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):
[18925, 19958, 21485, 23508, 25247, 26657, 30442]
Open Access
Abstract: Numerous viral genes are involved in the assembly of herpes simplex virus-1 (HSV-1), but their relative importance and function remain poorly characterised. Transmission electron microscopy has been used to study viral protein function in cells infected with HSV-1 mutants; however, these studies were usually conducted without correlative light microscopy to identify specific viral components. In this study, fluorescent capsid (eYFP-VP26) and envelope (gM-mCherry) proteins were imaged by structured illumination microscopy under cryogenic conditions (cryoSIM) and cellular ultrastructure was captured from the same infected cells using cryo-soft-X-ray tomography (cryoSXT). Nine fluorescent HSV-1 mutants, each lacking a different viral protein, were compared to assess the importance of viral proteins in different stages of HSV-1 morphogenesis. The relative importance of five viral proteins to nuclear egress were ranked (pUL34 >pUL21>VP16>pUL16>pUS3) according to the levels of attenuation observed for each virus. Correlative imaging also revealed the roles of five viral proteins in cytoplasmic envelopment. VP16 was found to be important in capsid delivery to envelopment compartments, while cytoplasmic clusters of virus particles plus features of stalled envelopment not previously described were observed in the absence of pUL11, pUL51, gK, and gE. Finally, this 3D imaging approach was used to capture different assembly stages during cytoplasmic envelopment and to determine that envelopment occurs by particle budding rather than wrapping. The findings demonstrate that tomographic 3D correlative imaging is an emerging technology that sheds new light on viral protein functions and virion morphogenesis.
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Dec 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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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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B24-Cryo Soft X-ray Tomography
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Simon
Leclerc
,
Alka
Gupta
,
Visa
Ruokolainen
,
Jian-Hua
Chen
,
Kari
Kunnas
,
Axel A.
Ekman
,
Henri
Niskanen
,
Ilya
Belevich
,
Helena
Vihinen
,
Paula
Turkki
,
Ana J.
Perez-Berna
,
Sergey
Kapishnikov
,
Elina
Mäntylä
,
Maria
Harkiolaki
,
Eric
Dufour
,
Vesa
Hytönen
,
Eva
Pereiro
,
Tony
Mcenroe
,
Kenneth
Fahy
,
Minna U.
Kaikkonen
,
Eija
Jokitalo
,
Carolyn A.
Larabell
,
Venera
Weinhardt
,
Salla
Mattola
,
Vesa
Aho
,
Maija
Vihinen-Ranta
Open Access
Abstract: iruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.
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Apr 2024
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[14508, 20326, 18833, 18312]
Open Access
Abstract: Protein misfolding is common to neurodegenerative diseases (NDs) including Alzheimer’s disease (AD), which is partly characterized by the self-assembly and accumulation of amyloid-beta in the brain. Lysosomes are a critical component of the proteostasis network required to degrade and recycle material from outside and within the cell and impaired proteostatic mechanisms have been implicated in NDs. We have previously established that toxic amyloid-beta oligomers are endocytosed, accumulate in lysosomes, and disrupt the endo-lysosomal system in neurons. Here, we use pioneering correlative cryo-structured illumination microscopy and cryo-soft X-ray tomography imaging techniques to reconstruct 3D cellular architecture in the native state revealing reduced X-ray density in lysosomes and increased carbon dense vesicles in oligomer treated neurons compared with untreated cells. This work provides unprecedented visual information on the changes to neuronal lysosomes inflicted by amyloid beta oligomers using advanced methods in structural cell biology.
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Mar 2024
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[30710, 32140]
Open Access
Abstract: Non-junctional connexin43 (Cx43) plasma membrane hemichannels have been implicated in several inflammatory diseases, particularly playing a role in ATP release that triggers activation of the inflammasome. Therapies targeting the blocking of the hemichannels to prevent the pathological release or uptake of ions and signalling molecules through its pores are of therapeutic interest. To date, there is no close-to-native, high-definition documentation of the impact of Cx43 hemichannel-mediated inflammation on cellular ultrastructure, neither is there a robust account of the ultrastructural changes that occur following treatment with selective Cx43 hemichannel blockers such as Xentry-Gap19 (XG19).
A combination of same-sample correlative high-resolution three-dimensional fluorescence microscopy and soft X-ray tomography at cryogenic temperatures, enabled in the identification of novel 3D molecular interactions within the cellular milieu when comparing behaviour in healthy states and during the early onset or late stages under inflammatory conditions. Notably, our findings suggest that XG19 blockage of connexin hemichannels under pro-inflammatory conditions may be crucial in preventing the direct degradation of connexosomes by lysosomes, without affecting connexin protein translation and trafficking. We also delineated fine and gross cellular phenotypes, characteristic of inflammatory insult or road-to-recovery from inflammation, where XG19 could indirectly prevent and reverse inflammatory cytokine-induced mitochondrial swelling and cellular hypertrophy through its action on Cx43 hemichannels. Our findings suggest that XG19 might have prophylactic and therapeutic effects on the inflammatory response, in line with functional studies.
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Mar 2024
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[33905, 31110, 33917, 31381]
Abstract: The early endosome serves as an essential sorting hub, enabling cells to process incoming signals and cues into responses. Recent studies have shown actin networks to be an increasingly important structure in the biogenesis of endocytic intermediates and their function in the sorting of internalized molecules. The ubiquity of actin in the cell underlines a need for a mechanism to polymerize actin in the appropriate cellular context. Currently, Annexin A2, a member of a highly conserved family of calcium and phospholipid binding proteins, and WASP and Scar homolog (WASH), belonging to the Class I group of nucleation-promoting factors (NPFs), have been found to be essential in actin patch formation on the surface of endosomes [1–3]. While both established to be key in linking actin dynamics with endosomal membrane dynamics, the mechanism with which Annexin A2 and WASH mediates the precise membrane rearrangements required of endosomes remains unclear. A more comprehensive view of the regulatory systems at play at the early endosome will have important implications for our understanding of endosome physiology and consequently how the cell adapts to environmental challenges.
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Aug 2023
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B24-Cryo Soft X-ray Tomography
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Abstract: Cryo-Soft X-ray Tomography (cryoSXT) is an emerging 3D imaging tool for the investigation of the cellular world under near physiological states in 3D and to a resolution of tens of nanometers. Imaging can take place over substantial fields of views (10–16 μ which can be sequentially stitched to form 3D mosaics of hundreds of microns in samples that only need to be snap frozen and therefore remain representative of the structures within fully hydrated living cells [1]. As cryoSXT exploits the natural absorption of a specified range soft X-rays by carbonaceous biological matter there is no requirement for chemical staining or otherwise processing of samples minimising therefore the risk for artefact formation [2].
As the methodology has matured over the past few years, the need has arisen for well mapped processes in sample preparation, data collection and data analyses as well as correlation with other imaging methods to allow us to harness the power of more than one technique for any one research endeavor. Concurrently the need to standardize and quantify the quality and content of cryoSXT data collected at different locations and over different experiments necessitates the design of archiving and reporting protocols that will democratise access to the accumulated data for the benefit of the broader scientific community and researchers that are not necessarily technique experts.
Here, the theory and current implementations of cryoSXT will be presented alongside the latest developments in the field and the research that has been enabled through its use. Data from the UK synchrotron beamline B24 will be used to exemplify applications including correlative 3D imaging (Figure 1) and method automation (Figure 2) that highlights the pressing need for cryoSXT availability in biomedical research. New, user-friendly decision matrices developed at B24 that aim to both simplify and standardize the use of this method will be also presented and the landscape which lies ahead will be explored.
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Aug 2023
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