B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[32901, 33390]
Abstract: Upon exposure to biological environments, nanoparticles are rapidly coated with biomolecules, predominantly proteins, which alter their colloidal stability, biodistribution, and cell interactions. Despite extensive efforts to investigate the nanoparticles' fate, only a few studies use high-resolution characterization methods that allow in-depth characterization, and the existing methodologies are unable to differentiate particles internalized at the onset of incubation from those taken up toward the end of an incubation period. In this study, these limitations related to incubation disparities are overcame and precisely monitored the spatiotemporal displacement of colloidally stable protein corona-coated nanoparticles within cells. An unprecedented application of cryogenic X-ray nanotomography, combined with high-resolution, super-resolution, and correlative microscopy techniques, revealed the migration of nanoparticles to the perinuclear region while monitoring the evolution of cellular organelles in fully hydrated cells under near-native conditions, without the need for contrasting agents. Notably, this tracking indicates the progressive fusion of vesicles carrying the nanoparticles intracellularly. This strategy demonstrates the potential for uncovering the temporal aspects of nanoparticle behavior within cells and can be adaptable to a wide range of nanoparticles and cell types, offering a versatile and powerful tool to follow nanoparticles in cellular environments.
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Dec 2024
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[34206]
Abstract: Cubosomes have emerged as a powerful platform for cancer treatment due to their biocompatibility and ability to encapsulate hydrophilic/lipophilic drugs, providing controlled drug release. While investigating these nanoparticles' stability and intracellular localization is essential for advancing them as clinically efficient nanomedicine, such studies are still lacking, and those available do not provide a reliable and comprehensive understanding. Here, we analyze cubosomes stability in complex media and conduct a pioneering study on visualizing their intracellular localization using a combination of correlative high-resolution three-dimensional fluorescence microscopy and soft X-ray tomography (synchrotron-based technique) at cryogenic temperatures, leveraging natural cellular contrast. Our studies revealed that cubosomes were stable in complex media, confirming their localization within lysosomes. In addition to being crucial for ensuring the advancement of cubosomes for therapeutic purposes, this study paves the way for defining the intracellular localization of other nanoparticles in greater detail, utilizing synchrotron-based 3D imaging techniques. Finally, we confirm the efficacy of doxorubicin-incorporated cubosomes against breast cancer cells.
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Dec 2024
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[14784]
Open Access
Abstract: Silicified peritidal carbonates of the Tonian Draken Formation, Spitsbergen, contain highly diverse and well-preserved microfossil assemblages dominated by filamentous microbial mats, but also including diverse benthic and/or allochthonous (possibly planktonic) microorganisms. Here, we characterize eight morphospecies in focused ion beam (FIB) ultrathin sections using transmission electron microscopy (TEM) and X-ray absorption near-edge structure (XANES) spectromicroscopy. Raman and XANES spectroscopies show the highly aromatic molecular structure of preserved organic matter. Despite this apparently poor molecular preservation, nano-quartz crystallization allowed for the preservation of various ultrastructures distinguished in TEM. In some filamentous microfossils (Siphonophycus) as well as in all cyanobacterial coccoids, extracellular polysaccharide sheaths appear as bands of dispersed organic nanoparticles. Synodophycus microfossils, made up of pluricellular colonies of coccoids, contain organic walls similar to the F-layers of pleurocapsalean cyanobacteria. In some fossils, internal content occurs as particulate organic matter, forming dense networks throughout ghosts of the intracellular space (e.g., in Salome svalbardensis filaments), or scarce granules (in some Chroococcales). In some chroococcalean microfossils (Gloeodiniopsis mikros, and also possibly Polybessurus), we find layered internal contents that are more continuous than nanoparticulate bands defining the sheaths, and with a shape that can be contracted, folded, or invaginated. We interpret these internal layers as the remains of cell envelope substructures and/or photosynthetic membranes thickened by additional cellular material. Some Myxococccoides show a thick (up to ~ 0.9 μm) wall ultrastructure displaying organic pillars that is best reconciled with a eukaryotic affinity. Finally, a large spheroid with ruptured wall, of uncertain affinity, displays a bi-layered envelope. Altogether, our nanoscale investigations provide unprecedented insights into the taphonomy and taxonomy of this well-preserved assemblage, which can help to assess the nature of organic microstructures in older rocks.
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Nov 2024
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[30471, 33090]
Open Access
Abstract: Iron is a crucial element integral to various fundamental biological molecular mechanisms, including magnetosome biogenesis in magnetotactic bacteria (MTB). Magnetosomes are formed through the internalization and biomineralization of iron into magnetite crystals. However, the interconnected mechanisms by which MTB uptake and regulate intracellular iron for magnetosome biomineralization remain poorly understood, particularly at the single-cell level. To gain insights we employed a holistic multiscale approach, i.e., from elemental iron species to bacterial populations, to elucidate the interplay between iron uptake dynamics and magnetosome formation in Magnetospirillum gryphiswaldense MSR-1 under near-native conditions. We combined a correlative microscopy approach integrating light and X-ray tomography with analytical techniques, such as flow cytometry and inductively coupled plasma spectroscopy, to evaluate the effects of iron and oxygen availability on cellular growth, magnetosome biogenesis, and intracellular iron pool in MSR-1. Our results revealed that increased iron availability under microaerobic conditions significantly promoted the formation of longer magnetosome chains and increased intracellular iron uptake, with a saturation point at 300 μM iron citrate. Beyond this threshold, additional iron did not further extend the magnetosome chain length or increase total intracellular iron levels. Moreover, our work reveals (i) a direct correlation between the labile Fe2+ pool size and magnetosome content, with higher intracellular iron concentrations correlating with increased magnetosome production, and (ii) the existence of an intracellular iron pool, distinct from magnetite, persisting during all stages of biomineralization. This study offers insights into iron dynamics in magnetosome biomineralization at a single-cell level, potentially enhancing the industrial biomanufacturing of magnetosomes.
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Oct 2024
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[29042, 24534]
Open Access
Abstract: Neuromelanin-pigmented neurons of the substantia nigra are selectively lost during the progression of Parkinson’s disease. These neurons accumulate iron in the disease state, and iron-mediated neuron damage is implicated in cell death. Animal models of Parkinson’s have evidenced iron loading inside the nucleoli of nigral neurons, however the nature of intranuclear iron deposition in the melanised neurons of the human substantia nigra is not understood. Here, scanning transmission x-ray microscopy (STXM) is used to probe iron foci in relation to the surrounding ultrastructure in melanised neurons of human substantia nigra from a confirmed Parkinson’s case. In addition to the expected neuromelanin-bound iron, iron deposits are also associated with the edge of the cell nucleolus. Speciation analysis confirms these deposits to be ferric (Fe3+) iron. The function of intranuclear iron in these cells remains unresolved, although both damaging and protective mechanisms are considered. This finding shows that STXM is a powerful label-free tool for the in situ, nanoscale chemical characterisation of both organic and inorganic intracellular components. Future applications are likely to shed new light on incompletely understood biochemical mechanisms, such as metal dysregulation and morphological changes to cell nucleoli, that are important in understanding the pathogenesis of Parkinson’s.
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Aug 2024
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[18925, 19958, 21485, 23508, 25247, 26657, 30442]
Abstract: For decades, when scientists have wanted to study cell architecture in granular detail and to study how viruses assemble inside the cell, they've turned to transmission electron microscopy (TEM), a high-resolution imaging technique. However, TEM studies are limited to 2D sections and offer no information regarding the 3D geometry of different features. They also look at thin slivers rather than a whole cell, and slow throughput means TEM studies are conducted on a small number of samples. On Diamond’s B24 beamline, researchers can perform correlative fluorescence microscopy and X-ray tomography, imaging proteins using structured illumination microscopy under cryogenic conditions (cryoSIM) and capturing cellular ultrastructure from the same cells using cryo-soft-X-ray tomography (cryoSXT). In a preprint recently published on bioRxiv, an international team of researchers used this correlative light X-ray tomography (CLXT) approach to study the roles of nine genes in virus assembly in herpes simplex virus-1 (HSV-1). This multi-modal imaging strategy allowed a 3D study of viral assembly, highlighting the contributions that key HSV-1 proteins make to virus assembly and underscoring the power of correlative fluorescence and X-ray tomography cryo-imaging for studies of this type.
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Jul 2024
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I08-Scanning X-ray Microscopy beamline (SXM)
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James
Everett
,
Jake
Brooks
,
Vindy Tjendana
Tjhin
,
Frederik
Lermyte
,
Ian
Hands-Portman
,
Germán
Plascencia-Villa
,
George
Perry
,
Peter J.
Sadler
,
Peter B.
O’connor
,
Joanna F.
Collingwood
,
Neil D.
Telling
Open Access
Abstract: The accumulation of amyloid plaques and increased brain redox burdens are neuropathological hallmarks of Alzheimer’s disease. Altered metabolism of essential biometals is another feature of Alzheimer’s, with amyloid plaques representing sites of disturbed metal homeostasis. Despite these observations, metal-targeting disease treatments have not been therapeutically effective to date. A better understanding of amyloid plaque composition and the role of the metals associated with them is critical. To establish this knowledge, the ability to resolve chemical variations at nanometer length scales relevant to biology is essential. Here, we present a methodology for the label-free, nanoscale chemical characterization of amyloid plaques within human Alzheimer’s disease tissue using synchrotron X-ray spectromicroscopy. Our approach exploits a C–H carbon absorption feature, consistent with the presence of lipids, to visualize amyloid plaques selectively against the tissue background, allowing chemical analysis to be performed without the addition of amyloid dyes that alter the native sample chemistry. Using this approach, we show that amyloid plaques contain elevated levels of calcium, carbonates, and iron compared to the surrounding brain tissue. Chemical analysis of iron within plaques revealed the presence of chemically reduced, low-oxidation-state phases, including ferromagnetic metallic iron. The zero-oxidation state of ferromagnetic iron determines its high chemical reactivity and so may contribute to the redox burden in the Alzheimer’s brain and thus drive neurodegeneration. Ferromagnetic metallic iron has no established physiological function in the brain and may represent a target for therapies designed to lower redox burdens in Alzheimer’s disease. Additionally, ferromagnetic metallic iron has magnetic properties that are distinct from the iron oxide forms predominant in tissue, which might be exploitable for the in vivo detection of amyloid pathologies using magnetically sensitive imaging. We anticipate that this label-free X-ray imaging approach will provide further insights into the chemical composition of amyloid plaques, facilitating better understanding of how plaques influence the course of Alzheimer’s disease.
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Mar 2024
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I08-Scanning X-ray Microscopy beamline (SXM)
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Edward
Harding
,
Tohru
Araki
,
Joseph
Askey
,
Matthew
Hunt
,
Arjen
Van Den Berg
,
David
Raftrey
,
Lucia
Aballe
,
Burkhard
Kaulich
,
Emyr
Macdonald
,
Peter
Fischer
,
Sam
Ladak
Open Access
Abstract: Artificial spin-ice systems are patterned arrays of magnetic nanoislands arranged into frustrated geometries and provide insight into the physics of ordering and emergence. The majority of these systems have been realized in two-dimensions, mainly due to the ease of fabrication, but with recent developments in advanced nanolithography, three-dimensional artificial spin ice (ASI) structures have become possible, providing a new paradigm in their study. Such artificially engineered 3D systems provide new opportunities in realizing tunable ground states, new domain wall topologies, monopole propagation, and advanced device concepts, such as magnetic racetrack memory. Direct imaging of 3DASI structures with magnetic force microscopy has thus far been key to probing the physics of these systems but is limited in both the depth of measurement and resolution, ultimately restricting measurement to the uppermost layers of the system. In this work, a method is developed to fabricate 3DASI lattices over an aperture using two-photon lithography, thermal evaporation, and oxygen plasma exposure, allowing the probe of element-specific structural and magnetic information using soft x-ray microscopy with x-ray magnetic circular dichroism (XMCD) as magnetic contrast. The suspended polymer–permalloy lattices are found to be stable under repeated soft x-ray exposure. Analysis of the x-ray absorption signal allows the complex cross section of the magnetic nanowires to be reconstructed and demonstrates a crescent-shaped geometry. Measurement of the XMCD images after the application of an in-plane field suggests a decrease in magnetic moment on the lattice surface due to oxidation, while a measurable signal is retained on sub-lattices below the surface.
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Feb 2024
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[30183, 31026]
Open Access
Abstract: Organic matter in extraterrestrial samples is a complex material that might have played an important role in the delivery of prebiotic molecules to the early Earth. We report here on the identification of nitrogen-containing compounds such as amino acids and N-heterocycles within the recent observed meteorite fall Winchcombe by high-spatial resolution spectroscopy techniques. Although nitrogen contents of Winchcombe organic matter are low (N/C ~ 1–3%), we were able to detect the presence of these compounds using a low-noise direct electron detector. These biologically relevant molecules have therefore been tentatively found within a fresh, minimally processed meteorite sample by high spatial resolution techniques conserving the overall petrographic context. Carbon functional chemistry investigations show that sizes of aromatic domains are small and that abundances of carboxylic functional groups are low. Our observations demonstrate that Winchcombe represents an important addition to the collection of carbonaceous chondrites and still preserves pristine extraterrestrial organic matter.
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Jan 2024
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B16-Test Beamline
Optics
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Diamond Proposal Number(s):
[32391, 30943, 20983, 19945]
Open Access
Abstract: Diffracting crystals are extensively used at synchrotron beamlines as x-ray monochromators and phase retarders. Imperfect growth processes, surface damage occurring during fabrication, and strain caused by poor clamping methods can all degrade the quality of these crystals and the x-ray beams diffracted by them. Because x-ray topography of these crystals can reveal both the location and the magnitude of these defects, it is now regularly used as an acceptance test for diffracting crystal optics at the Diamond Light Source synchrotron. Before installation on beamlines, crystal optics are inspected at the versatile bending-magnet B16 Test Beamline, where a variety of topographic techniques have been implemented with both white and monochromatic x-ray beams. A set of digital detectors permits rocking curve imaging with a choice of fields of view and spatial resolution down to 2 μm. Test crystals may be mounted in a variety of geometries according to need. For inspecting monochromator crystals fabricated for imaging applications, both on-the-fly scans and stitching techniques have been used to compose maps of surface defects. First crystals of multi-crystal monochromators have been tested under realistic cryocooled conditions, and their design has been improved to minimize strain. The Diamond Light Source’s x-ray topography program serves not only its own beamlines, but also industrial users and other x-ray synchrotron facilities.
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Oct 2023
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