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1. Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

Author

Alexandra Botté, Jeanne Lainé, Laura Xicota, Xavier Heiligenstein, Gaëlle Fontaine, Amal Kasri, Isabelle Rivals, Pollyanna Goh, Orestis Faklaris, Jack-Christophe Cossec, Etienne Morel, Anne-Sophie Rebillat, Dean Nizetic, Graça Raposo, and Marie-Claude Potier

Subjects
Down syndrome, Alzheimer’s disease, Early endosomes, Endocytosis, Electron microscopy, Super-resolution microscopy, Neurology. Diseases of the nervous system, RC346-429
Abstract

Abstract Enlarged early endosomes have been visualized in Alzheimer’s disease (AD) and Down syndrome (DS) using conventional confocal microscopy at a resolution corresponding to endosomal size (hundreds of nm). In order to overtake the diffraction limit, we used super-resolution structured illumination microscopy (SR-SIM) and transmission electron microscopies (TEM) to analyze the early endosomal compartment in DS. By immunofluorescence and confocal microscopy, we confirmed that the volume of Early Endosome Antigen 1 (EEA1)-positive puncta was 13–19% larger in fibroblasts and iPSC-derived neurons from individuals with DS, and in basal forebrain cholinergic neurons (BFCN) of the Ts65Dn mice modelling DS. However, EEA1-positive structures imaged by TEM or SR-SIM after chemical fixation had a normal size but appeared clustered. In order to disentangle these discrepancies, we imaged optimally preserved High Pressure Freezing (HPF)-vitrified DS fibroblasts by TEM and found that early endosomes were 75% denser but remained normal-sized. RNA sequencing of DS and euploid fibroblasts revealed a subgroup of differentially-expressed genes related to cargo sorting at multivesicular bodies (MVBs). We thus studied the dynamics of endocytosis, recycling and MVB-dependent degradation in DS fibroblasts. We found no change in endocytosis, increased recycling and delayed degradation, suggesting a “traffic jam” in the endosomal compartment. Finally, we show that the phosphoinositide PI (3) P, involved in early endosome fusion, is decreased in DS fibroblasts, unveiling a new mechanism for endosomal dysfunctions in DS and a target for pharmacotherapy.

Published
2020
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2. Zika virus enhances monocyte adhesion and transmigration favoring viral dissemination to neural cells

Author

Nilda Vanesa Ayala-Nunez, Gautier Follain, François Delalande, Aurélie Hirschler, Emma Partiot, Gillian L. Hale, Brigid C. Bollweg, Judith Roels, Maxime Chazal, Florian Bakoa, Margot Carocci, Sandrine Bourdoulous, Orestis Faklaris, Sherif R. Zaki, Anita Eckly, Béatrice Uring-Lambert, Frédéric Doussau, Sarah Cianferani, Christine Carapito, Frank M. J. Jacobs, Nolwenn Jouvenet, Jacky G. Goetz, and Raphael Gaudin

Subjects
Science
Abstract

Zika virus (ZIKV) can infect the central nervous system, but it is not clear how it reaches the brain. Here, Ayala-Nunez et al. show in ex vivo and in vivo models that ZIKV can hitch a ride in monocytes in a Trojan Horse manner to cross the endothelium and disseminate the virus.

Published
2019
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3. The PAX-FOXO1s trigger fast trans-differentiation of chick embryonic neural cells into alveolar rhabdomyosarcoma with tissue invasive properties limited by S phase entry inhibition.

Author

Gloria Gonzalez Curto, Audrey Der Vartanian, Youcef El-Mokhtar Frarma, Line Manceau, Lorenzo Baldi, Selene Prisco, Nabila Elarouci, Frédéric Causeret, Daniil Korenkov, Muriel Rigolet, Frédéric Aurade, Aurélien De Reynies, Vincent Contremoulins, Frédéric Relaix, Orestis Faklaris, James Briscoe, Pascale Gilardi-Hebenstreit, and Vanessa Ribes

Subjects
Genetics, QH426-470
Abstract

The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS.

Published
2020
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4. Mapping molecular assemblies with fluorescence microscopy and object-based spatial statistics

Author

Thibault Lagache, Alexandre Grassart, Stéphane Dallongeville, Orestis Faklaris, Nathalie Sauvonnet, Alexandre Dufour, Lydia Danglot, and Jean-Christophe Olivo-Marin

Subjects
Science
Abstract

Elucidating molecular organisation requires precise localisation and analysis. Here the authors develop SODA software for automatic and quantitative mapping of statistically coupled molecules, and use it to unravel spatial organisation of thousands of synaptic proteins in SIM and 3DSTORM microscopy.

Published
2018
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5. Distinct molecular cues ensure a robust microtubule-dependent nuclear positioning in the Drosophila oocyte

Author

Nicolas Tissot, Jean-Antoine Lepesant, Fred Bernard, Kevin Legent, Floris Bosveld, Charlotte Martin, Orestis Faklaris, Yohanns Bellaïche, Maïté Coppey, and Antoine Guichet

Subjects
Science
Abstract

Asymmetric nuclear positioning in the fruit fly oocyte is essential for the correct localization of axis determinants. Here, the authors show that different microtubule-dependent mechanisms contribute to nuclear transport and ensure the robustness of nuclear positioning.

Published
2017
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6. Original Fluorescent Ligand-Based Assays Open New Perspectives in G-Protein Coupled Receptor Drug Screening

Author

Thierry Durroux, Jurriaan M. Zwier, Eric Trinquet, Orestis Faklaris, Martin Cottet, and Jean-Philippe Pin

Subjects
screening assay, fluorescence, lanthanides, time resolved FRET, GPCR, Medicine, Pharmacy and materia medica, RS1-441
Abstract

The identification of new drugs exhibiting reduced adverse side-effects constitutes a great challenge for the next decade. Various steps are needed to screen for good ligand candidates and one of them is the evaluation of their binding properties. New strategies based on fluorescence measurement constitute excellent alternatives to the traditional radioactive assays. Less hazardous, faster and cheaper, these methods also exhibit very good sensitivity and can be used on various biological models such as heterologous expression systems or native tissues.

Published
2011
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8. Towards community-driven metadata standards for light microscopy: tiered specifications extending the OME model

Author

Maximiliaan Huisman, James J. Chambers, Alex Laude, Peter Bajcsy, Ulrike Boehm, Caterina Strambio-De-Castillia, Damir Sudar, Orestis Faklaris, Glyn Nelson, Mathias Hammer, Nathalie Gaudreault, Farzin Farzam, Carlas Smith, Claire M. Brown, Roland Nitschke, Alexander D. Corbett, Judith Lacoste, Jaime A. Pimentel, Alison J. North, Alessandro Rigano, and David Grunwald

Subjects
Quality Control, Societies, Scientific, Biomedical Research, Computer science, Data management, media_common.quotation_subject, Biochemistry, Article, User-Computer Interface, Software, Animals, Data Mining, Humans, Quality (business), Instrumentation (computer programming), Namespace, Molecular Biology, media_common, Metadata, Microscopy, business.industry, Data Collection, Quality control, Reproducibility of Results, Cell Biology, Data science, Systems Integration, Data model (ArcGIS), Calibration, business, Biotechnology
Abstract

1 -ABSTRACTDigital light microscopy provides powerful tools for quantitatively probing the real-time dynamics of subcellular structures. While the power of modern microscopy techniques is undeniable, rigorous record-keeping and quality control are required to ensure that imaging data may be properly interpreted (quality), reproduced (reproducibility), and used to extract reliable information and scientific knowledge which can be shared for further analysis (value). Keeping notes on microscopy experiments and quality control procedures ought to be straightforward, as the microscope is a machine whose components are defined and the performance measurable. Nevertheless, to this date, no universally adopted community-driven specifications exist that delineate the required information about the microscope hardware and acquisition settings (i.e., microscopy “data provenance” metadata) and the minimally accepted calibration metrics (i.e., microscopy quality control metadata) that should be automatically recorded by both commercial microscope manufacturers and customized microscope developers. In the absence of agreed guidelines, it is inherently difficult for scientists to create comprehensive records of imaging experiments and ensure the quality of resulting image data or for manufacturers to incorporate standardized reporting and performance metrics. To add to the confusion, microscopy experiments vary greatly in aim and complexity, ranging from purely descriptive work to complex, quantitative and even sub-resolution studies that require more detailed reporting and quality control measures.To solve this problem, the4D Nucleome Initiative (4DN) (1, 2) Imaging Standards Working Group (IWG), working in conjunction with theBioImagingNorthAmerica (BINA) Quality Control and Data Management Working Group (QC-DM-WG) (3), here propose light Microscopy Metadata specifications that scale with experimental intent and with the complexity of the instrumentation and analytical requirements. They consist of a revision of the Core of the Open Microscopy Environment (OME) Data Model, which forms the basis for the widely adopted Bio-Formats library (4–6), accompanied by a suite of three extensions, each with three tiers, allowing the classification of imaging experiments into levels of increasing imaging and analytical complexity (7, 8). Hence these specifications not only provide an OME-based comprehensive set of metadata elements that should be recorded, but they also specify which subset of the full list should be recorded for a given experimental tier. In order to evaluate the extent of community interest, an extensive outreach effort was conducted to present the proposed metadata specifications to members of several core-facilities and international bioimaging initiatives including theEuropeanLightMicroscopyInitiative (ELMI),GlobalBioImaging (GBI), andEuropeanMolecularBiologyLaboratory (EMBL) -EuropeanBioinformaticsInstitute (EBI). Consequently, close ties were established between our endeavour and the undertakings of the recently establishedQUAlity Assessment andREProducibility for Instruments and Images inLightMicroscopy global community initiative (9). As a result this flexible 4DN-BINA-OME (NBO namespace) framework (7, 8) represents a turning point towards achieving community-driven Microscopy Metadata standards that will increase data fidelity, improve repeatability and reproducibility, ease future analysis and facilitate the verifiable comparison of different datasets, experimental setups, and assays, and it demonstrates the method for future extensions. Such universally accepted microscopy standards would serve a similar purpose as the Encode guidelines successfully adopted by the genomic community (10, 11). The intention of this proposal is therefore to encourage participation, critiques and contributions from the entire imaging community and all stakeholders, including research and imaging scientists, facility personnel, instrument manufacturers, software developers, standards organizations, scientific publishers, and funders.

Published
2021

9. Structure and mechanics of the human Nuclear Pore Complex basket

Author

Anthony Vial, Luca Costa, Patrice Dosset, Pietro Rosso, Gaëlle Boutières, Orestis Faklaris, Heiko Haschke, Pierre-Emmanuel Milhiet, Christine M. Doucet, Centre de Biologie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Montpellier Ressources Imagerie, Biocampus, CNRS, INSERM, Universite Montpellier, Montpellier, France, BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Bruker, and DOUCET, Christine

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio]
Abstract

Nuclear pore complexes (NPCs) are the only gateways between the nucleus and cytoplasm in eukaryotic cells. They restrict free diffusion to molecules below 5 nm while facilitating the active transport of selected cargoes, sometimes as large as the pore itself. This versatility implies an important pore plasticity. Recently, cryo-EM and AI-based protein modeling revealed with acute precision how most NPC constituents are arranged. But the basket, a fish trap-like structure capping the nucleoplasmic side of the pore, remains the missing piece in this puzzle. Here by Atomic Force Microscopy (AFM) coupled to Single Molecule Localization Microscopy (SMLM) we revealed that the basket is very soft and explores a large conformational landscape: apart from its canonical shape, it dives into the central pore channel or opens, with filaments reaching to the pore sides. Our observations enlighten how this structure can adapt and let morphologically diverse cargoes shuttling through NPCs.

Published
2022

10. Quality assessment in light microscopy for routine use through simple tools and robust metrics

Author

Orestis Faklaris, Leslie Bancel-Vallée, Aurélien Dauphin, Baptiste Monterroso, Perrine Frère, David Geny, Tudor Manoliu, Sylvain de Rossi, Fabrice P. Cordelières, Damien Schapman, Roland Nitschke, Julien Cau, and Thomas Guilbert

Subjects
Microscopy, Fluorescence, Image Processing, Computer-Assisted, Reproducibility of Results, Cell Biology, Software
Abstract

Although there is a need to demonstrate reproducibility in light microscopy acquisitions, the lack of standardized guidelines monitoring microscope health status over time has so far impaired the widespread use of quality control (QC) measurements. As scientists from 10 imaging core facilities who encounter various types of projects, we provide affordable hardware and open source software tools, rigorous protocols, and define reference values to assess QC metrics for the most common fluorescence light microscopy modalities. Seven protocols specify metrics on the microscope resolution, field illumination flatness, chromatic aberrations, illumination power stability, stage drift, positioning repeatability, and spatial-temporal noise of camera sensors. We designed the MetroloJ_QC ImageJ/Fiji Java plugin to incorporate the metrics and automate analysis. Measurements allow us to propose an extensive characterization of the QC procedures that can be used by any seasoned microscope user, from research biologists with a specialized interest in fluorescence light microscopy through to core facility staff, to ensure reproducible and quantifiable microscopy results.

Published
2022

11. QUAREP-LiMi: a community endeavor to advance quality assessment and reproducibility in light microscopy

Author

John E Eriksson, Arne Seitz, Ian M. Dobbie, Caterina Strambio-De-Castillia, Jason R. Swedlow, Ali Gheisari, Miso Mitkovski, Alexia Ferrand, Roland Nitschke, Steve Bagley, Tobias M. Rasse, Glyn Nelson, Alex Laude, Peter Bajcsy, Alison J. North, Laurent Gelman, Julia Fernandez-Rodriguez, Ute Resch-Genger, Christian Kukat, Sebastian Munck, Claire M. Brown, Johanna Bischof, Ulrike Boehm, Hella Hartmann, Aurelien Dauphin, Lucas C Schuetz, and Orestis Faklaris

Subjects
Quality Control, medicine.medical_specialty, Biomedical Research, media_common.quotation_subject, Biochemistry, Article, 03 medical and health sciences, Microscopy, Image Processing, Computer-Assisted, medicine, Humans, Image acquisition, Medical physics, Quality (business), Molecular Biology, Reliability (statistics), 030304 developmental biology, Research data, media_common, Metadata, 0303 health sciences, Reproducibility, business.industry, Quality assessment, Reproducibility of Results, cell-line authentication, Cell Biology, crisis, Calibration, business, Quality assurance, Software, Biotechnology
Abstract

The community-driven initiative Quality Assessment and Reproducibility for Instruments & Images in Light Microscopy (QUAREP-LiMi) wants to improve reproducibility for light microscopy image data through quality control (QC) management of instruments and images. It aims for a common set of QC guidelines for hardware calibration and image acquisition, management and analysis.

Published
2021

12. Occludin stalls HCV particle dynamics apart from hepatocyte tight junctions, promoting virion internalization

Author

Maika S. Deffieu, Camille M. H Clément, Cristina M. Dorobantu, Emma Partiot, Yonis Bare, Orestis Faklaris, Benjamin Rivière, Nilda Vanesa Ayala‐Nunez, Thomas F. Baumert, Philippe Rondé, Yves Mély, Vincent Lucansky, Raphael Gaudin, Institut de Recherche en Infectiologie de Montpellier (IRIM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Institut de Recherche sur les Maladies Virales et Hépatiques (IVH), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Laboratoire de Bioimagerie et Pathologies (LBP), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), European Project: 667273,H2020,H2020-PHC-2015-two-stage,HEP-CAR(2016), and European Project: 609102,EC:FP7:PEOPLE,FP7-PEOPLE-2013-COFUND,PRESTIGE(2014)

Subjects
Hepatology, Virion, [SDV.MHEP.HEM]Life Sciences [q-bio]/Human health and pathology/Hematology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Hepacivirus, Sciences du Vivant [q-bio]/Médecine humaine et pathologie, Virus Internalization, Hepatitis C, Tight Junctions, live cell imaging, flavivirus, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, CRISPR, CRISPR-Associated Protein 9, Occludin, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Claudin-1, Hepatocytes, Humans, human liver, virus-host interactions
Abstract

International audience; Background and Aims: Numerous HCV entry factors have been identified, and yet information regarding their spatiotemporal dynamics is still limited. Specifically, one of the main entry factors of HCV is occludin (OCLN), a protein clustered at tight junctions (TJs), away from the HCV landing site. Thus, whether HCV particles slide toward TJs or, conversely, OCLN is recruited away from TJs remain debated.Approach and Results: Here, we generated CRISPR/CRISPR-associated protein 9 edited Huh7.5.1 cells expressing endogenous levels of enhanced green fluorescent protein/OCLN and showed that incoming HCV particles recruit OCLN outside TJs, independently of claudin 1 (CLDN1) expression, another important HCV entry factor located at TJs. Using ex vivo organotypic culture of hepatic slices obtained from human liver explants, a physiologically relevant model that preserves the overall tissue architecture, we confirmed that HCV associates with OCLN away from TJs. Furthermore, we showed, by live cell imaging, that increased OCLN recruitment beneath HCV particles correlated with lower HCV motility. To decipher the mechanism underlying virus slow-down upon OCLN recruitment, we performed CRISPR knockout (KO) of CLDN1, an HCV entry factor proposed to act upstream of OCLN. Although CLDN1 KO potently inhibits HCV infection, OCLN kept accumulating underneath the particle, indicating that OCLN recruitment is CLDN1 independent. Moreover, inhibition of the phosphorylation of Ezrin, a protein involved in HCV entry that links receptors to the actin cytoskeleton, increased OCLN accumulation and correlated with more efficient HCV internalization.Conclusions: Together, our data provide robust evidence that HCV particles interact with OCLN away from TJs and shed mechanistic insights regarding the manipulation of transmembrane receptor localization by extracellular virus particles.

Published
2022

13. Illumination Power and illumination stability v1

Author

Nathalie Gaudreault, Steve Bagley, Rodrigo R Bammann, Fabio Barachati, Laszlo Barna, Veronika Boczonadi, Ulrike Boehm, Manel Bosch, Craig Brideau, Mariana T Carvalho, Pina Colarusso, Richard Cole, Nasser Darwish-Miranda, Sam Duwé, Frank Eismann, Orestis Faklaris, Andreas Felscher, Manfred Gonnert, David Grunwald, Marcel Kirchner, Birgit Hoffmann, Gabriel Krens, Alex J Laude, Jeffrey M LeDue, Pascal Lorentz, Miso Mitkovski, Michael S Nelson, Britta Schroth-Diez, Stanley Schwartz, Sathya Srinivasan, Roland Thuenauer, Tse-Luen Wee, Kees van der Oord, Chloë van Oostende-Triplet, Roland Nitschke, and Laurent Gelman

Subjects
ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION
Abstract

To obtain accurate, reproducible, and interpretable data when conducting imaging experiments, it is critical to consider external factors affecting data acquisition at various steps of the experimental workflow. Illumination power and stability represent two critical factors, especially when comparing fluorescence intensities between images during a time-lapse experiment or experiments performed at different times or on other microscopes. The fluorescence signal can be generated by different types of light sources. These light sources and their coupling elements (e.g., fibers) can display varying performances over time as they age, move, or as environmental conditions change. Unfortunately, microscope users can often only set illumination power as a percentage of its maximal output and, may therefore, not be aware of potential performance changes. It is important to recognize that a set percentage will not always yield the same illumination power in Watts at the objective over the course of an experiment, not to mention between days or systems. This means that selecting for example 10% output may lead to different experimental results over time and will not necessarily be comparable to outputs obtained from other lasers or microscopes, even those of the very same model. If you are responsible for system maintenance, routinely measuring the illumination power, stability, and linearity over time can help you detect issues that affect the integrity of the system and thus the reproducibility of an experiment. This protocol describes how to measure the stability and linearity of the illumination power using calibrated external power sensors. This protocol is at the moment intended for confocal systems (raster scanning and spinning disks), but will be extended later to other imaging modalities. It represents the collective experience of 60 imaging scientists. Measurements made by our working group with this protocol are available in a public database, which will be updated with further contributions from the community.

Published
2021

14. Long-term quality assessment and monitoring of light microscope performance through accessible and reliable protocols, tools and metrics

Author

Orestis Faklaris, Geny D, Thomas Guilbert, Frere P, Cau J, Roland Nitschke, Damien Schapman, Manoliu T, Fabrice P. Cordelières, Monterroso B, de Rossi S, Aurelien Dauphin, and Bancel-Vallee L

Subjects
Data processing, Microscope, Computer science, Automated data processing, Real-time computing, ComputingMethodologies_IMAGEPROCESSINGANDCOMPUTERVISION, Quality control, computer.software_genre, law.invention, law, Robustness (computer science), Microscopy, Data analysis, Plug-in, computer
Abstract

Reliable, reproducible and comparable results are what biology requires from microscopy. To achieve that level of confidence, monitoring the stability of the microscope performance over time with standardized quality testing routines is essential for mining quantitative data. Three levels of microscope quality control procedures should be considered: i) usage of accessible and affordable tools and samples, ii) execution of easy and fast, preferably automatized, acquisition protocols, iii) analysis of data in the most automated way possible with adequate metrics for long-term monitoring. In this paper, we test the acquisition protocols on the mainly used microscope techniques (wide-field, spinning disk and confocal microscopy) with simple quality control tools. Seven protocols specify metrics on measuring the lateral and axial resolution (Point-Spread Function) of the system, field flatness, chromatic aberrations and co-registration, illumination power monitoring and stability, stage drift and positioning repeatability and finally temporal and spatial noise sources of camera detectors. We designed an ImageJ/FiJi java plugin named MetroloJ_QC to incorporate the identified metrics and automatize the data processing for the analysis. After processing and comparing the data of microscopes from more than ten imaging facilities, we test the robustness of the metrics and the protocols by determining experimental limit values. Our results give a first extensive characterization of the quality control procedures of a light microscope, with an automated data processing and experimental limit values that can be used by core facility staff and researchers to monitor the microscope performance over time.

Published
2021

15. Micro-Meta App: an interactive tool for collecting microscopy metadata based on community specifications

Author

James J. Chambers, Karl D. Bellve, Glyn Nelson, Claire M. Brown, Serkan Utku Öztürk, Willa Y. Ma, Jaime A. Pimentel, Orestis Faklaris, Michelle S. Itano, Mathias Hammer, Daniel P. Keeley, Marco Marcello, David Grunwald, Ulrike Boehm, Alessandro Rigano, Koray Kirli, Caterina Strambio-De-Castillia, Alexander Balashov, Alex Laude, Shannon Ehmsen, Judith Lacoste, Joel Ryan, Robert A. Coleman, Burak H. Alver, Stefanie Weidtkamp-Peters, Anna B Hamacher, Susanne Kunis, Roland Nitschke, Paula Montero-Llopis, Andrea Cosolo, Thomas Guilbert, Peter J. Park, and Kevin E. Fogarty

Subjects
Quality Control, Standards, Computer science, media_common.quotation_subject, Software tool, Interoperability, Context (language use), Brief Communication, Biochemistry, Data publication and archiving, Cell Line, Pattern Recognition, Automated, Workflow, Mice, User-Computer Interface, Microscopy, Image Processing, Computer-Assisted, Animals, Humans, Quality (business), Molecular Biology, media_common, Metadata, Information retrieval, Microscopy, Confocal, business.industry, Computational Biology, Reproducibility of Results, Cell Biology, Mobile Applications, Confocal microscopy, Wide-field fluorescence microscopy, Microscopy, Fluorescence, Programming Languages, business, Quality assurance, Software, Biotechnology
Abstract

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes., Micro-Meta App is an intuitive, highly interoperable, open-source software tool designed to facilitate the extraction and collection of relevant microscopy metadata as specified by recent community guidelines.

Published
2021

16. Micro-Meta App: an interactive software tool to facilitate the collection of microscopy metadata based on community-driven specifications

Author

Willa Y. Ma, Alexander Balashov, Michelle S. Itano, Judith Lacoste, James J. Chambers, Daniel P. Keeley, Karl A. Bellvé, Orestis Faklaris, Grunwald D, Anna B Hamacher, Joel Ryan, Andrea Cosolo, Koray Kirli, Claire M. Brown, Alex Rigano, Marco Marcello, Mathias Hammer, Stefanie Weidtkamp-Peters, Serkan Utku Öztürk, Alex Laude, Peter J. Park, Glyn Nelson, Roland Nitschke, Burak H. Alver, Kunis S, Thomas Guilbert, Paula Montero-Llopis, Ulrike Boehm, Shannon Ehmsen, Caterina Strambio-De-Castillia, Kevin E. Fogarty, Robert A. Coleman, and Jaime A. Pimentel

Subjects
Information retrieval, business.industry, Computer science, Interface (computing), Context (language use), computer.file_format, Metadata modeling, Metadata, Software, Documentation, Data quality, Image file formats, business, computer
Abstract

For the information content of microscopy images to be appropriately interpreted, reproduced, and meet FAIR (Findable Accessible Interoperable and Reusable) principles, they should be accompanied by detailed descriptions of microscope hardware, image acquisition settings, image pixel and dimensional structure, and instrument performance. Nonetheless, the thorough documentation of imaging experiments is significantly impaired by the lack of community-sanctioned easy-to-use software tools to facilitate the extraction and collection of relevant microscopy metadata. Here we presentMicro-Meta App, an intuitive open-source software designed to tackle these issues that was developed in the context of nascent global bioimaging community organizations, includingBioImagingNorthAmerica (BINA) andQUAlity Assessment andREProducibility inLightMicroscopy (QUAREP-LiMi), whose goal is to improve reproducibility, data quality and sharing value for imaging experiments. The App provides a user-friendly interface for building comprehensive descriptions of the conditions utilized to produce individual microscopy datasets as specified by the recently proposed 4DN-BINA-OME tiered-system of Microscopy Metadata model. To achieve this goal the App provides a visual guide for a microscope-user to: 1) interactively build diagrammatic representations of hardware configurations of given microscopes that can be easily reused and shared with colleagues needing to document similar instruments. 2) Automatically extracts relevant metadata from image files and facilitates the collection of missing image acquisition settings and calibration metrics associated with a given experiment. 3) Output all collected Microscopy Metadata to interoperable files that can be used for documenting imaging experiments and shared with the community. In addition to significantly lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training users that have limited knowledge of the intricacies of light microscopy experiments. To ensure wide-adoption by microscope-users with different needs Micro-Meta App closely interoperates withMethodsJ2andOMERO.mde, two complementary tools described in parallel manuscripts.

Published
2021

17. RNA labelling in live plants reveals single cell transcriptional dynamics: application to phosphate signaling

Author

Edouard Bertrand, Laura Cuyas, Marie-Christine Thibaud, Eric Maréchal, Nathalie Pochon, Sahar Hani, Florian Mueller, Laurent Nussaume, Hélène Javot, Rémy Merret, James Whelan, Pascale David, Orestis Faklaris, and David Secco

Subjects
chemistry.chemical_compound, medicine.anatomical_structure, Chemistry, Labelling, Dynamics (mechanics), Cell, medicine, RNA, Phosphate, Cell biology
Abstract

Plants are sessile organisms constantly adapting to ambient fluctuations through spatial and temporal transcriptional responses. Here, we implemented the latest generation RNA imaging system and combined it with microfluidics to visualize transcriptional regulation in living Arabidopsis plants. This enabled quantitative measurements of the transcriptional activity of single loci in single cells, real time and changing environmental conditions. Using phosphate responsive genes as model, we found that active genes displayed high transcription initiation rates (∼3s) and frequently clustered together in endoreplicated cells. We observed gene bursting and large allelic differences in single cells, revealing that at steady-state, intrinsic noise dominated extrinsic variations. Moreover, we established that transcriptional repression triggered in roots by phosphate, a crucial macronutrient limiting plant development, occurred with unexpected fast kinetics (∼minutes) and striking heterogeneity between neighboring cells. Access to single cell RNA polymerase II dynamics within live plants will benefit future studies of signaling processes.

Published
2021

18. QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy

Author

Glyn Nelson, Ulrike Boehm, Steve Bagley, Peter Bajcsy, Johanna Bischof, Brown, Claire M., Aurelien Dauphin, Dobbie, Ian M., Eriksson, John E., Orestis Faklaris, Julia Fernandez-Rodriguez, Alexia Ferrand, Laurent Gelman, Ali Gheisari, Hella Hartmann, Christian Kukat, Alex Laude, Miso Mitkovski, Sebastian Munck, North, Alison J., Rasse, Tobias M., Ute Resch-Genger, Schuetz, Lucas C., Arne Seitz, Caterina Strambio-De-Castillia, Swedlow, Jason R., Ioannis Alexopoulos, Karin Aumayr, Sergiy Avilov, Gert-Jan Bakker, Bammann, Rodrigo R., Andrea Bassi, Hannes Beckert, Sebastian Beer, Yury Belyaev, Jakob Bierwagen, Birngruber, Konstantin A., Manel Bosch, Juergen Breitlow, Cameron, Lisa A., Joe Chalfoun, Chambers, James J., Chieh-Li Chen, Eduardo Conde-Sousa, Corbett, Alexander D., Cordelieres, Fabrice P., Elaine del Nery, Ralf Dietzel, Frank Eismann, Elnaz Fazeli, Andreas Felscher, Hans Fried, Nathalie Gaudreault, Wah Ing Goh, Thomas Guilbert, Roland Hadleigh, Peter Hemmerich, Holst, Gerhard A., Itano, Michelle S., Jaffe, Claudia B., Jambor, Helena K., Jarvis, Stuart C., Antje Keppler, David Kirchenbuechler, Marcel Kirchner, Norio Kobayashi, Gabriel Krens, Susanne Kunis, Judith Lacoste, Marco Marcello, Martins, Gabriel G., Metcalf, Daniel J., Mitchell, Claire A., Joshua Moore, Tobias Mueller, Nelson, Michael S., Stephen Ogg, Shuichi Onami, Palmer, Alexandra L., Perrine Paul-Gilloteaux, Pimentel, Jaime A., Laure Plantard, Santosh Podder, Elton Rexhepaj, Michael Royeck, Arnaud Royon, Saari, Markku A., Damien Schapman, Vincent Schoonderwoert, Britta Schroth-Diez, Stanley Schwartz, Michael Shaw, Martin Spitaler, Stoeckl, Martin T., Damir Sudar, Jeremie Teillon, Stefan Terjung, Roland Thuenauer, Wilms, Christian D., Wright, Graham D., Roland Nitschke, Paul-Gilloteaux, Perrine, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, Structure fédérative de recherche François Bonamy (SFR François Bonamy), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Santé de l'Université de Nantes (IRS-UN), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), and ANR-10-INBS-04-01/10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010)

Subjects
minimum information, Technology, Histology, Physics - Instrumentation and Detectors, quality assessment, [SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, media_common.quotation_subject, Cancer development and immune defence Radboud Institute for Molecular Life Sciences [Radboudumc 2], 0206 medical engineering, confocal, light microscopy, metadata, quality assessment, quality control, reproducibility, widefield, Bioimage informatics, FOS: Physical sciences, 02 engineering and technology, MINIMUM INFORMATION, Imaging, Pathology and Forensic Medicine, 03 medical and health sciences, White paper, ddc:570, Quality (business), quality control, reproducibility, 030304 developmental biology, media_common, Microscopy, 0303 health sciences, Science & Technology, business.industry, metadata, Principal (computer security), resolution, Reproducibility of Results, Quality control, Instrumentation and Detectors (physics.ins-det), Reference Standards, widefield, Other Quantitative Biology (q-bio.OT), Data science, Quantitative Biology - Other Quantitative Biology, Metadata, Reference data, [SDV.IB.IMA] Life Sciences [q-bio]/Bioengineering/Imaging, confocal, RESOLUTION, FOS: Biological sciences, business, Quality assurance, 020602 bioinformatics, light microscopy
Abstract

In April 2020, the QUality Assessment and REProducibility for Instruments and Images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models, and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper 1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; 2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers, and observers of such; 3) outlines the current actions of the QUAREP-LiMi initiative, and 4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics., Comment: 17 pages, 3 figures, shortened abstract, Co-Lead Authors: Glyn Nelson and Ulrike Boehm, Corresponding author: Roland Nitschke

Published
2021
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19. The PAX-FOXO1s trigger fast trans-differentiation of chick embryonic neural cells into alveolar rhabdomyosarcoma with tissue invasive properties limited by S phase entry inhibition

Author

Muriel Rigolet, Pascale Gilardi-Hebenstreit, Frédéric Relaix, Gloria Gonzalez Curto, Orestis Faklaris, Frédéric Causeret, Aurélien de Reyniès, Daniil Korenkov, James Briscoe, Selene Prisco, Audrey Der Vartanian, Youcef El-Mokhtar Frarma, Vincent Contremoulins, Nabila Elarouci, Line Manceau, Vanessa Ribes, Lorenzo Baldi, Frédéric Auradé, Institut Jacques Monod (IJM (UMR_7592)), Université de Paris (UP)-Centre National de la Recherche Scientifique (CNRS), École nationale vétérinaire d'Alfort (ENVA), Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), (le programme) Cartes d'identité des tumeurs (CIT), Ligue Nationales Contre le Cancer (LNCC), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Institut de psychiatrie et neurosciences de Paris (IPNP - U1266 Inserm), Centre de Recherche en Myologie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), ImagoSeine core facility, Université de Paris (UP)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Centre National de la Recherche Scientifique (CNRS), The Francis Crick Institute [London], Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), École nationale vétérinaire - Alfort (ENVA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Centre de recherche en Myologie – U974 SU-INSERM, ImagoSeine, Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Martinez Rico, Clara, Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), and Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)

Subjects
Cancer Research, Oncogene Proteins, Fusion, Molecular biology, Biopsy, PAX3, Datasets as Topic, Gene Expression, Chick Embryo, QH426-470, Lung and Intrathoracic Tumors, S Phase, Cell Fusion, 0302 clinical medicine, Neural Stem Cells, Animal Cells, Medicine and Health Sciences, Paired Box Transcription Factors, Cyclin D1, Cell Cycle and Cell Division, Child, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Genetics (clinical), Neurons, Immunodetection, 0303 health sciences, N-Myc Proto-Oncogene Protein, PAX7 Transcription Factor, food and beverages, musculoskeletal system, 3. Good health, Chromatin, Cell biology, Gene Expression Regulation, Neoplastic, medicine.anatomical_structure, Cell Transformation, Neoplastic, Oncology, Cell Processes, 030220 oncology & carcinogenesis, embryonic structures, Alveolar rhabdomyosarcoma, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Cellular Types, Research Article, Neural Tube, Cell Physiology, endocrine system, Mesenchyme, Surgical and Invasive Medical Procedures, Biology, DNA construction, Research and Analysis Methods, 03 medical and health sciences, [SDV.BDD] Life Sciences [q-bio]/Development Biology, medicine, Genetics, Animals, Humans, Neoplasm Invasiveness, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Progenitor cell, Transcription factor, PAX3 Transcription Factor, Ecology, Evolution, Behavior and Systematics, Rhabdomyosarcoma, Alveolar, 030304 developmental biology, Gene Expression Profiling, Biology and Life Sciences, Cancers and Neoplasms, Cell Biology, medicine.disease, Embryonic stem cell, Disease Models, Animal, Molecular biology techniques, Cellular Neuroscience, Cell Transdifferentiation, Plasmid Construction, Immunologic Techniques, PAX7, Neuroscience
Abstract

The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS., Author summary The fusion-positive subtype of rhabdomyosarcoma (FP-RMS) is a rare malignant paediatric cancer, whose induction and evolution still remain to be deciphered. Out of the gross genetic aberrations found in these cancers, t(2:13) and t(1,13) chromosome translocations are the first to appear and lead to the expression of fusion proteins made of the DNA binding domains of either PAX3 or PAX7 and the transactivation domain of FOXO1. Both PAX3-FOXO1 and PAX7-FOXO1 have a strong impact on gene transcription, yet they only inefficiently promote the transformation of healthy cells into tumorigenic cells. To address this issue, we have used chick embryos to monitor in vivo the early response of cells to PAX-FOXO1 chimeric proteins. We showed that both proteins, but not the normal PAX3 and PAX7, transform neural cells into cells with FP-RMS molecular features. The PAX-FOXO1s also force polarized epithelial neural cells to adopt a mesenchymal phenotype with tissue invasive properties. However, the PAX-FOXO1s inhibit cell division and hence tumour growth. Genetically re-activating core cell cycle regulators rescues PAX-FOXO1 mediated cell cycle inhibition. Together, our findings bring further support to the idea that the PAX-FOXO1s are stricto sensu oncoproteins, whose oncogenicity is limited by negative effects on cell cycle.

Published
2020

20. Author Correction: Towards community-driven metadata standards for light microscopy: tiered specifications extending the OME model

Author

Mathias Hammer, Maximiliaan Huisman, Alessandro Rigano, Ulrike Boehm, James J. Chambers, Nathalie Gaudreault, Alison J. North, Jaime A. Pimentel, Damir Sudar, Peter Bajcsy, Claire M. Brown, Alexander D. Corbett, Orestis Faklaris, Judith Lacoste, Alex Laude, Glyn Nelson, Roland Nitschke, Farzin Farzam, Carlas S. Smith, David Grunwald, and Caterina Strambio-De-Castillia

Subjects
Cell Biology, Molecular Biology, Biochemistry, Biotechnology
Published
2022

21. Author Correction: QUAREP-LiMi: a community endeavor to advance quality assessment and reproducibility in light microscopy

Author

Ulrike Boehm, Glyn Nelson, Claire M. Brown, Steve Bagley, Peter Bajcsy, Johanna Bischof, Aurelien Dauphin, Ian M. Dobbie, John E. Eriksson, Orestis Faklaris, Julia Fernandez-Rodriguez, Alexia Ferrand, Laurent Gelman, Ali Gheisari, Hella Hartmann, Christian Kukat, Alex Laude, Miso Mitkovski, Sebastian Munck, Alison J. North, Tobias M. Rasse, Ute Resch-Genger, Lucas C. Schuetz, Arne Seitz, Caterina Strambio-De-Castillia, Jason R. Swedlow, and Roland Nitschke

Subjects
Cell Biology, Molecular Biology, Biochemistry, Biotechnology
Published
2022

22. The PAXFOXO1s trigger fast trans-differentiation of chick embryonic neural cells into alveolar rhabdomyosarcoma with tissue invasive properties limited by S phase entry inhibition

Author

Vanessa Ribes, Gloria Gonzalez Curto, Frédéric Relaix, Orestis Faklaris, Pascale Gilardi-Hebenstreit, Youcef El-Mokhtar Frarma, Line Manceau, Aurélien de Reyniès, Lorenzo Baldi, Frédéric Causeret, Nabila Elarouci, James Briscoe, Audrey Der Vartanian, Vincent Contremoulins, Selene Prisco, Frédéric Auradé, and Muriel Rigolet

Subjects
medicine.anatomical_structure, Mesenchyme, Alveolar rhabdomyosarcoma, medicine, PAX3, PAX7, Biology, Progenitor cell, medicine.disease, Embryonic stem cell, Malignant transformation, Cell biology, Chromatin
Abstract

The chromosome translocations generating PAX3FOXO1 and PAX7FOXO1 chimeric proteins are the primary hallmarks of the paediatric cancer, Alveolar Rhabdomyosarcoma (ARMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformationin vivo. The reason for this is unclear. To address this, we developed anin ovomodel to follow the response of spinal cord progenitors to PAXFOXO1s. Our data demonstrate that PAXFOXO1s, but not wild-type PAX3 and PAX7, trigger the trans-differentiation of neural cells into ARMS-like cells with myogenic characteristics. In parallel expression of PAXFOXO1s remodels the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, gain for PAXFOXO1s, as for wild-type PAX3/7, reduces the levels of CDK-CYCLIN activity and arrests cells in G1. Introduction of CYCLIN D1 or MYCN overcomes PAXFOXO1s mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism underpinning the apparent limited oncogenicity of PAXFOXO1 fusion transcription factors and support a neural origin for ARMS.

Published
2020

23. Additional file 2 of Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

Author

Botté, Alexandra, Lainé, Jeanne, Xicota, Laura, Heiligenstein, Xavier, Fontaine, Gaëlle, Kasri, Amal, Rivals, Isabelle, Pollyanna Goh, Orestis Faklaris, Jack-Christophe Cossec, Morel, Etienne, Anne-Sophie Rebillat, Nizetic, Dean, Raposo, Graça, and Potier, Marie-Claude

Abstract

Additional file 2: Supplementary Table. Demographic description of fibroblasts from euploid individuals and individuals with DS.

Published
2020
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24. Additional file 1 of Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

Author

Botté, Alexandra, Lainé, Jeanne, Xicota, Laura, Heiligenstein, Xavier, Fontaine, Gaëlle, Kasri, Amal, Rivals, Isabelle, Pollyanna Goh, Orestis Faklaris, Jack-Christophe Cossec, Morel, Etienne, Anne-Sophie Rebillat, Nizetic, Dean, Raposo, Graça, and Potier, Marie-Claude

Abstract

Additional file 1: Supplentary Figure S1. Representative image of a cluster of EEA1-DAB labelled endosomes in a LCL cell from an individual with DS. Electron micrographs along 8 serial 50 nm-thick sections. Note that sections 3 and 6 are missing. Individual endosomes are identified by differently colored dots, and most of them can be seen in 2 successive sections. The cluster contains at least 17 distinct endosomes and would appear as polycyclic if it was fluorescently labelled. Note that the DAB precipitate accumulating around surrounding small vesicles makes them appear as pseudo-coated.

Published
2020
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25. Author Correction: Micro-Meta App: an interactive tool for collecting microscopy metadata based on community specifications

Author

Alessandro Rigano, Shannon Ehmsen, Serkan Utku Öztürk, Joel Ryan, Alexander Balashov, Mathias Hammer, Koray Kirli, Ulrike Boehm, Claire M. Brown, Karl Bellve, James J. Chambers, Andrea Cosolo, Robert A. Coleman, Orestis Faklaris, Kevin E. Fogarty, Thomas Guilbert, Anna B. Hamacher, Michelle S. Itano, Daniel P. Keeley, Susanne Kunis, Judith Lacoste, Alex Laude, Willa Y. Ma, Marco Marcello, Paula Montero-Llopis, Glyn Nelson, Roland Nitschke, Jaime A. Pimentel, Stefanie Weidtkamp-Peters, Peter J. Park, Burak H. Alver, David Grunwald, and Caterina Strambio-De-Castillia

Subjects
Cell Biology, Molecular Biology, Biochemistry, Biotechnology
Published
2021

26. Zika virus enhances monocyte adhesion and transmigration favoring viral dissemination to neural cells

Author

Emma Partiot, Nilda Vanesa Ayala-Nunez, Jacky G. Goetz, Gautier Follain, Judith R.E. Roels, Anita Eckly, Béatrice Uring-Lambert, Maxime Chazal, Raphael Gaudin, Gillian Hale, Frank M. J. Jacobs, Sandrine Bourdoulous, Orestis Faklaris, Sherif R. Zaki, Aurélie Hirschler, Nolwenn Jouvenet, Margot Carocci, Florian Bakoa, Sarah Cianférani, Christine Carapito, Frédéric Doussau, Brigid C. Bollweg, François Delalande, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA), Institut National de la Santé et de la Recherche Médicale (INSERM), Immuno-Rhumatologie Moléculaire, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Strasbourg (UNISTRA), Fédération de Médecine Translationnelle de Strasbourg (FMTS), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centers for Disease Control and Prevention [Atlanta] (CDC), Centers for Disease Control and Prevention, Swammerdam Institute for Life Sciences, University of Amsterdam [Amsterdam] (UvA)-Center for Neurosciences, Génomique virale et vaccination, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Université Paris Descartes - Paris 5 (UPD5), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), BioCampus Montpellier (BCM), Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Biologie et pharmacologie des plaquettes sanguines: hémostase, thrombose, transfusion, Université de Strasbourg (UNISTRA)-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), Les Hôpitaux Universitaires de Strasbourg (HUS), Institut des Neurosciences Cellulaires et Intégratives (INCI), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), This work has received funding from the French State via the French National Research Agency (ANR) as part of the program Investissements d’avenir (IdEx Université de Strasbourg) to RG. This work was supported by an ATIP-AVENIR starting grant to RG. The research leading to these results has received funding from the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program (FP7/2007–2013) under REA grant agreement no. PCOFUND-GA-2013-609102, through the PRESTIGE program coordinated by Campus France, and from the French Agency for Research on AIDS and Viral Hepatitis (ANRS), both attributed to NVAN. Experiments conducted by G.F. and J.G.G. were funded by Plan Cancer (OptoMetaTrap), Ligue contre le Cancer, Idex Attractivités (University of Strasbourg), and by institutional funds from INSERM and University of Strasbourg. G.F. was supported by a doctoral fellowship from Ligue Contre le Cancer. Mass spectrometry experiments were supported by the French Proteomic Infrastructure (ProFI, ANR-10-INBS-08-03). F.B. is a recipient of a PhD grant provided by ANRT (Association Nationale de la Recherche et de la Technologie). N.J. is funded by Agence Nationale pour la Recherche (ANR-16-CE15-0025-01), Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur. CDC contributions to this paper written and edited by G.L.H. and B.C.B. in their private capacity. No official support or endorsement by the Centers for Disease Control and Prevention, Department of Health and Human Services is intended, nor should be inferred. All authors read, provided feedback, and approved the paper. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 NL4-3 AD8 Infectious Molecular Clone (pNL(AD8)) from Dr. Eric O. Freed (cat# 11346)., ANR-16-CE15-0025,Viro-Storm,Mécanismes de production incontrôlée de cytokines au cours de l'infection virale(2016), European Project: 609102,EC:FP7:PEOPLE,FP7-PEOPLE-2013-COFUND,PRESTIGE(2014), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), EFS-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), jouvenet, nolwenn, Mécanismes de production incontrôlée de cytokines au cours de l'infection virale - - Viro-Storm2016 - ANR-16-CE15-0025 - AAPG2016 - VALID, PRES Towards International Gain of Excellence - PRESTIGE - - EC:FP7:PEOPLE2014-09-01 - 2019-08-31 - 609102 - VALID, Equipe Direction scientifique, Sciences et Technologies de la Musique et du Son (STMS), Université Pierre et Marie Curie - Paris 6 (UPMC)-IRCAM-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-IRCAM-Centre National de la Recherche Scientifique (CNRS), École pratique des hautes études (EPHE), Neuro-Immunologie Virale, Virologie UMR1161 (VIRO), École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Laboratoire de Photonique Quantique et Moléculaire (LPQM), Centre National de la Recherche Scientifique (CNRS)-CentraleSupélec-École normale supérieure - Cachan (ENS Cachan), Biologie et pharmacologie des plaquettes sanguines: hémostase, thrombose, transfusion (http://www.u949.inserm.fr/), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-EFS, Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Spectrométrie de Masse BioOrganique, Département des Sciences Analytiques, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France (LSMBO-DSA-IPHC), Centre National de la Recherche Scientifique (CNRS), Immunorhumathologie moléculaire, Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), and Molecular Neuroscience (SILS, FNWI)

Subjects
Central Nervous System, 0301 basic medicine, MESH: Neurons, General Physics and Astronomy, MESH: Organoids, MESH: Monocytes, Monocytes, Zika virus, 0302 clinical medicine, MESH: Transendothelial and Transepithelial Migration, Cerebellum, MESH: Animals, lcsh:Science, MESH: Embryonic Stem Cells, ComputingMilieux_MISCELLANEOUS, Zebrafish, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Neurons, Multidisciplinary, Zika Virus Infection, Cell adhesion molecule, food and beverages, Sciences du Vivant [q-bio]/Microbiologie et Parasitologie, Phenotype, 3. Good health, Cell biology, Organoids, Mechanisms of disease, medicine.anatomical_structure, MESH: Cell Survival, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Female, Endothelium, Cell Survival, Science, MESH: Zika Virus, Biology, MESH: Endothelium, Article, General Biochemistry, Genetics and Molecular Biology, Virus, MESH: Cell Adhesion, 03 medical and health sciences, Virology, medicine, Animals, Humans, MESH: Central Nervous System, Cellular microbiology, Cell adhesion, MESH: Zebrafish, Embryonic Stem Cells, MESH: Humans, Monocyte, fungi, Transendothelial and Transepithelial Migration, Zika Virus, General Chemistry, biology.organism_classification, Embryonic stem cell, MESH: Cerebellum, Disease Models, Animal, 030104 developmental biology, lcsh:Q, MESH: Disease Models, Animal, Cell Adhesion Molecules, MESH: Female, 030217 neurology & neurosurgery
Abstract

Zika virus (ZIKV) invades and persists in the central nervous system (CNS), causing severe neurological diseases. However the virus journey, from the bloodstream to tissues through a mature endothelium, remains unclear. Here, we show that ZIKV-infected monocytes represent suitable carriers for viral dissemination to the CNS using human primary monocytes, cerebral organoids derived from embryonic stem cells, organotypic mouse cerebellar slices, a xenotypic human-zebrafish model, and human fetus brain samples. We find that ZIKV-exposed monocytes exhibit higher expression of adhesion molecules, and higher abilities to attach onto the vessel wall and transmigrate across endothelia. This phenotype is associated to enhanced monocyte-mediated ZIKV dissemination to neural cells. Together, our data show that ZIKV manipulates the monocyte adhesive properties and enhances monocyte transmigration and viral dissemination to neural cells. Monocyte transmigration may represent an important mechanism required for viral tissue invasion and persistence that could be specifically targeted for therapeutic intervention., Zika virus (ZIKV) can infect the central nervous system, but it is not clear how it reaches the brain. Here, Ayala-Nunez et al. show in ex vivo and in vivo models that ZIKV can hitch a ride in monocytes in a Trojan Horse manner to cross the endothelium and disseminate the virus.

Published
2019

27. Embryogenesis of the peristaltic reflex

Author

Cécile Jacques, Lucas Langlois, Chloé Guedj, Nicolas Chevalier, Orestis Faklaris, Nicolas Dacher, Matière et Systèmes Complexes (MSC (UMR_7057)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Institut Jacques Monod (IJM (UMR_7592))

Subjects
0301 basic medicine, Embryo, Nonmammalian, Physiology, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Myogenic contraction, Chick Embryo, Tetrodotoxin, Enteric Nervous System, 03 medical and health sciences, symbols.namesake, Mice, 0302 clinical medicine, Reflex, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Premovement neuronal activity, Animals, ComputingMilieux_MISCELLANEOUS, Migrating motor complex, Zebrafish, Peristalsis, Chemistry, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Smooth muscle contraction, Embryo, Mammalian, Interstitial cell of Cajal, Cell biology, Intestines, 030104 developmental biology, embryonic structures, symbols, Enteric nervous system, Nitric Oxide Synthase, Gastrointestinal Motility, 030217 neurology & neurosurgery, Neuroscience, Muscle Contraction
Abstract

Key points Neurogenic gut movements start after longitudinal smooth muscle differentiation in three species (mouse, zebrafish, chicken), and at E16 in the chicken embryo. The first activity of the chicken enteric nervous system is dominated by inhibitory neurons. The embryonic enteric nervous system electromechanically couples circular and longitudinal spontaneous myogenic contractions, thereby producing a new, rostro-caudally directed bolus transport pattern: the migrating motor complex. The response of the embryonic gut to mechanical stimulation evolves from a symmetric, myogenic response at E12, to a neurally mediated, polarized, descending inhibitory, 'law of the intestine'-like response at E16. High resolution, whole-mount 3D reconstructions are presented of the enteric nervous system of the chicken embryo at the neural-control stage E16 with the iDISCO+ tissue clarification technique. Abstract Gut motility is a complex transport phenomenon involving smooth muscle, enteric neurons, glia and interstitial cells of Cajal. Because these different cells differentiate and become active at different times during embryo development, studying the ontogenesis of motility offers a unique opportunity to 'time-reverse-engineer' the peristaltic reflex. Working on chicken embryo intestinal explants in vitro, we found by spatio-temporal mapping and signal processing of diameter and position changes that motility follows a characteristic sequence of increasing complexity: (1) myogenic circular smooth muscle contractions from E6 to E12 that propagate as waves along the intestine, (2) overlapping and independent, myogenic, low-frequency, bulk longitudinal smooth muscle contractions around E14, and (3) tetrodotoxin-sensitive coupling of longitudinal and circular contractions by the enteric nervous system as from E16. Inhibition of nitric oxide synthase neurons shows that the coupling consists in nitric oxide-mediated relaxation of circular smooth muscle when the longitudinal muscle layer is contracted. This mechanosensitive coupling gives rise to a directional, cyclical, propagating bolus transport pattern: the migrating motor complex. We further reveal a transition to a polarized, descending, inhibitory reflex response to mechanical stimulation after neuronal activity sets in at E16. This asymmetric response is the elementary mechanism responsible for peristaltic transport. We finally present unique high-resolution 3D reconstructions of the chicken enteric nervous system at the neural-control stage based on confocal imaging of iDISCO+ clarified tissues. Our study shows that the enteric nervous system gives rise to new peristaltic transport patterns during development by coupling spontaneous circular and longitudinal smooth muscle contraction waves.

Published
2019

28. Post-production modifications of murine mesenchymal stem cell (mMSC) derived extracellular vesicles (EVs) and impact on their cellular interaction

Author

Sarah Le Saux, Simon George, Orestis Faklaris, Patrick Bron, Hanna Aarrass, Jean Armengaud, Justine Bertrand-Michel, Jean-Marie Devoisselle, Emeric Dubois, Philippe Legrand, Joséphine Lai-Kee-Him, Marie Morille, Guylaine Miotello, Joël Chopineau, Institut Charles Gerhardt Montpellier - Institut de Chimie Moléculaire et des Matériaux de Montpellier (ICGM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biochimie des Systèmes Perturbés (LBSP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Innovations technologiques pour la Détection et le Diagnostic (LI2D), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), MetaToul Lipidomics, Institut des Maladies Métaboliques et Cardiovasculaires (I2MC), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-MetaboHUB-MetaToul, MetaboHUB-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université de Toulouse (UT)-Université de Toulouse (UT)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-MetaboHUB-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Génomique Fonctionnelle - Montpellier GenomiX (IGF MGX), Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-BioCampus (BCM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), BioCampus (BCM), Université de Nîmes (UNIMES), Institut Charles Gerhardt Montpellier - Institut de Chimie Moléculaire et des Matériaux de Montpellier (ICGM ICMMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut de Chimie du CNRS (INC), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateau MetaToul-LIPIDOMIQUE = MetaToul-Lipidomics, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-MetaToul-MetaboHUB, Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-MetaboHUB-MetaToul, Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Ecole Nationale Vétérinaire de Toulouse (ENVT), CCSD, Accord Elsevier, Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-MetaToul-MetaboHUB, and Centre National de la Recherche Scientifique (CNRS)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 1 (UM1)-Centre National de la Recherche Scientifique (CNRS)-Université Montpellier 2 - Sciences et Techniques (UM2)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 1 (UM1)

Subjects
[SDV]Life Sciences [q-bio], Endocytic cycle, Biophysics, Bioengineering, Endocytosis Pathway, 02 engineering and technology, Biomaterials, Extracellular Vesicles, Mice, 03 medical and health sciences, Drug Delivery Systems, Caveolae, Animals, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Liposome, Chemistry, Vesicle, Mesenchymal stem cell, Mesenchymal Stem Cells, 021001 nanoscience & nanotechnology, Microvesicles, Cell biology, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV] Life Sciences [q-bio], Freeze Drying, Mechanics of Materials, Liposomes, Drug delivery, Ceramics and Composites, 0210 nano-technology
Abstract

International audience; In regards to their key role in intercellular communication, extracellular vesicles (EVs) have a strong potential as bio-inspired drug delivery systems (DDS). With the aim of circumventing some of their well-known issues (production yield, drug loading yield, pharmacokinetics), we specifically focused on switching the biological vision of these entities to a more physico-chemical one, and to consider and fine-tune EVs as synthetic vectors. To allow a rational use, we first performed a full physico-chemical (size, concentration, surface charge, cryoTEM), biochemical (western blot, proteomics, lipidomics, transcriptomics) and biological (cell internalisation) characterisation of murine mesenchymal stem cell (mMSC)-derived EVs. A stability study based on evaluating the colloidal behaviour of obtained vesicles was performed in order to identify optimal storage conditions. We evidenced the interest of using EVs instead of liposomes, in regards to target cell internalisation efficiency. EVs were shown to be internalised through a caveolae and cholesterol-dependent pathway, following a different endocytic route than liposomes. Then, we characterised the effect of physical methods scarcely investigated with EVs (extrusion through 50 nm membranes, freeze-drying, sonication) on EV size, concentration, structure and cell internalisation properties. Our extensive characterisation of the effect of these physical processes highlights their promise as loading methods to make EVs efficient delivery vehicles.

Published
2020

29. MEIOB and SPATA22 resemble RPA subunits and interact with the RPA complex to promote meiotic recombination

Author

Xavier Veaute, Gabriel Livera, Carole Saintomé, Orestis Faklaris, Duquenne C, Martini E, Devanshi Jain, Didier Busso, Ribeiro J, Scott Keeney, Raphael Guerois, Pauline Dupaigne, and Petrillo C

Subjects
0303 health sciences, biology, DNA repair, Chemistry, Protein subunit, 030302 biochemistry & molecular biology, RAD51, Helicase, complex mixtures, Cell biology, Chromosome segregation, enzymes and coenzymes (carbohydrates), 03 medical and health sciences, Meiosis, biology.protein, DMC1, Homologous recombination, 030304 developmental biology
Abstract

Homologous recombination is a conserved DNA repair process mandatory for chromosome segregation during meiosis. RPA, a ubiquitous complex essential to recombination, is thought to play a similar role during mitotic and meiotic recombination. MEIOB, a meiosis-specific factor with unknown molecular function, ressembles a RPA subunit. Here we usein vivoapproaches to show that in mouse spermatocytes, DMC1 and RAD51 appear to be normally loaded in the absence of MEIOB but are prematurely lost from unrepaired recombination sites. This loss correlates with an accumulation of the BLM helicase on meiotic chromosomes. We also show that MEIOB alters the immunodetection of RPA subunits at meiotic recombination sites. Using electron microscopy and purified proteins, we demonstrate that the MEIOB-SPATA22 complex associates with and modifies the conformation of RPA-coated ssDNA. Finally, we identify structural homology between MEIOB, SPATA22 and RPA subunits, and show that MEIOB and SPATA22 interact through C-terminal OB-fold containing domains (OBCDs) like RPA subunits. Moreover, MEIOB and SPATA22 cooperate to interact with RPA through their OBCDs. Our results suggest that MEIOB, SPATA22 and RPA work together to ensure proper processing of meiotic recombination intermediates.

Published
2018

30. Ultrabright and Fluorogenic Probes for Multicolor Imaging and Tracking of Lipid Droplets in Cells and Tissues

Author

Tkhe Kyong Fam, Thierry Galli, Andrey S. Klymchenko, Pichandi Ashokkumar, Lydia Danglot, Mayeul Collot, Orestis Faklaris, Laboratoire de Bioimagerie et Pathologies (LBP), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Biophotonique et Pharmacologie (CNRS UMR 7213), Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biophotonique et Pharmacologie - UMR 7213 (LBP), Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, and Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))

Subjects
Models, Molecular, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Indoles, multicolor imaging, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, KB Cells, chemistry.chemical_compound, Mice, Colloid and Surface Chemistry, Lipid droplet, Microscopy, Organelle, Moiety, Animals, Humans, Merocyanine, Benzopyrans, Fluorescent Dyes, tissue imaging, communication, Chemistry, Optical Imaging, Nile red, dioxaborine, General Chemistry, Lipid Droplets, 021001 nanoscience & nanotechnology, Fluorescence, Two-Photon Excitation imaging, 0104 chemical sciences, Mice, Inbred C57BL, Adipose Tissue, Liver, Microscopy, Fluorescence, Biophysics, 0210 nano-technology, Intracellular
Abstract

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders, and diseases including obesity, diabetes, and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenine moiety and a dioxaborine barbiturate derivative. These so-called StatoMerocyanines (SMCy) fluoresce from yellow to the near-infrared (NIR) in oil with an impressive fluorescence enhancement compared to aqueous media. Additionally, SMCy display remarkably high molar extinction coefficients (up to 390 000 M–1 cm–1) and high quantum yield values (up to 100%). All the members of this new family specifically stain the LDs in live cells with very low background noise. Unlike Nile Red, a well-known lipid droplet marker, SMCy dyes possess narrow absorption and emission bands in the visible, thus allowing multicolor imaging. SMCy proved to be compatible with fixation and le...

Published
2018

31. Mapping molecular assemblies with fluorescence microscopy and object-based spatial statistics

Author

Stephane Dallongeville, Jean-Christophe Olivo-Marin, Alexandre Grassart, Alexandre Dufour, Nathalie Sauvonnet, Orestis Faklaris, Lydia Danglot, Thibault Lagache, Analyse d'images biologiques - Biological Image Analysis (BIA), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Pathogénie microbienne moléculaire, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre de Psychiatrie et Neurosciences (U894), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), We acknowledge the ImagoSeine core facility of the Institut Jacques Monod, member of IBiSA and the France-BioImaging infrastructure, supported by ANR grant (ANR-10-INBS-04). We thank the NeurImag imaging facility of the Centre for Psychiatry and Neuroscience, the labex ‘Who am I’ and the national research group in Microscopy of the living (GDR ImaBio). This work was financially supported by the Institut Pasteur, the CNRS, the INSERM, ANR grant ANR-10-LABX-62-IBEID and a grant from the Institut pour la Recherche sur la Moelle épinière et l’Encéphale. T.L. was funded by a Bourse Roux from Institut Pasteur. A.G. is funded by a Bourse Roux from Institut Pasteur. Finally, we warmly thank Fabrice de Chaumont and Anna Raffaela Damato for their critical reading of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., We deeply thank Dr. Carl G. Ebeling (Worldwide Application Scientist for Bruker Fluorescence Microscopy), and Jeff Stuckey (Product Marketing Manager, Bruker) for their help and the use of the Bruker Vutara system and its SRX visualisation and analysis software for rendering localisation data, ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-11-LABX-0071,WHO AM I,Determinants de l'Identité : de la molécule à l'individu(2011), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de psychiatrie et neurosciences (U894 / UMS 1266), Analyse d'images biologiques - BioImage Analysis (AIQ), ANR-10-INBS-04-01/10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR: 11-LABX-0071,WHO AM I,Determinants de l'Identité : de la molécule à l'individu(2011), and ANR-10-LABX-62-IBEID,IBEID,Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases'(2010)

Subjects
0301 basic medicine, Physics, Multidisciplinary, Spatial organisation, Science, Object based, General Physics and Astronomy, General Chemistry, Synapsin, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Synaptic vesicle, Article, General Biochemistry, Genetics and Molecular Biology, Synapse, 03 medical and health sciences, 030104 developmental biology, [INFO.INFO-TI]Computer Science [cs]/Image Processing [eess.IV], Microscopy, Fluorescence microscope, lcsh:Q, lcsh:Science, Biological system, Spatial analysis
Abstract

Elucidating protein functions and molecular organisation requires to localise precisely single or aggregated molecules and analyse their spatial distributions. We develop a statistical method SODA (Statistical Object Distance Analysis) that uses either micro- or nanoscopy to significantly improve on standard co-localisation techniques. Our method considers cellular geometry and densities of molecules to provide statistical maps of isolated and associated (coupled) molecules. We use SODA with three-colour structured-illumination microscopy (SIM) images of hippocampal neurons, and statistically characterise spatial organisation of thousands of synapses. We show that presynaptic synapsin is arranged in asymmetric triangle with the 2 postsynaptic markers homer and PSD95, indicating a deeper localisation of homer. We then determine stoichiometry and distance between localisations of two synaptic vesicle proteins with 3D-STORM. These findings give insights into the protein organisation at the synapse, and prove the efficiency of SODA to quantitatively assess the geometry of molecular assemblies., Elucidating molecular organisation requires precise localisation and analysis. Here the authors develop SODA software for automatic and quantitative mapping of statistically coupled molecules, and use it to unravel spatial organisation of thousands of synaptic proteins in SIM and 3DSTORM microscopy.

Published
2018

32. Nuclear pore complex plasticity during developmental process as revealed by super-resolution microscopy

Author

Julien Sellés, May Penrad-Mobayed, Cyndélia Guillaume, Alica Fuger, Loïc Auvray, Orestis Faklaris, and Fabien Montel

Subjects
Nuclear Pore Complex Proteins, Microscopy, Stochastic Processes, Xenopus laevis, lcsh:R, Nuclear Pore, Oocytes, lcsh:Medicine, Animals, lcsh:Q, lcsh:Science, Article
Abstract

Nuclear Pore Complex (NPC) is of paramount importance for cellular processes since it is the unique gateway for molecular exchange through the nucleus. Unraveling the modifications of the NPC structure in response to physiological cues, also called nuclear pore plasticity, is key to the understanding of the selectivity of this molecular machinery. As a step towards this goal, we use the optical super-resolution microscopy method called direct Stochastic Optical Reconstruction Microscopy (dSTORM), to analyze oocyte development impact on the internal structure and large-scale organization of the NPC. Staining of the FG-Nups proteins and the gp210 proteins allowed us to pinpoint a decrease of the global diameter by measuring the mean diameter of the central channel and the luminal ring of the NPC via autocorrelation image processing. Moreover, by using an angular and radial density function we show that development of the Xenopus laevis oocyte is correlated with a progressive decrease of the density of NPC and an ordering on a square lattice.

Published
2017

33. Original Fluorescent Ligand-Based Assays Open New Perspectives in G-Protein Coupled Receptor Drug Screening

Author

Orestis Faklaris, Jurriaan M. Zwier, Thierry Durroux, Martin Cottet, Eric Trinquet, and Jean-Philippe Pin

Subjects
Drug, media_common.quotation_subject, lcsh:Medicine, lcsh:RS1-441, Pharmaceutical Science, Review, Computational biology, Biology, Bioinformatics, 01 natural sciences, lcsh:Pharmacy and materia medica, 03 medical and health sciences, GPCR, Drug Discovery, lanthanides, 030304 developmental biology, media_common, G protein-coupled receptor, 0303 health sciences, lcsh:R, Binding properties, time resolved FRET, Screening assay, Ligand (biochemistry), Fluorescence, 0104 chemical sciences, 010404 medicinal & biomolecular chemistry, Molecular Medicine, fluorescence, Heterologous expression, screening assay
Abstract

The identification of new drugs exhibiting reduced adverse side-effects constitutes a great challenge for the next decade. Various steps are needed to screen for good ligand candidates and one of them is the evaluation of their binding properties. New strategies based on fluorescence measurement constitute excellent alternatives to the traditional radioactive assays. Less hazardous, faster and cheaper, these methods also exhibit very good sensitivity and can be used on various biological models such as heterologous expression systems or native tissues.

Published
2011

34. Photoluminescent nanodiamonds: Comparison of the photoluminescence saturation properties of the NV color center and a cyanine dye at the single emitter level, and study of the color center concentration under different preparation conditions

Author

Jacques Botsoa, François Treussart, Thierry Sauvage, Jean-François Roch, and Orestis Faklaris

Subjects
Materials science, Photoluminescence, Synthetic diamond, Mechanical Engineering, Nanoparticle, Diamond, Nanotechnology, General Chemistry, engineering.material, Fluorescence, Electronic, Optical and Magnetic Materials, law.invention, chemistry.chemical_compound, chemistry, Nanocrystal, law, Materials Chemistry, engineering, Electrical and Electronic Engineering, Cyanine, Nanodiamond
Abstract

Nitrogen-Vacancy (NV) color centers in diamond nanoparticles (NanoDiamonds, NDs) have a great number of applications, in particular in quantum information processing and as cellular fluorescent labels. In this work we compare the photoluminescence properties of a single NV color center embedded in a 30 nm diameter nanodiamond to that of a single organic fluorescent molecule. We also study the photoluminescence properties of NDs prepared under different conditions in order to find the optimal parameters to achieve a high fluorescence yield, which is still an open question in the case of nanocrystals compared to bulk diamond. The results of this study are of interest for the applications of small and bright diamond nanoparticles in nanophotonics and biology.

Published
2010

35. Oxidative stress-induced assembly of PML nuclear bodies controls sumoylation of partner proteins

Author

Umut Sahin, Valérie Lallemand-Breitenbach, Florence Jollivet, Michiko Niwa-Kawakita, Caroline Berthier, Marion Jeanne, Omar Ferhi, Niclas Setterblad, Shirine Benhenda, Orestis Faklaris, Pathologie cellulaire : aspects moléculaires et viraux / Pathologie et Virologie Moléculaire, Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), INSERM, CNRS, Université Paris-Diderot, Institut Universitaire de France, Ligue Contre le Cancer, Institut National du Cancer, Association pour la Recherche contre le Cancer (GriffuelAwardto H. de Thé), Canceropôle Ile de France, the European Research Council (STEMAPL advanced grant to H. de Thé), PACRI, Saint Louis Institute, French National Research Agency (ANR), Fondation pour la Recherche Medicale, INSERM U944 Laboratoire de Pathologie et Virologie Moléculaire, Laboratoire de Pathologie et Virologie Moléculaire, Laboratoire de Photonique Quantique et Moléculaire (LPQM), École normale supérieure - Cachan (ENS Cachan)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS), Plate-forme d'imagerie, Hopital Saint-Louis [AP-HP] (AP-HP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Université Paris Diderot - Paris 7 (UPD7), Service de Biochimie [AP-HP Hôpital Saint-Louis, Paris], Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Centre National de la Recherche Scientifique (CNRS), and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Protein sumoylation, viruses, Ubiquitin-Protein Ligases, SUMO protein, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], [SDV.BC]Life Sciences [q-bio]/Cellular Biology, CHO Cells, Ubiquitin-conjugating enzyme, Promyelocytic Leukemia Protein, Article, Promyelocytic leukemia protein, Mice, Cricetulus, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], hemic and lymphatic diseases, Cricetinae, Chlorocebus aethiops, Animals, Humans, Nuclear protein, ComputingMilieux_MISCELLANEOUS, Research Articles, Cellular Senescence, Cell Nucleus, biology, RNF4, Tumor Suppressor Proteins, Nuclear Proteins, Sumoylation, Cell Biology, 3. Good health, Cell biology, Transport protein, Oxidative Stress, Protein Transport, Biochemistry, COS Cells, Ubiquitin-Conjugating Enzymes, biology.protein, Small Ubiquitin-Related Modifier Proteins, Reactive Oxygen Species, Cell aging, HeLa Cells, Transcription Factors
Abstract

PML multimerization into nuclear bodies following its oxidation promotes sumoylation and sequestration of partner proteins in these structures., The promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are stress-responsive domains where many partner proteins accumulate. Here, we clarify the basis for NB formation and identify stress-induced partner sumoylation as the primary NB function. NB nucleation does not rely primarily on intermolecular interactions between the PML SUMO-interacting motif (SIM) and SUMO, but instead results from oxidation-mediated PML multimerization. Oxidized PML spherical meshes recruit UBC9, which enhances PML sumoylation, allow partner recruitment through SIM interactions, and ultimately enhance partner sumoylation. Intermolecular SUMO–SIM interactions then enforce partner sequestration within the NB inner core. Accordingly, oxidative stress enhances NB formation and global sumoylation in vivo. Some NB-associated sumoylated partners also become polyubiquitinated by RNF4, precipitating their proteasomal degradation. As several partners are protein-modifying enzymes, NBs could act as sensors that facilitate and confer oxidative stress sensitivity not only to sumoylation but also to other post-translational modifications, thereby explaining alterations of stress response upon PML or NB loss.

Published
2014
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36. Superresolution imaging of albumin-conjugated fluorescent nanodiamonds in cells by stimulated emission depletion

Author

Huan-Cheng Chang, Yung Kuo, Jui-Hung Hsu, Be-Ming Chang, Yan-Kai Tzeng, and Orestis Faklaris

Subjects
Microscopy, Confocal, Chemistry, Albumin, STED microscopy, Nanoparticle, Nanotechnology, Serum Albumin, Bovine, General Medicine, General Chemistry, Conjugated system, Endocytosis, Fluorescence, Catalysis, Nanodiamonds, Microscopy, Fluorescence, Lactalbumin, Animals, Humans, Cattle, Stimulated emission, Nanodiamond, Fluorescent Dyes, HeLa Cells
Published
2010

37. Comparison of the photoluminescence properties of semiconductor quantum dots and non-blinking diamond nanoparticles. Observation of the diffusion of diamond nanoparticles in living cells

Author

Jean-Paul Boudou, Damien Garrot, Patrick A. Curmi, Thierry Sauvage, Orestis Faklaris, Fran c cois Treussart, Vandana Joshi, Laboratoire de Photonique Quantique et Moléculaire (LPQM), Centre National de la Recherche Scientifique (CNRS)-CentraleSupélec-École normale supérieure - Cachan (ENS Cachan), Structure et activité des biomolécules normales et pathologiques (SABNP), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de mécanique des sols, structures et matériaux (MSSMat), Centre National de la Recherche Scientifique (CNRS)-CentraleSupélec, Conditions Extrêmes et Matériaux : Haute Température et Irradiation (CEMHTI), Centre National de la Recherche Scientifique (CNRS)-Université d'Orléans (UO), Financement européen: 'Nano4Drugs' (contrat LSHB-2005-CT-019102), Financement région 'Ile de France'(C'Nano IdF): projet 'Biodiam'., NaDia (contrat ANR-2007-PNANO-045),NaDia (contrat ANR-2007-PNANO-045), École normale supérieure - Cachan (ENS Cachan)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS), Université d'Orléans (UO)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), ANR-07-NANO-0045,NADIA,Nanocristaux de diamant, photoluminescence et croissance de couches ultraminces de diamant(2007), CentraleSupélec-Centre National de la Recherche Scientifique (CNRS), Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS), Treussart, François, and Nanosciences et nanotechnologies - Nanocristaux de diamant, photoluminescence et croissance de couches ultraminces de diamant - - NADIA2007 - ANR-07-NANO-0045 - NANO - VALID

Subjects
[PHYS.PHYS.PHYS-OPTICS] Physics [physics]/Physics [physics]/Optics [physics.optics], Photoluminescence, Materials science, Fluorophore, FOS: Physical sciences, Nanoparticle, quantum dots, 02 engineering and technology, 010402 general chemistry, photostability, 01 natural sciences, chemistry.chemical_compound, [PHYS.QPHY]Physics [physics]/Quantum Physics [quant-ph], Molecule, [PHYS.QPHY] Physics [physics]/Quantum Physics [quant-ph], Condensed Matter - Materials Science, Quantum Physics, [PHYS.PHYS.PHYS-OPTICS]Physics [physics]/Physics [physics]/Optics [physics.optics], business.industry, Materials Science (cond-mat.mtrl-sci), 021001 nanoscience & nanotechnology, Photobleaching, [PHYS.COND.CM-MS] Physics [physics]/Condensed Matter [cond-mat]/Materials Science [cond-mat.mtrl-sci], Atomic and Molecular Physics, and Optics, 0104 chemical sciences, chemistry, Quantum dot, nanodiamonds, [PHYS.COND.CM-MS]Physics [physics]/Condensed Matter [cond-mat]/Materials Science [cond-mat.mtrl-sci], Optoelectronics, Particle, biomarker, photoluminescence, Quantum Physics (quant-ph), 0210 nano-technology, business, Excitation, Optics (physics.optics), Physics - Optics
Abstract

International audience; Long-term observations of photoluminescence at the single-molecule level were until recently very difficult, due to the photobleachingof organic fluorophore molecules. Although inorganic semiconductor nanocrystals can overcome this difficulty showing very low photobleachingyield, they suffer from photoblinking. A new marker has been recently introduced, relying on diamond nanoparticles containingphotoluminescent color centers. In this work we compare the photoluminescence of single quantum dots (QDs) to the one of nanodiamondscontaining a single-color center. Contrary to other markers, photoluminescent nanodiamonds present a perfect photostability andno photoblinking. At saturation of their excitation, nanodiamonds photoluminescence intensity with a single NV-color center is only threetimes smaller than one of the single QDs. Moreover, the bright and stable photoluminescence of nanodiamonds allows wide field observationsof single nanoparticles motion. We demonstrate the possibility of recording the trajectory of such single particle in culture cells.

Published
2009

38. Near-field optical microscopy with a nanodiamond-based single photon tip

Author

Orestis Faklaris, A. Drezet, François Treussart, Yannick Sonnefraud, Aurélien Cuche, Serge Huant, and Jean-François Roch

Subjects
Photon, Materials science, Transducers, Physics::Optics, FOS: Physical sciences, Near and far field, Microscopy, Scanning Probe, Sensitivity and Specificity, law.invention, Condensed Matter::Materials Science, Optical microscope, law, Microscopy, Particle Size, Nanodiamond, Quantum, Plasmon, business.industry, Reproducibility of Results, Equipment Design, Image Enhancement, Atomic and Molecular Physics, and Optics, Nanostructures, Equipment Failure Analysis, Nanocrystal, Computer-Aided Design, Optoelectronics, Diamond, business, Physics - Optics, Optics (physics.optics)
Abstract

We introduce a point-like scanning single-photon source that operates at room temperature and offers an exceptional photostability (no blinking, no bleaching). This is obtained by grafting in a controlled way a diamond nanocrystal (size around 20 nm) with single nitrogen-vacancy color-center occupancy at the apex of an optical probe. As an application, we image metallic nanostructures in the near-field, thereby achieving a near-field scanning single-photon microscopy working at room temperature on the long term. Our work may be of importance to various emerging fields of nanoscience where an accurate positioning of a quantum emitter is required such as for example quantum plasmonics., Comment: submitted to Optics Express

Published
2009
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39. Diamond nanoparticles as photoluminescent nanoprobes for biology and near-field optics

Author

François Treussart, Damien Garrot, Orestis Faklaris, Aurélien Cuche, Jean-François Roch, Thierry Sauvage, Yannick Sonnefraud, Jean-Paul Boudou, Serge Huant, Nano-Optique et Forces (NOF), Institut Néel (NEEL), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Photonique Quantique et Moléculaire (LPQM), École normale supérieure - Cachan (ENS Cachan)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS), Biogéochimie et écologie des milieux continentaux (Bioemco), Centre National de la Recherche Scientifique (CNRS)-AgroParisTech-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Conditions Extrêmes et Matériaux : Haute Température et Irradiation (CEMHTI), Université d'Orléans (UO)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Nano-Optique et Forces (NEEL - NOF), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Recherche pour le Développement (IRD)-Institut National de la Recherche Agronomique (INRA)-Université Pierre et Marie Curie - Paris 6 (UPMC)-AgroParisTech-Centre National de la Recherche Scientifique (CNRS), NOF - Nano-Optique et Forces, Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Centre National de la Recherche Scientifique (CNRS)-CentraleSupélec-École normale supérieure - Cachan (ENS Cachan), Centre National de la Recherche Scientifique (CNRS)-Université d'Orléans (UO), and Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Materials science, Photoluminescence, Biophysics, Physics::Optics, Nanoparticle, FOS: Physical sciences, Nanotechnology, 02 engineering and technology, 01 natural sciences, Biochemistry, law.invention, 010309 optics, Optical microscope, law, fluorescent probes, 0103 physical sciences, Condensed Matter - Materials Science, Near-field optics, Materials Science (cond-mat.mtrl-sci), nitrogen-vacancy (NV) colour centres, General Chemistry, 021001 nanoscience & nanotechnology, Condensed Matter Physics, Atomic and Molecular Physics, and Optics, near-field scanning optical microscopy, 3. Good health, PACS: 78.67.Bf, 07.79.Fc, 33.50.- j, nanodiamonds, [PHYS.COND.CM-MS]Physics [physics]/Condensed Matter [cond-mat]/Materials Science [cond-mat.mtrl-sci], Near-field scanning optical microscope, Diamond nanoparticles, 0210 nano-technology, active tips
Abstract

We present results showing the potential of diamond nanoparticles with size less than 50 nm as photoluminescent nanoprobes for serving as stable point-like emitters attached at the tip apex of a near-field optical microscope to achieve enhanced spatial resolution., Comment: Keynote talk by F. Treussart at the 15th Int. Conf. on Luminescence and Optical Spectroscopy of Condensed Matter (ICL 08), Lyon, July 2008. Proceedings to appear in J. Luminescence

Published
2009

40. Photoluminescent diamond nanoparticles for cell labeling: study of the uptake mechanism in mammalian cells

Author

Alain Thorel, Theano Irinopoulou, Hugues A. Girard, Jean-Paul Boudou, Céline Gesset, Patrick A. Curmi, François Treussart, Patrick Tauc, Mohamed Senour, Jean-Charles Arnault, Orestis Faklaris, Vandana Joshi, Faklaris, Orestis, Nanosciences et nanotechnologies - Nanocristaux de diamant, photoluminescence et croissance de couches ultraminces de diamant - - NADIA2007 - ANR-07-NANO-0045 - NANO - VALID, Laboratoire de Photonique Quantique et Moléculaire (LPQM), École normale supérieure - Cachan (ENS Cachan)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS), Structure et activité des biomolécules normales et pathologiques (SABNP), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut du Fer à Moulin, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Intégration des Systèmes et des Technologies (LIST (CEA)), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre des Matériaux (CDM), Mines Paris - PSL (École nationale supérieure des mines de Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), 'Nano4Drugs' (contract LSHB-2005-CT-019102), ANR-07-NANO-0045,NADIA,Nanocristaux de diamant, photoluminescence et croissance de couches ultraminces de diamant(2007), Centre National de la Recherche Scientifique (CNRS)-CentraleSupélec-École normale supérieure - Cachan (ENS Cachan), Laboratoire d'Intégration des Systèmes et des Technologies (LIST), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Centre des Matériaux (MAT), Centre National de la Recherche Scientifique (CNRS)-PSL Research University (PSL)-MINES ParisTech - École nationale supérieure des mines de Paris, 'NaDia' (contract ANR-2007-PNANO-045),'NaDia' (contract ANR-2007-PNANO-045), and MINES ParisTech - École nationale supérieure des mines de Paris

Subjects
[PHYS.PHYS.PHYS-OPTICS] Physics [physics]/Physics [physics]/Optics [physics.optics], Macromolecular Substances, Surface Properties, Molecular Conformation, FOS: Physical sciences, General Physics and Astronomy, Nanoparticle, 02 engineering and technology, 010402 general chemistry, Endocytosis, 01 natural sciences, Clathrin, diamond, Materials Testing, Humans, Nanotechnology, endocytosis, General Materials Science, Particle Size, chemistry.chemical_classification, [PHYS.PHYS.PHYS-OPTICS]Physics [physics]/Physics [physics]/Optics [physics.optics], Staining and Labeling, biology, Vesicle, Biomolecule, General Engineering, Colocalization, biolabel, 021001 nanoscience & nanotechnology, Nanostructures, 0104 chemical sciences, Endocytic vesicle, chemistry, Transmission electron microscopy, Luminescent Measurements, biology.protein, Biophysics, nanoparticles, photoluminescence, Crystallization, 0210 nano-technology, Optics (physics.optics), Physics - Optics, HeLa Cells
Abstract

International audience; Diamond nanoparticles (nanodiamonds) have been recently proposed as new labels for cellular imaging. For small nanodiamonds (size

Published
2009

41. Detection of single photoluminescent diamond nanoparticles in cell and study of the internalization pathway

Author

Orestis Faklaris, Vandana Joshi, Patrick Georges, François Treussart, Jean-Paul Boudou, Frédéric Druon, Patrick A. Curmi, Thierry Sauvage, Damien Garrot, Laboratoire de Photonique Quantique et Moléculaire (LPQM), École normale supérieure - Cachan (ENS Cachan)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS), Structure et activité des biomolécules normales et pathologiques (SABNP), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire Charles Fabry de l'Institut d'Optique / Elsa, Laboratoire Charles Fabry de l'Institut d'Optique (LCFIO), Université Paris-Sud - Paris 11 (UP11)-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), Biogéochimie et écologie des milieux continentaux (Bioemco), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Recherche pour le Développement (IRD)-Institut National de la Recherche Agronomique (INRA)-Université Pierre et Marie Curie - Paris 6 (UPMC)-AgroParisTech-Centre National de la Recherche Scientifique (CNRS), Conditions Extrêmes et Matériaux : Haute Température et Irradiation (CEMHTI), Université d'Orléans (UO)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), ANR-07-NANO-0045,NADIA,Nanocristaux de diamant, photoluminescence et croissance de couches ultraminces de diamant(2007), European Project, Centre National de la Recherche Scientifique (CNRS)-Institut d'Optique Graduate School (IOGS)-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Institut d'Optique Graduate School (IOGS)-Université Paris-Sud - Paris 11 (UP11), Centre National de la Recherche Scientifique (CNRS)-AgroParisTech-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Treussart, François, Nanosciences et nanotechnologies - Nanocristaux de diamant, photoluminescence et croissance de couches ultraminces de diamant - - NADIA2007 - ANR-07-NANO-0045 - NANO - VALID, Nano4Drugs : contrat LSHB-2005-CT-019102 - INCOMING, Centre National de la Recherche Scientifique (CNRS)-CentraleSupélec-École normale supérieure - Cachan (ENS Cachan), Centre National de la Recherche Scientifique (CNRS)-Université d'Orléans (UO), NaDia : contrat ANR-2007-PNANO-045,NaDia : contrat ANR-2007-PNANO-045, Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Recherche Agronomique (INRA)-Université Pierre et Marie Curie - Paris 6 (UPMC)-AgroParisTech-Centre National de la Recherche Scientifique (CNRS)

Subjects
[PHYS.PHYS.PHYS-OPTICS] Physics [physics]/Physics [physics]/Optics [physics.optics], Luminescence, Materials science, Photochemistry, Endosome, media_common.quotation_subject, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Fluorescent Antibody Technique, Nanoparticle, Nanotechnology, Endosomes, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, law.invention, Biomaterials, diamond, Confocal microscopy, law, Humans, General Materials Science, Internalization, ComputingMilieux_MISCELLANEOUS, media_common, [PHYS.PHYS.PHYS-OPTICS]Physics [physics]/Physics [physics]/Optics [physics.optics], [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Colocalization, General Chemistry, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Autofluorescence, Drug delivery, cytotoxicity, biomarker, nanoparticles, photoluminescence, 0210 nano-technology, Intracellular, HeLa Cells, Biotechnology
Abstract

International audience; Diamond nanoparticles are promising photoluminescent probes for tracking intracellular processes, due to embedded perfectly photostable color centers. In this work, we investigate the spontaneous internalization of such nanoparticles (diameter 25 nm) in HeLa cells by confocal microscopy and time-resolved techniques. Nanoparticles are observed inside the cell cytoplasm at the single particle and single color center level, assessed by time correlation intensity measurements. Improvement of the nanoparticles signal-to-noise ratio inside cell is achieved using a pulsed excitation laser and a time resolved detection taking advantage of the long radiative lifetime of the color center excited state, compared to cell auto-fluorescence. The internalization pathways are also investigated, with endosomal marking and colocalization analyses. The low colocalization ratio observed proves that nanodiamonds are not trapped in endosomes, a promising result in prospect of drug delivery by these nanoparticles. We also prove a low cytotoxicity of these nanoparticles in this cell line. Submitted to-2

Published
2008

42. Diamond nanoparticles as photoluminescent nanoprobes

Author

Thierry Sauvage, Orestis Faklaris, Jean-Paul Boudou, Vandana Joshi, Patrick A. Curmi, François Treussart, Serge Huant, Aurélien Cuche, Jean-François Roch, Yannick Sonnefraud, Laboratoire de Photonique Quantique et Moléculaire (LPQM), Centre National de la Recherche Scientifique (CNRS)-CentraleSupélec-École normale supérieure - Cachan (ENS Cachan), NOF - Nano-Optique et Forces, Institut Néel (NEEL), Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Centre d'Études et de Recherches par Irradiation (CERI), Centre National de la Recherche Scientifique (CNRS), Structure et activité des biomolécules normales et pathologiques (SABNP), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biogéochimie et écologie des milieux continentaux (Bioemco), Centre National de la Recherche Scientifique (CNRS)-AgroParisTech-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Paris (ENS Paris), École normale supérieure - Cachan (ENS Cachan)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS), Nano-Optique et Forces (NOF), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Recherche Agronomique (INRA)-Université Pierre et Marie Curie - Paris 6 (UPMC)-AgroParisTech-Centre National de la Recherche Scientifique (CNRS), Nano-Optique et Forces (NEEL - NOF), École normale supérieure - Paris (ENS-PSL), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Recherche pour le Développement (IRD)-Institut National de la Recherche Agronomique (INRA)-Université Pierre et Marie Curie - Paris 6 (UPMC)-AgroParisTech-Centre National de la Recherche Scientifique (CNRS)

Subjects
Photoluminescence, General Physics and Astronomy, markers, Nanotechnology, 02 engineering and technology, Living cell, engineering.material, 01 natural sciences, law.invention, color centers, Optical microscope, diamond, law, near-field optics, 0103 physical sciences, 010302 applied physics, Physics, chemistry.chemical_classification, Biomolecule, Near-field optics, Diamond, 021001 nanoscience & nanotechnology, chemistry, [PHYS.COND.CM-GEN]Physics [physics]/Condensed Matter [cond-mat]/Other [cond-mat.other], engineering, Diamond nanoparticles, 0210 nano-technology
Abstract

Proceedings of COLOQ'10 - 10ème COlloque sur les Lasers et l'Optique Quantique, Grenoble, July 2007; We present preliminary results showing the potential of diamond nanoparticles with size

Published
2007

43. Quenching nitrogen–vacancy center photoluminescence with an infrared pulsed laser

Author

V. Jacques, Ngoc Diep Lai, Dingwei Zheng, François Treussart, Huan-Cheng Chang, Orestis Faklaris, Jean-François Roch, Laboratoire de Photonique Quantique et Moléculaire (LPQM), and École normale supérieure - Cachan (ENS Cachan)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)

Subjects
Materials science, Photoluminescence, Infrared, FOS: Physical sciences, General Physics and Astronomy, 02 engineering and technology, engineering.material, 01 natural sciences, [PHYS.QPHY]Physics [physics]/Quantum Physics [quant-ph], 0103 physical sciences, 010306 general physics, Absorption (electromagnetic radiation), ComputingMilieux_MISCELLANEOUS, Quenching, [PHYS.PHYS.PHYS-OPTICS]Physics [physics]/Physics [physics]/Optics [physics.optics], business.industry, Diamond, 021001 nanoscience & nanotechnology, Nanocrystal, Picosecond, engineering, Optoelectronics, 0210 nano-technology, business, Nitrogen-vacancy center, Optics (physics.optics), Physics - Optics
Abstract

Diamond nanocrystals containing Nitrogen-Vacancy (NV) color centers have been used in recent years as fluorescent probes for near-field and cellular imaging. In this work we report that an infrared (IR) pulsed excitation beam can quench the photoluminescence of NV color center in a diamond nanocrystal (size < 50 nm) with an extinction ratio as high as ~90%. We attribute this effect to the heating of the nanocrystal consecutive to multi-photon absorption by the diamond matrix. This quenching is reversible: the photoluminescence intensity goes back to its original value when the IR laser beam is turned off, with a typical response time of hundred picoseconds, allowing for a fast control of NV color center photoluminescence. We used this effect to achieve sub-diffraction limited imaging of fluorescent diamond nanocrystals on a coverglass. For that, as in Ground State Depletion super-resolution technique, we combined the green excitation laser beam with the control IR depleting one after shaping its intensity profile in a doughnut form, so that the emission comes only from the sub-wavelength size central part., 5 figures, 15 pages

Published
2013

44. Diamond nanocrystals hosting single nitrogen-vacancy color centers sorted by photon-correlation near-field microscopy

Author

Yannick Sonnefraud, Thierry Sauvage, François Treussart, Jean-François Roch, Orestis Faklaris, Serge Huant, Aurélien Cuche, and Jean-Paul Boudou

Subjects
Optics and Photonics, Time Factors, Materials science, Photoluminescence, Nitrogen, Annealing (metallurgy), Molecular Probe Techniques, 02 engineering and technology, engineering.material, 01 natural sciences, 010309 optics, Materials Testing, 0103 physical sciences, Microscopy, Nanotechnology, Particle Size, Nanodiamond, Photons, business.industry, Resolution (electron density), Diamond, Equipment Design, 021001 nanoscience & nanotechnology, Atomic and Molecular Physics, and Optics, Nanostructures, Nanocrystal, engineering, Nanoparticles, Optoelectronics, Near-field scanning optical microscope, Crystallization, 0210 nano-technology, business
Abstract

Diamond nanocrystals containing highly photoluminescent color centers are attractive, nonclassical, and near-field light sources. For near-field applications, the size of the nanocrystal is crucial, since it defines the optical resolution. Nitrogen-vacancy (NV) color centers are efficiently created by proton irradiation and annealing of a nanodiamond powder. Using near-field microscopy and photon statistics measurements, we show that nanodiamonds with sizes down to 25 nm can hold a single NV color center with bright and stable photoluminescence.

Published
2008

45. Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

Author

Cécile Leduc, Audrey Salles, Sandrine Etienne-Manneville, Spencer L. Shorte, lp2n-04,lp2n-12, Laboratoire Photonique, Numérique et Nanosciences (LP2N), Université Sciences et Technologies - Bordeaux 1-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS)-Université Sciences et Technologies - Bordeaux 1-Institut d'Optique Graduate School (IOGS)-Centre National de la Recherche Scientifique (CNRS), Technologie et Service BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], Imagerie Dynamique (Plate-Forme) (PFID), Institut Pasteur [Paris], Polarité cellulaire, Migration et Cancer / Cell Polarity, Migration and Cancer, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Polarité cellulaire, Migration et Cancer - Cell Polarity, Migration and Cancer, BioImagerie Photonique – Photonic BioImaging (UTechS PBI), We gratefully acknowledge the UtechS Photonic BioImaging (Imagopole) Citech of Institut Pasteur (Paris, France) as well as the France-BioImaging infrastructure network supported by the French National Research Agency (ANR-10-INSB-04, Investments for the Future), and the Région Ile-de-France (program Domaine d'Intérêt Majeur-Malinf) for the use of the Elyra microscope. This work was supported by the the Ligue Contre le Cancer and the French National Research Agency (ANR-16-CE13-0019), We thank Mickael Lelek, Orestis Faklaris and Nicolas Bourg for fruitful discussion, Andrey Aristov and Elena Rensen for help with the super- resolution technique and Shailaja Seetharaman for careful reading of the manuscript., ANR-16-CE13-0019,SiFi2Net,Filaments intermédiaires: du filament unique au réseau(2016), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)

Subjects
0301 basic medicine, General Chemical Engineering, [SDV]Life Sciences [q-bio], STORM, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Microtubules, General Biochemistry, Genetics and Molecular Biology, Issue 133, 03 medical and health sciences, 0302 clinical medicine, vimentin, Microtubule, super-resolution microscopy, S Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct, Etienne-Manneville, Fluorescence microscope, Molecular motor, Animals, Intermediate filaments, Intermediate filament, Cytoskeleton, Biology, Actin, General Immunology and Microbiology, Super-resolution microscopy, General Neuroscience, Salles, SL, cytoskeleton, 030104 developmental biology, Microscopy, Fluorescence, Cytoplasm, Shorte, 030220 oncology & carcinogenesis, cytoskeleton Citation: Leduc, Biophysics, microtubule
Abstract

International audience; The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

Published
2018

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