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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