I08-Scanning X-ray Microscopy beamline (SXM)
I14-Hard X-ray Nanoprobe
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Diamond Proposal Number(s):
[15230, 15854, 20809, 24526, 24531]
Abstract: Parkinson’s disease causes the loss of a particular group of brain cells, the neurons that produce the neurotransmitter dopamine. As these cells contain a dark pigment, neuromelanin, the change is evident from the loss of pigment in this brain region. Characterisation of neuromelanin in tissue remains dependent on visible pigmentation. Faint pigmentation may be interpreted as cell loss, and so contrast-
enhancing stains are commonly used. However, this staining constrains further chemical analysis of the tissue.
Researchers explored the use of synchrotron X-ray microscopy to visualise neuromelanin without relying on visible pigmentation or chemical staining. They performed combined imaging and spectroscopy (spectromicroscopy) on Diamond Light Source’s Scanning X-ray Microscopy beamline (I08), allowing the creation of images from distinct X-ray absorption features. Nanoscale spatial resolution using soft (low energy) X-rays allowed the researchers to probe the organic structure of neuromelanin to seek distinguishing spectral features. This revealed a characteristic feature in the absorption spectrum for neuromelanin. The team used this feature to create maps of neuromelanin distributions, which matched those observed in stained tissue sections.
The team also used nanoscale X-ray Fluorescence (XRF) with hard (high energy) X-rays on the Hard X-ray Nanoprobe beamline (I14) to discover a signature for identifying neuromelanin. This showed that neuromelanin could be identified by its elevated sulfur content. However, this approach is not as specific to neuromelanin as the soft X-ray method. The discovery of the soft X-ray neuromelanin signature offers significant potential for non-destructive studies of the relationships between depigmentation, metal binding and neurodegeneration in Parkinson’s disease.
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Jul 2021
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I08-Scanning X-ray Microscopy beamline (SXM)
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James
Everett
,
Frederik
Lermyte
,
Jake
Brooks
,
Vindy
Tjendana-Tjhin
,
Germán
Plascencia-Villa
,
Ian
Hands-Portman
,
Jane M.
Donnelly
,
Kharmen
Billimoria
,
George
Perry
,
Xiongwei
Zhu
,
Peter J.
Sadler
,
Peter B.
O'Connor
,
Joanna F.
Collingwood
,
Neil D.
Telling
Diamond Proposal Number(s):
[15854]
Open Access
Abstract: The chemistry of copper and iron plays a critical role in normal brain function. A variety of enzymes and proteins containing positively charged Cu+, Cu2+, Fe2+, and Fe3+ control key processes, catalyzing oxidative metabolism and neurotransmitter and neuropeptide production. Here, we report the discovery of elemental (zero–oxidation state) metallic Cu0 accompanying ferromagnetic elemental Fe0 in the human brain. These nanoscale biometal deposits were identified within amyloid plaque cores isolated from Alzheimer’s disease subjects, using synchrotron x-ray spectromicroscopy. The surfaces of nanodeposits of metallic copper and iron are highly reactive, with distinctly different chemical and magnetic properties from their predominant oxide counterparts. The discovery of metals in their elemental form in the brain raises new questions regarding their generation and their role in neurochemistry, neurobiology, and the etiology of neurodegenerative disease.
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Jun 2021
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Open Access
Abstract: Iron (Fe) is an essential trace element required for healthy brain function. Yet, disrupted iron neurochemistry, and the associated formation of aberrantly aggregated protein lesions has been implicated in the development of multiple degenerative brain disorders including Alzheimer's disease (AD). Here, nanoscale resolution soft X-ray spectromicroscopy is used to examine the interaction of β-amyloid (Aβ), a peptide fundamentally implicated in the development of Alzheimer's, and ferric (Fe3+) iron. Crucially, by probing the carbon K (280–320 eV) and iron L2,3 (700–740 eV) edges, both the organic and inorganic (iron) sample chemistry was established. The co-aggregation of Aβ and iron is known to influence iron chemistry, resulting in the chemical reduction of Fe3+ into reactive and potentially toxic ferrous (Fe2+) and zero-oxidation (Fe0) states. Here, nanoscale (i.e. sub-micron) variations in both iron oxidation state and the organic composition of Aβ were observed, replicating in vitro the diverse iron chemistry documented in amyloid plaques from human brain, with the chemical state of iron linked to the conformation state of Aβ. Furthermore, aggregates were formed that were morphologically and chemically distinct dependent on the treatment of Aβ prior to the addition of ferric iron. These findings support the hypothesis that Aβ is responsible for altering iron neurochemistry, and that this altered chemistry is a factor in neurodegenerative processes documented in AD. The methods applied here, combining nanoscale-resolution imaging and high chemical sensitivity, enabled discovery of the nanoscale heterogeneity in the iron and carbon chemistry of in vitro aggregates, and these approaches have scope for wider application in metallomics.
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Mar 2021
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I08-Scanning X-ray Microscopy beamline (SXM)
I10-Beamline for Advanced Dichroism
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Open Access
Abstract: Atypical low-oxidation-state iron phases in Alzheimer’s disease (AD) pathology are implicated in disease pathogenesis, as they may promote elevated redox activity and convey toxicity. However, the origin of low-oxidation-state iron and the pathways responsible for its formation and evolution remain unresolved. Here we investigate the interaction of the AD peptide β-amyloid (Aβ) with the iron storage protein ferritin, to establish whether interactions between these two species are a potential source of low-oxidation-state iron in AD. Using X-ray spectromicroscopy and electron microscopy we found that the co-aggregation of Aβ and ferritin resulted in the conversion of ferritin’s inert ferric core into more reactive low-oxidation-states. Such findings strongly implicate Aβ in the altered iron handling and increased oxidative stress observed in AD pathogenesis. These amyloid-associated iron phases have biomarker potential to assist with disease diagnosis and staging, and may act as targets for therapies designed to lower oxidative stress in AD tissue.
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Jun 2020
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[15230]
Open Access
Abstract: Background: Neuromelanin-pigmented neurons, which are highly susceptible to neurodegeneration in the Parkinson’s disease substantia nigra, harbour elevated iron levels in the diseased state. Whilst it is widely believed that neuronal iron is stored in an inert, ferric form, perturbations to normal metal homeostasis could potentially generate more reactive forms of iron capable of stimulating toxicity and cell death. However, non-disruptive analysis of brain metals is inherently challenging, since use of stains or chemical fixatives, for example, can significantly influence metal ion distributions and/or concentrations in tissues. Aims: The aim of this study was to apply synchrotron soft x-ray spectromicroscopy to the characterisation of iron deposits and their local environment within neuromelanin-containing neurons of Parkinson’s disease substantia nigra. Methods: Soft x-ray spectromicroscopy was applied in the form of Scanning Transmission X-ray Microscopy (STXM) to analyse resin-embedded tissue, without requirement for chemically disruptive processing or staining. Measurements were performed at the oxygen and iron K-edges in order to characterise both organic and inorganic components of anatomical tissue using a single label-free method. Results: STXM revealed evidence for mixed oxidation states of neuronal iron deposits associated with neuromelanin clusters in Parkinson’s disease substantia nigra. The excellent sensitivity, specificity and spatial resolution of these STXM measurements showed that the iron oxidation state varies across sub-micron length scales. Conclusions: The label-free STXM approach is highly suited to characterising the distributions of both inorganic and organic components of anatomical tissue, and provides a proof-of-concept for investigating trace metal speciation within Parkinson’s disease neuromelanin-containing neurons.
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May 2020
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I08-Scanning X-ray Microscopy beamline (SXM)
I14-Hard X-ray Nanoprobe
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Diamond Proposal Number(s):
[15230, 15854, 20809, 24526, 24531]
Abstract: A hallmark of Parkinson’s disease is the death of neuromelanin‐pigmented neurons, but the role of neuromelanin is unclear. Lack of a neuromelanin‐specific marker was highlighted over 30 years ago, yet in‐situ characterization of neuromelanin remains dependent on detectable pigmentation, rather than direct quantification of neuromelanin. We show that direct, label‐free nanoscale visualization of neuromelanin and associated metal ions in human brain tissue can be achieved using synchrotron Scanning Transmission X‐ray Microscopy (STXM), via a characteristic feature in the neuromelanin x‐ray absorption spectrum at 287.4 eV that is also present in iron‐free and iron‐laden synthetic neuromelanin. This is confirmed in consecutive brain sections by correlating STXM neuromelanin imaging with silver nitrate‐stained neuromelanin. Analysis suggests that the 1s ‐ σ* (C‐S) transition in benzothiazine groups accounts for this feature. This advance in visualizing neuromelanin illustrates the wider potential of STXM as a label‐free spectromicroscopy technique applicable to both organic and inorganic materials.
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Mar 2020
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I18-Microfocus Spectroscopy
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Open Access
Abstract: Iron is an essential element, and cornflake-style cereals are typically fortified with iron to a level up to 14 mg iron per 100 g. Even single cornflakes exhibit magnetic behaviour. We extracted iron microparticles from samples of two own-brand supermarket cornflakes using a strong permanent magnet. Synchrotron iron K-edge X-ray absorption near-edge spectroscopic data were consistent with identification as metallic iron, and X-ray diffraction studies provided unequivocal identification of the extracted iron as body-centred cubic (BCC) α-iron. Magnetometry measurements were also consistent with ca. 14 mg per 100 g BCC iron. These findings emphasise that attention must be paid to the speciation of trace elements, in relation to their bioavailability. To mimic conditions in the stomach, we suspended the iron extract in dilute HCl (pH 1.0–2.0) at 310 K (body temperature) and found by ICP-MS that over a period of 5 hours, up to 13% of the iron dissolved. This implies that despite its metallic form in the cornflakes, the iron is potentially bioavailable for oxidation and absorption into the body.
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Mar 2020
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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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