B24-Cryo Soft X-ray Tomography
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Guiyu
Song
,
Zihan
Ma
,
Matthew
Fan
,
Liying
He
,
Yulong
Lan
,
Weihao
Li
,
Zhengtao
Jiang
,
Quan
Jiang
,
Dylan P.
Noone
,
Andrea
Nans
,
Kamal L.
Nahas
,
Mahsa Nouri
Barkestani
,
Shaoxun
Wang
,
Qianxun
Wang
,
Pengwei
Ren
,
Jolin
Cheng
,
Yinuo
Zang
,
Haitian
Zhou
,
Justin
Johnson
,
Clancy
Mullan
,
Xiangyu
Gong
,
Doryen
Bubeck
,
Gilbert
Moeckel
,
Michael
Mak
,
George
Tellides
,
Dan
Jane-Wit
Diamond Proposal Number(s):
[40765]
Open Access
Abstract: Immune effects of membrane attack complexes (MAC) have been widely attributed to their abilities to cause cell death. Here, we show that the MAC component, C9, forms non-cytolytic aggregates with pro-inflammatory effects. Intracellular aggregates of C9 are detected within inflamed tissues of patients in association with endothelial cell (EC) activation but not increased cell death. We identify NUMBL as a Rab35 effector that directly binds surface-bound C9 to promote C9 internalization and entry into the endolysosomal pathway. Within acidified endolysosomes, C9 forms insoluble aggregates that are targeted for degradative aggrephagy in a process that activates NF-κB. For C9 aggrephagy to occur, ZFYVE21, a Rab5 effector, complexes with RNF34 to bridge C9 aggregates to LC3B+ aggresome membranes. We detect C9 aggregates in vivo, and we show that a ZFYVE21-RNF34 signaling axis is required for C9 aggrephagy and NF-κB -dependent EC activation in three separate mouse models. Mice with conditional loss of ZFYVE21 in ECs show reduced aggregraphy, resulting in attenuated systemic inflammation and reduced tissue injury following skin transplantation. Our data show that the C9 component of MACs forms intracellular aggregates with alarmin-like properties.
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Jul 2026
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B16-Test Beamline
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Diamond Proposal Number(s):
[36299, 34545]
Open Access
Abstract: This study investigates the lattice strain induced by Ge:Sb alloy films on Ge substrates. Metastable films are formed by UV pulsed laser melting (PLM) of a Sb-coated Ge substrate. We fabricate thin Ge:Sb layers, systematically varying processing parameters and crystal orientation to study strain and strain-relaxation-induced defects. High-resolution X-Ray diffraction and electrical characterization revealed extremely high strain values as well as ultra-low resistivity induced by Sb. Maximum strain before the onset of strain relaxation was found to depend on crystal orientation with the Ge (1 1 1) orientation yielding the highest strain values. By combining structural as well as electrical information, we estimated Sb contribution to lattice expansion, separating electronically active from inactive fractions. Strain optimization was applied to an innovative application that is the production of bent crystals for high energy particle beam deflection and radiation production. Bending tests on thin Ge substrates confirmed the method, with controlled PLM processing allowing inducing quantifiable curvature with smallest achievable radii of 4.5 m. Exploiting non-equilibrium doping/alloying to exceed equilibrium Sb solubility is promising for applications ranging from ultra-low-resistivity layers in scaled nano-electronic devices to bent crystals for advanced systems like crystal-based undulators, enabling new approaches to high-energy photon production.
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Jun 2026
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B24-Cryo Soft X-ray Tomography
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Edward C.
Lant
,
Archana C.
Jadhav
,
Annabel
Sumeray
,
Gustavo F.
Trindade
,
Luca
Craciunescu
,
Andrew W.
Prentice
,
Juliusz A.
Wolny
,
Jaspreet S.
Grewal
,
Robert
Dallmann
,
Guy J.
Clarkson
,
Ann M.
Dixon
,
Volker
Schünemann
,
Ian S.
Gilmore
,
Martin J.
Paterson
,
Maria
Harkiolaki
,
Peter J.
Sadler
Diamond Proposal Number(s):
[32778]
Open Access
Abstract: An integrated multimodal imaging workflow of cryogenic super-resolution fluorescence microscopy and soft X-ray tomography, Orbitrap secondary ion mass spectrometry, and inductively coupled plasma-mass spectrometry has revealed the unexpected targeting of a half-sandwich cyclopentadienyl Rh(III) phenylazopyridine anticancer complex to cellular lipid membranes and lipid droplets. The complex accumulates in plasma membranes with a surprisingly intense switch-on luminescence in living cancer cells, drives remodeling of lipid droplet architecture, and penetrates deeply into lipid-rich tissue environments. DFT modeling shows strong supramolecular interactions between the complex and glycerophosphorylcholine lipids.
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Mar 2026
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[32901, 33390]
Open Access
Abstract: Understanding the intracellular fate of nanoparticles (NPs) is essential for advancing nanomedicine, particularly in targeted drug delivery for cancer therapy. Here, we present a complementary cryogenic microscopy workflow across scales to investigate the uptake and subcellular localization of zirconyl-containing inorganic–organic hybrid nanoparticles (IOH-NPs) in murine breast cancer cells. Our approach integrates cryogenic fluorescence microscopy (cryo-FM), cryo-focused ion beam scanning electron microscopy (cryo-FIBSEM), and cryo-soft X-ray tomography (cryo-SXT), enabling molecular specificity, high-resolution imaging, and volumetric ultrastructural analysis in near-native cellular states. We demonstrate that the cryogenic workflow provides enough contrast and resolution across all modalities for quantifying the IOH-NP uptake: NPs are internalized within 2 h of incubation and progressively accumulate in endolysosomes over time, as confirmed by fluorescence labeling and SXT. Quantitative analysis reveals a marked increase in endolysosomal accumulation of IOH-NPs from 2 to 24 h. Our findings help to establish multimodal cryogenic microscopy as a powerful tool for nanoscale imaging and quantitative analysis of NP uptake within close-to-native cells, offering new insights into NP trafficking and cellular responses relevant to nanomedicine development.
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Sep 2025
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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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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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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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