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Quantitative Imaging of the Cell

Team Leader : Jean-Baptiste Sibarita

General objective

General objectives

We develop cutting-edge quantitative imaging methods to decipher protein organization and dynamics with high spatial and temporal resolution and compatible with high-content screening standards. Over the last 10 years, we have successfully developed and combined single-molecule based nanoscopy, dedicated image analysis methods and bioengineering techniques to tackle important biological questions. With no relevant biological question to address within the team itself, our developments are applied in very close collaboration with biology research groups.

Our team has an important academic and industrial technology transfer activity. We develop software and microscopy solutions which we valorize through scientific publications, patents, industrial technology transfers, academic Material Transfer Agreements, and free/collaborative/open source distribution.

Team organization

The Quantitative imaging of the cell team is a R&D team composed of people coming from various disciplines: microscopy, computer science and bioengineering.

Project leaders

Jean-Baptiste Sibarita

Florian Levet

Rémi Galland

 

Research Projects

3D high- and super-resolution imaging at different scales

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Quantitative analysis of super-resolution data

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Single-molecule-based High Content Screening

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Correlative platform for SMLM & STED nanoscopy

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High-content screening imaging of living organoids

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Combining deep-learning with geometric and image processing for analysis of microscopy data

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PalmTracer

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Expertise

  • Single-Molecule Localization Microscopy
  • Light-Sheet Fluorescence Microscopy
  • High Content Screening
  • Bioimage Analysis
  • Geometry Processing
  • Deep Learning
  • News

    Multi-dimensional spectral Single Molecule Localization Microscopy

    Single molecule localization (SML) and tracking (SPT) techniques, such as (spt)PALM, (u/DNA)PAINT and quantum dot tracking, have given unprecedented insight into the nanoscale molecular organization and dynamics in living cells. They allow monitoring individual proteins with millisecond temporal resolution and high spatial resolution (<30 nm) by precisely localizing the point spread function (PSF) of individual emitters and tracking their position over time. While SPT methods have been extended to study the temporal dynamics and co-organization of multiple proteins, conventional experimental setups are restricted in the number of proteins they can probe simultaneously and usually have to tradeoff between the number of colors, the spatio-temporal resolution, and the field of view. Yet, localizing and tracking several proteins simultaneously at high spatial and temporal resolution within large field of views can provide important biological insights. By employing a dual-objective spectral imaging configuration compatible with live cell imaging combined with dedicated computation tools, we demonstrate simultaneous 3D single particle localization and tracking of multiple distinct species over large field of views to be feasible without compromising spatio-temporal resolution. The dispersive element introduced into the second optical path induces a spectrally dependent displacement, which we used to analytically separate up to five different fluorescent species of single emitters based on their emission spectra. We used commercially available microscope bodies aligned one on top of the other, offering biologists with a very ergonomic and flexible instrument covering a broad range of SMLM applications. Finally, we developed a powerful freely available software, called PALMTracer, which allows to quantitatively assess 3D + t + λ SMLM data. We illustrate the capacity of our approach by performing multi-color 3D DNA-PAINT of fixed samples, and demonstrate simultaneous tracking of multiple receptors in live fibroblast and neuron cultures.

    Multi-dimensional spectral Single Molecule Localization Microscopy
    Authors: Corey Butler, G. Ezequiel Saraceno, Adel Kechkar, Vincent Studer, Julien P. Dupuis, Laurent Groc, Rémi Galland, Jean-Baptiste Sibarita

    Front. Bioinform., 04 March 2022 | https://doi.org/10.3389/fbinf.2022.813494

    Contacts: Rémi Galland and Jean-Baptiste Sibarita

    + Cf. Bordeaux Neurocampus website here

    Automated high-speed 3D imaging of organoid cultures - Nature Methods, June 2022

    Current imaging approaches limit the ability to perform multi-scale characterization of three-dimensional (3D) organotypic cultures (organoids) in large numbers. Here, we present an automated multi-scale 3D imaging platform synergizing high-density organoid cultures with rapid and live 3D single-objective light-sheet imaging. It is composed of disposable microfabricated organoid culture chips, termed JeWells, with embedded optical components and a laser beam-steering unit coupled to a commercial inverted microscope. It permits streamlining organoid culture and high-content 3D imaging on a single user-friendly instrument with minimal manipulations and a throughput of 300 organoids per hour. We demonstrate that the large number of 3D stacks that can be collected via our platform allows training deep learning-based algorithms to quantify morphogenetic organizations of organoids at multi-scales, ranging from the subcellular scale to the whole organoid level. We validated the versatility and robustness of our approach on intestine, hepatic, neuroectoderm organoids and oncospheres.

    Authors: Anne Beghin, Gianluca Grenci, Geetika Sahni, Su Guo, Harini Rajendiran, Tom Delaire, Saburnisha Binte Mohamad Raffi, Damien Blanc, Richard de Mets, Hui Ting Ong, Xareni Galindo, Anais Monet, Vidhyalakshmi Acharya, Victor Racine, Florian Levet, Remi Galland, Jean-Baptiste Sibarita and Virgile Viasnoff

    Cf. Nature Methods - June 2022 here

    Contact: Jean-Baptiste Sibarita

    Selected Publications

  • F. Levet, J. Tonnesen, U.V. Nagerl, J.B. Sibarita
  • SpineJ: A software tool for quantitative analysis of nanoscale spine morphology Methods (2020)
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  • S. Kedia, P. Ramakrishna, P.R. Netrakanti, M. Jose, J.B. Sibarita, S. Nadkarni, D. Nair
  • Real-time nanoscale organization of amyloid precursor protein Nanoscale (2020)
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  • D. Jullie, M. Stoeber, J.B. Sibarita, H.L. Zieger, T.M. Bartol, S. Arttamangkul, T.J. Sejnowski, E. Hosy, M. von Zastrow
  • A Discrete Presynaptic Vesicle Cycle for Neuromodulator Receptors Neuron (2020)
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  • J. Goncalves, T.M. Bartol, C. Camus, F. Levet, A.P. Menegolla, T.J. Sejnowski, J.B. Sibarita, M. Vivaudou, D. Choquet, E. Hosy
  • Nanoscale co-organization and coactivation of AMPAR, NMDAR, and mGluR at excitatory synapses PNAS (Proceedings of the National Academy of Sciences) (2020)
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  • J.S. Ferreira, J.P. Dupuis, B. Kellermayer, N. Benac, C. Manso, D. Bouchet, F. Levet, C. Butler, J.B. Sibarita, L. Groc
  • Distance-dependent regulation of NMDAR nanoscale organization along hippocampal neuron dendrites PNAS (Proceedings of the National Academy of Sciences) (2020)
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  • E. Dondi, J.B. Sibarita, N. Varin-Blank, L. Velazquez
  • The adaptor protein APS modulates BCR signalling in mature B cells Cellular Signaling (2020)
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  • L. Aoun, A. Farutin, N. Garcia-Seyda, P. Negre, M.S. Rizvi, S. Tlili, S. Song, X. Luo, M. Biarnes-Pelicot, R. Galland, J.B. Sibarita, A. Michelot, C. Hivroz, S. Rafai, M.P. Valignat, C. Misbah, O. Theodoly
  • Amoeboid Swimming Is Propelled by Molecular Paddling in Lymphocytes Biophysical Journal (2020)
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  • D. Sage, T.A. Pham, H. Babcock, T. Lukes, T. Pengo, J. Chao, R. Velmurugan, A. Herbert, A. Agrawal, S. Colabrese, A. Wheeler, A. Archetti, B. Rieger, R. Ober, G.M. Hagen, J.B. Sibarita, J. Ries, R. Henriques, M. Unser, S. Holden
  • Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software Nature Methods (2019)
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  • A. Mehidi, O. Rossier, M. Schaks, A. Chazeau, F. Biname, A. Remorino, M. Coppey, Z. Karatas, J.B. Sibarita, K. Rottner, V. Moreau, G. Giannone
  • Transient Activations of Rac1 at the Lamellipodium Tip Trigger Membrane Protrusion Current Biology (2019)
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  • F. Levet, G. Julien, R. Galland, C. Butler, A. Beghin, A. Chazeau, P. Hoess, J. Ries, G. Giannone, J.B. Sibarita
  • A tessellation-based colocalization analysis approach for single-molecule localization microscopy Nature Communications (2019)
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  • V. Inavalli, M.O. Lenz, C. Butler, J. Angibaud, B. Compans, F. Levet, J. Tonnesen, O. Rossier, G. Giannone, O. Thoumine, E. Hosy, D. Choquet, J.B. Sibarita*, U.V. Nagerl*
  • A super-resolution platform for correlative live single-molecule imaging and STED microscopy Nature Methods (2019)
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  • M. Letellier, F. Levet, O. Thoumine, Y. Goda
  • Differential role of pre- and postsynaptic neurons in the activity-dependent control of synaptic strengths across dendrites PLoS Biology (2019)
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    Members

    « Researcher »

    GALLAND Remi Researcher remi.galland@u-bordeaux.fr +33533514748

    « Technical Staff »

    BARRY Thierno Technical staff thierno.barry.1@u-bordeaux.fr +33533514700
    LEVET Florian Technical staff florian.levet@inserm.fr +33533514747
    NEUHAUS Abdelghani Technical staff abdelghani.neuhaus@u-bordeaux.fr +33533514700
    SIBARITA Jean-Baptiste Technical staff jean-baptiste.sibarita@u-bordeaux.fr +33533514706

    « PhD student »

    DELAIRE Tom PhD student tom.delaire@u-bordeaux.fr +33533514700
    GALINDO Xareni PhD student xareni.galindo@u-bordeaux.fr +33533514700
    LOHK Christer PhD student christer.lohk@u-bordeaux.fr +33533514700

    « Student »

    BETTAREL Laetitia Student laetitia.bettarel@etu.u-bordeaux.fr +33533514700
    PEIRO Rémy Student remy.peiro@u-bordeaux.fr +33533514700