, putamen, globus pallidus and substantia nigra are analysed prior to imaging of total iron distribution with synchrotron X-ray fluorescence mapping. Iron fluorescence calibration is achieved with adjacent tissue blocks, analysed by inductively coupled plasma mass spectrometry or graphite furnace atomic absorption spectroscopy. Results: Correlated MR images and fluorescence maps indicate linear dependence of R2, R2* and R2’ on iron at 9.4 T, for both disease and control, as follows: [R2(s−1) = 0.072[Fe] + 20]; [R2*(s−1) = 0.34[Fe] + 37]; [R2’(s−1) = 0.26[Fe] + 16] for Fe in μg/g tissue (wet weight). Comparison with Existing Methods: This method permits simultaneous non-destructive imaging of most bioavailable elements. Iron is the focus of the present study as it offers strong scope for clinical evaluation; the approach may be used more widely to evaluate the impact of chemical elements on clinical imaging parameters. Conclusion: The results at 9.4 T are in excellent quantitative agreement with predictions from experiments performed at lower magnetic fields.
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Mar 2019
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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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I08-Scanning X-ray Microscopy beamline (SXM)
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Aug 2018
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[15854, 19779]
Open Access
Abstract: Altered metabolism of biometals in the brain is a key feature of Alzheimer’s disease, and biometal interactions
with amyloid-β are linked to amyloid plaque formation. Iron-rich aggregates, including evidence
for the mixed-valence iron oxide magnetite, are associated with amyloid plaques. To test the hypothesis
that increased chemical reduction of iron, as observed in vitro in the presence of aggregating amyloid-β,
may occur at sites of amyloid plaque formation in the human brain, the nanoscale distribution and
physicochemical states of biometals, particularly iron, were characterised in isolated amyloid plaque cores
from human Alzheimer’s disease cases using synchrotron X-ray spectromicroscopy. In situ X-ray magnetic
circular dichroism revealed the presence of magnetite: a finding supported by ptychographic observation
of an iron oxide crystal with the morphology of biogenic magnetite. The exceptional sensitivity and
specificity of X-ray spectromicroscopy, combining chemical and magnetic probes, allowed enhanced
differentiation of the iron oxides phases present. This facilitated the discovery and speciation of ferrousrich
phases and lower oxidation state phases resembling zero-valent iron as well as magnetite.
Sequestered calcium was discovered in two distinct mineral forms suggesting a dynamic process of
amyloid plaque calcification in vivo. The range of iron oxidation states present and the direct observation
of biogenic magnetite provide unparalleled support for the hypothesis that chemical reduction of iron
arises in conjunction with the formation of amyloid plaques. These new findings raise challenging questions
about the relative impacts of amyloid-β aggregation, plaque formation, and disrupted metal homeostasis
on the oxidative burden observed in Alzheimer’s disease.
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Apr 2018
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Abstract: A signature characteristic of Alzheimer’s disease (AD) is aggregation of amyloid-beta (Ab) fibrils in the brain. Nevertheless, the links between Ab and AD pathology remain incompletely understood. It has been proposed that neurotoxicity arising from aggregation of the Ab1-42 peptide can in part be explained by metal ion binding interactions. Using advanced X-ray microscopy techniques at submicron resolution, we investigated relationships between iron biochemistry and AD pathology in intact cortex from an established mouse model over-producing Ab. We found a direct correlation of amyloid plaque morphology with iron, and evidence for the formation of an iron-amyloid complex. We also show that iron biomineral deposits in the cortical tissue contain the mineral magnetite, and provide evidence that Ab-induced chemical reduction of iron could occur in vivo. Our observations point to the specific role of iron in amyloid deposition and AD pathology, and may impact development of iron-modifying therapeutics for AD.
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Sep 2017
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[4911]
Abstract: Cells employ various metal and metalloid ions to augment the structure and the function of proteins and to assist with vital biological processes. In the brain they mediate biochemical processes, and disrupted metabolism of metals may be a contributing factor in neurodegenerative disorders. In this tutorial review we will discuss the particular role of X-ray methods for elemental imaging analysis of accumulated metal species and metal-containing compounds in biological materials, in the context of post-mortem brain tissue. X-rays have the advantage that they have a short wavelength and can penetrate through a thick biological sample. Many of the X-ray microscopy techniques that provide the greatest sensitivity and specificity for trace metal concentrations in biological materials are emerging at synchrotron X-ray facilities. Here, the extremely high flux available across a wide range of soft and hard X-rays, combined with state-of-the-art focusing techniques and ultra-sensitive detectors, makes it viable to undertake direct imaging of a number of elements in brain tissue. The different methods for synchrotron imaging of metals in brain tissues at regional, cellular, and sub-cellular spatial resolution are discussed. Methods covered include X-ray fluorescence for elemental imaging, X-ray absorption spectrometry for speciation imaging, X-ray diffraction for structural imaging, phase contrast for enhanced contrast imaging and scanning transmission X-ray microscopy for spectromicroscopy. Two- and three-dimensional (confocal and tomographic) imaging methods are considered as well as the correlation of X-ray microscopy with other imaging tools.
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Feb 2017
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[1125]
Abstract: This chapter has provided an overview of XRM methods as they
have developed, and here we discuss practical aspects concerning
SR excited X-ray microscopy. This section includes notes relevant
for XRF microscopy and XAS near-edge structure mapping, and
also higher resolution STXM work and sample preparation. This
overview encapsulates X-ray microscopy to detect metals in the
brain on length scales from whole brain sections down to the cellular
and subcellular level.
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Jan 2017
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I02-Macromolecular Crystallography
I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[7670, 8731, 388, 612]
Open Access
Abstract: There is evidence for iron dysregulation in many forms of disease, including a broad spectrum of neurodegenerative disorders. In order to advance our understanding of the pathophysiological role of iron, it is helpful to be able to determine in detail the distribution of iron as it relates to metabolites, proteins, cells, and tissues, the chemical state and local environment of iron, and its relationship with other metal elements. Synchrotron light sources, providing primarily X-ray beams accompanied by access to longer wavelengths such as infra-red, are an outstanding tool for multi-modal non-destructive analysis of iron in these systems. The micro- and nano-focused X-ray beams that are generated at synchrotron facilities enable measurement of iron and other transition metal elements to be performed with outstanding analytic sensitivity and specificity. Recent developments have increased the scope for methods such as X-ray fluorescence mapping to be used quantitatively rather than semi-quantitatively. Burgeoning interest, coupled with technical advances and beamline development at synchrotron facilities, has led to substantial improvements in resources and methodologies in the field over the past decade. In this paper we will consider how the field has evolved with regard to the study of iron in proteins, cells, and brain tissue, and identify challenges in sample preparation and analysis. Selected examples will be used to illustrate the contribution, and future potential, of synchrotron X-ray analysis for the characterization of iron in model systems exhibiting iron dysregulation, and for human cases of neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease, Friedreich’s ataxia, and amyotrophic lateral sclerosis.
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Aug 2014
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