B24-Cryo Soft X-ray Tomography
|
Mohamed A.
Koronfel
,
Ilias
Kounatidis
,
Dennis M.
Mwangangi
,
Nina
Vyas
,
Chidinma
Okolo
,
Archana
Jadhav
,
Tom
Fish
,
Phatcharin
Chotchuang
,
Albert
Schulte
,
Robert
Robinson
,
Maria
Harkiolaki
Diamond Proposal Number(s):
[23033, 23073]
Open Access
Abstract: Imaging of actin filaments is crucial due to the integral role that they play in many cellular functions such as intracellular transport, membrane remodelling and cell motility. Visualizing actin filaments has so far relied on fluorescence microscopy and electron microscopy/tomography. The former lacks the capacity to capture the overall local ultrastructure, while the latter requires rigorous sample preparation that can lead to potential artefacts, and only delivers relatively small volumes of imaging data at the thinnest areas of a cell. In this work, a correlative approach utilizing in situ super-resolution fluorescence imaging and cryo X-ray tomography was used to image bundles of actin filaments deep inside cells under near-native conditions. In this case, fluorescence 3D imaging localized the actin bundles within the intracellular space, while X-ray tomograms of the same areas provided detailed views of the local ultrastructure. Using this new approach, actin trails connecting vesicles in the perinuclear area and hotspots of actin presence within and around multivesicular bodies were observed. The characteristic prevalence of filamentous actin in cytoplasmic extensions was also documented.
|
Dec 2021
|
|
B24-Cryo Soft X-ray Tomography
|
Open Access
Abstract: Chlamydiae are strict intracellular pathogens residing within a specialised membrane-bound compartment called the inclusion. Therefore, each infected cell can, be considered as a single entity where bacteria form a community within the inclusion. It remains unclear as to how the population of bacteria within the inclusion influences individual bacterium. The life cycle of Chlamydia involves transitioning between the invasive elementary bodies (EBs) and replicative reticulate bodies (RBs). We have used cryo-soft X-ray tomography to observe individual inclusions, an approach that combines 40 nm spatial resolution and large volume imaging (up to 16 µm). Using semi-automated segmentation pipeline, we considered each inclusion as an individual bacterial niche. Within each inclusion, we identifyed and classified different forms of the bacteria and confirmed the recent finding that RBs have a variety of volumes (small, large and abnormal). We demonstrate that the proportions of these different RB forms depend on the bacterial concentration in the inclusion. We conclude that each inclusion operates as an autonomous community that influences the characteristics of individual bacteria within the inclusion.
|
Aug 2021
|
|
B24-Cryo Soft X-ray Tomography
|
Diamond Proposal Number(s):
[21046, 18314]
Abstract: Researchers have developed a new technique for studying cells in their native state. The goal was to obtain high-quality imaging data from cells without the need for sectioning or chemical fixation. The new method avoids any treatment that would disturb cell structure, so that no artefacts (errors) are introduced into the images.
To demonstrate this novel correlative microscopy platform’s effectiveness, the team studied the early stages of cell infection by reoviruses. Although the specific viruses have been studied extensively, there is a debate regarding the method of infection. This research focused on the way that the virus escapes from vesicles, a required step for replication. At beamline B24, using a correlative imaging approach by combining soft X-ray tomography with super resolution microscopy in cryogenic conditions, the team tracked the infection mechanism. The results revealed that the virus had already escaped from the host vesicles two hours after infection, with the vesicles preserving their circular shape, suggesting a gentle, pore-based exit mechanism for the virus.
Reoviruses are valuable tools that could be engineered to express proteins and have the potential to be used in vaccines. Knowing the infection mechanism will facilitate their handling and manipulation for biomedical purposes. The new imaging platform has also been used to validate anticancer compounds, study cell structure during development and investigate clearance of human pathogenic microorganism by immune host cells. The work is the outcome of a collective effort between Diamond Light Source and research groups and facilities across Europe, including the University of Oxford, Heidelberg University Hospital, the Université de Nantes and CryoCapCell.
|
Jul 2021
|
|
B24-Cryo Soft X-ray Tomography
Krios II-Titan Krios II at Diamond
|
Luiza
Mendonca
,
Andrew
Howe
,
James B.
Gilchrist
,
Yuewen
Sheng
,
Dapeng
Sun
,
Michael L.
Knight
,
Laura C.
Zanetti-Domingues
,
Benji
Bateman
,
Anna-Sophia
Krebs
,
Long
Chen
,
Julika
Radecke
,
Vivian D.
Li
,
Tao
Ni
,
Ilias
Kounatidis
,
Mohamed A.
Koronfel
,
Marta
Szynkiewicz
,
Maria
Harkiolaki
,
Marisa
Martin-Fernandez
,
William
James
,
Peijun
Zhang
Diamond Proposal Number(s):
[21004, 26987]
Open Access
Abstract: Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events – e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.
|
Jul 2021
|
|
B24-Cryo Soft X-ray Tomography
|
Chidinma
Okolo
,
Ilias
Kounatidis
,
Johannes
Groen
,
Kamal
Nahas
,
Stefan
Balint
,
Thomas M.
Fish
,
Mohamed A.
Koronfel
,
Aitziber L.
Cortajarena
,
Ian M.
Dobbie
,
Eva
Pereiro
,
Maria
Harkiolaki
Diamond Proposal Number(s):
[18737, 20321, 22274, 23046, 25162]
Abstract: 3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2–50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).
|
May 2021
|
|
B24-Cryo Soft X-ray Tomography
|
Nina
Vyas
,
Nina
Perry
,
Chidinma A.
Okolo
,
Ilias
Kounatidis
,
Thomas M.
Fish
,
Kamal L.
Nahas
,
Archana
Jadhav
,
Mohamed A.
Koronfel
,
Johannes
Groen
,
Eva
Pereiro
,
Ian M.
Dobbie
,
Maria
Harkiolaki
Diamond Proposal Number(s):
[25512]
Open Access
Abstract: Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron.
It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.
|
May 2021
|
|
B24-Cryo Soft X-ray Tomography
|
Diamond Proposal Number(s):
[23046]
Abstract: Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 µm thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.
|
Mar 2021
|
|
|
|
Open Access
Abstract: Introduction: Hypoxia inducible factors (HIF) and prolyl hydroxylase domain (PHD) enzymes play a central role in tumor progression in clear cell renal cell carcinoma (ccRCC). However, there are currently no data regarding the behavior of this pathway (HIF/PHD) in a large number of benign renal tumors, the oncocytomas. The aim of the present study was to compare the expression levels of these factors between ccRCC and oncocytoma tumors. Material and methods: A total of 56 fresh frozen specimens from patients with ccRCC and 14 oncocytoma specimens were analyzed via reverse transcription-quantitative polymerase chain reaction in order to assess the expression levels of HIF-1α, HIF-2α, PHD1, PHD2, and PHD3. The analysis involved both fresh frozen tumor samples as well as adjacent normal kidney tissues. Results: In ccRCC, HIF-1α and HIF-2α levels were upregulated in 65.5% and 71.4% of cases, respectively. PHD3 was downregulated only in 15.4% of the ccRCC cases, in contrast with oncocytoma cases, which exhibited low expression levels in the majority. The upregulation of PHD3 messenger RNA (mRNA) levels in ccRCC when compared with oncocytoma was statistically significant (P<0.001). No other comparisons (HIF-1α, HIF-2α, PHD1, and PHD2) were significantly different. HIF-2α and PHD3 mRNA expression levels were negatively correlated with Fuhrman Grade (P=0.029 and P=0.026, respectively) in ccRCC. Conclusion: To the best of our knowledge, this is the first time that the HIF/PHD pathway was compared between ccRCC and a common benign tumor, identifying the upregulation of PHD3 as the possible underlying factor guiding the difference in the behavior of ccRCC.
|
Oct 2020
|
|
B24-Cryo Soft X-ray Tomography
|
Michael A.
Phillips
,
Maria
Harkiolaki
,
David Miguel
Susano Pinto
,
Richard M.
Parton
,
Ana
Palanca
,
Manuel
Garcia-Moreno
,
Ilias
Kounatidis
,
John W.
Sedat
,
David I.
Stuart
,
Alfredo
Castello
,
Martin J.
Booth
,
Ilan
Davis
,
Ian
Dobbie
Diamond Proposal Number(s):
[18319, 18757]
Open Access
Abstract: Rapid cryopreservation of biological specimens is the gold standard for visualizing cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo-electron microscopy or cryo-soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly, open-source Python control software. Therefore, cryoSIM democratizes the ability to detect molecules using super-resolution fluorescence imaging of cryopreserved specimens for correlation with their cellular ultrastructure.
|
Jul 2020
|
|
B24-Cryo Soft X-ray Tomography
|
Ilias
Kounatidis
,
Megan L.
Stanifer
,
Michael A.
Phillips
,
Perrine
Paul-Gilloteaux
,
Xavier
Heiligenstein
,
Hongchang
Wang
,
Chidinma
Okolo
,
Thomas M.
Fish
,
Matthew C.
Spink
,
David I.
Stuart
,
Ilan
Davis
,
Steeveh
Boulant
,
Jonathan M.
Grimes
,
Ian M.
Dobbie
,
Maria
Harkiolaki
Diamond Proposal Number(s):
[21046, 18314]
Open Access
Abstract: Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.
|
Jun 2020
|
|
