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Open Access
Abstract: Correlation of 3D images acquired on different microscopes can be a daunting prospect even for experienced users. This protocol describes steps for registration of images from soft X-ray absorption contrast imaging and super-resolution fluorescence imaging of hydrated biological materials at cryogenic temperatures. Although it is developed for data generated at synchrotron beamlines that offer the above combination of microscopies, it is applicable to all analogous imaging systems where the same area of a sample is examined using successive non-destructive imaging techniques.
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Jun 2021
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B24-Cryo Soft X-ray Tomography
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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).
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May 2021
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B24-Cryo Soft X-ray Tomography
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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.
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May 2021
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Ugis
Sarkans
,
Wah
Chiu
,
Lucy M.
Collinson
,
Michele C.
Darrow
,
Jan
Ellenberg
,
David
Grunwald
,
Jean-Karim
Hériché
,
Andrii
Iudin
,
Gabriel G.
Martins
,
Terry
Meehan
,
Kedar
Narayan
,
Ardan
Patwardhan
,
Matthew Robert Geoffrey
Russell
,
Helen R.
Saibil
,
Caterina
Strambio-De-Castillia
,
Jason R.
Swedlow
,
Christian
Tischer
,
Virginie
Uhlmann
,
Paul
Verkade
,
Mary
Barlow
,
Omer
Bayraktar
,
Ewan
Birney
,
Cesare
Catavitello
,
Christopher
Cawthorne
,
Stephan
Wagner-Conrad
,
Elizabeth
Duke
,
Perrine
Paul-Gilloteaux
,
Emmanuel
Gustin
,
Maria
Harkiolaki
,
Pasi
Kankaanpää
,
Thomas
Lemberger
,
Jo
Mcentyre
,
Josh
Moore
,
Andrew W.
Nicholls
,
Shuichi
Onami
,
Helen
Parkinson
,
Maddy
Parsons
,
Marina
Romanchikova
,
Nicholas
Sofroniew
,
Jim
Swoger
,
Nadine
Utz
,
Lenard M.
Voortman
,
Frances
Wong
,
Peijun
Zhang
,
Gerard J.
Kleywegt
,
Alvis
Brazma
Abstract: Bioimaging data have significant potential for reuse, but unlocking this potential requires systematic archiving of data and metadata in public databases. We propose draft metadata guidelines to begin addressing the needs of diverse communities within light and electron microscopy. We hope this publication and the proposed Recommended Metadata for Biological Images (REMBI) will stimulate discussions about their implementation and future extension.
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May 2021
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Open Access
Abstract: This protocol illustrates the steps necessary to deposit correlated 3D cryo-imaging data from cryo-structured illumination microscopy and cryo-soft X-ray tomography with the BioStudies and EMPIAR deposition databases of the European Bioinformatics Institute. There is currently a real need for a robust method of data deposition to ensure unhindered access to and independent validation of correlative light and X-ray microscopy data to allow use in further comparative studies, educational activities, and data mining.
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Mar 2021
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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.
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Mar 2021
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B24-Cryo Soft X-ray Tomography
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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.
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Jul 2020
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B24-Cryo Soft X-ray Tomography
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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.
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Jun 2020
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[23000]
Abstract: Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes from CTLs to target cells, termed supramolecular attack particles (SMAPs). SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis and CRISPR editing identified a C-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nm diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo based on specificity of shell components.
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May 2020
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[18339, 19615]
Open Access
Abstract: The organoiridium complex Ir[(C,N)2(O,O)] (1) where C, N = 1-phenylisoquinoline and O,O = 2,2,6,6-tetramethyl-3,5-heptanedionate is a promising photosensitiser for Photo-Dynamic Therapy (PDT). 1 is not toxic to cells in the dark. However, irradiation of the compound with one-photon blue or two-photon red light generates high levels of singlet oxygen (1O2) (in Zhang et al. Angew Chem Int Ed Engl 56 (47):14898-14902 https://doi.org/10.1002/anie.201709082,2017), both within cell monolayers and in tumour models. Moreover, photo-excited 1 oxidises key proteins, causing metabolic alterations in cancer cells with potent antiproliferative activity. Here, the tomograms obtained by cryo-Soft X-ray Tomography (cryo-SXT) of human PC3 prostate cancer cells treated with 1, irradiated with blue light, and cryopreserved to maintain them in their native state, reveal that irradiation causes extensive and specific alterations to mitochondria, but not other cellular components. Such new insights into the effect of 1O2 generation during PDT using iridium photosensitisers on cells contribute to a detailed understanding of their cellular mode of action.
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Mar 2020
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