Transcriptomic heterogeneity of cells in organisms over time and space necessitates state-of-the-art technologies to access this variability and its dynamics in situ. Hence, the Spatial-Cell-ID facility aims to enhance the spatial resolution of MERFISH for pinpointing transcripts of specific genes (ranging from a few to thousands) in situ, achieving cellular and subcellular precision over time. To achieve this, we employ a microfluidics device enabling multiple rounds of hybridization (smFISH) synchronized with an advanced 3D STED microscope, providing precise spatial localization of RNA spots with a resolution of 50 nm. This technology is tailored for whole-mount samples across diverse organisms, spanning from mammalian cells to invertebrate systems such as Drosophila and C. elegans. Supported by EquipEx+ funding, Spatial-Cell-ID will be nationally accessible through the LyMIC core facility. Our facility complements commercial systems focused on imaging larger samples at lower 2D resolution. Our bespoke solution not only surpasses current state-of-the-art capabilities but also maintains its position at the forefront of technology, with potential future integration of genomic and spatial proteomic techniques.

Publications

Bouchet M., Urdy S., Guan W., Kabir C., Garvis S., Enriquez J. A simple smiFISH pipeline to quantify mRNA at the single-cell level in 3D. (2023). STAR Protocols. Volume 4, Issue 2, 2023, 102316.

The development occurring in the platform MEC is tighly associated with joint effort with the “platform of nanocaractérisation” (PFNC) from CEA, leading to have a unique expertise in 3D electron microscopy of large tissue volumes reaching tens of thousands of cubic micrometers at the nanometer scale. This is essential to analyze rare events and true 3D morphological details in neurobiology to study finely cell connectivity, the characterization of membrane structures in chloroplasts from algae and plants, or to study nanoparticles toxicity. Indeed, EM stacks are acquired with much less efforts than by serial sectioning and in a relatively short time (preparation of the sample 3 days, acquisition of the stack 1 day due to high quality stabilization of the samples) new biological questions of interest to the FBI community can be addressed. 

Moreover, we have developed a unique pipe-line of analysis of large stacks of 3D EM images. This pipe-line is composed of segmentation steps followed by Ilastik approach for recognition of objects of interests coupled with a serie of home-made plugin specific for quantification. As requested by the network of French electronic microscopists, we are actively collaborating to make available our process to make this pipe-line available for others colleagues and especially the FBI users, as requested by the network of French electronic microscopists. Our work allowed us to develop international collaboration with Andrea Volterra, a world-class leader in the field of FIB-SEM in Neurobiology.

Team 4 of the Inserm research unit 1245 aims to characterize the contribution of neurovascular dysfunction in the pathophysiology of neonatal brain lesions while keeping in mind brain immaturity. Thereby, research projects paid attention to molecular, cellular and integrated processes leading to angiogenesis defects and neurodevelopmental consequences such as vessel-associated migration. The team’s research activity is deeply committed into translational research validated by patents and clinical protocols with the objective to develop diagnosis tools and neuroprotection strategies. To reach these objectives, the research team developed mouse perinatal models of white matter injury, in utero gain and loss of functions, and environmental models of fetal toxicity such as FASD. Endothelial and neuro-vascular dysfunctions are apprehended in the developing brains and retinas by imaging approaches (light sheet, FLIM-STED, time-lapse migration, in situ zymography, OCT) on ex vivo tissues (brain organotypic slices, cultured retinas). Image processing and analysis through notably machine learning is developed in partnership with several members of the Normandy FBI node.

The “tPA and neurovascular disorders” team within the UMR-S U1237 – INSERM UNICAEN and GIS Blood and Brain @ Caen-Normandie Institute” (BB@C – INSERM – UNICAEN- CHU CAEN) is focused on a better understanding of the physiopathology of thrombosis/ischemic neurovascular disorders by developing and applying advanced methods (from molecular to multimodal cell and in vivo imaging). Mainly dedicated to live-cell imaging, we will provide access to dynamic imaging, super-resolution microscopy (STED). We developed innovative neuroimaging tools such as biocompatible nanoparticles, multimodal probes to detect vesicles trafficking, autophagy, neuronal activity, inflammation in the field of vascular disorders. Additionally, we used multiphoton imaging to observe in vivo vascular events including inflammation, diapedesis of immune cells within the parenchyma as well as microthrombosis. Functional ultrasound localization microscopy enables us to assess brain-wide neurovascular activity at a microscopic scale.

The platform is attached to ITI Neurostra. It is located at the Institut des Neurosciences Cellulaires et Intégrées (INCI) and managed administratively by the CNRS UAR 3156 headed by P. Darbon.
The expertise of the in vitro imaging platform applies to the study of biological materials, tissues and isolated cells on a structural and ultrastructural scale. It extends to the immunocytochemical visualisation of molecules and the phenotypic detection of gene expression.
The platform is involved in a wide variety of interdisciplinary projects (biology, chemistry, agronomy, medicine). Around two-thirds of users come from the CNRS INCI UPR3212 (6 groups), with the remainder coming from other Strasbourg institutes (IBMC, IBMP, ISIS, IPHC, ECPM, CRBS, etc.).
A number of developments have taken place in recent years, including cryofixation (Wohlwend system) and super-resolution microscopy (Stellaris 8 confocal microscope with a STED module).

Microscopy systems available @Plateforme Imagerie In Vitro INCI

PIC-STRA aims to support the 10 CRBS research units, as well as external teams from both the academic and private sectors. Opened in October 2020, this 350 m2 imaging platform provides users with around ten imaging systems (stereomicroscopes, wide-field and confocal single- and multiphoton microscopes with super-resolution module) for multi-scale observation, from whole small animals to sub-cellular details. It provides various solutions for the observation of fixed and living samples (videomicroscopy) and is equipped for image processing and analysis (IMARIS, Fiji/ImageJ, ICY, iLastik). The platform is part of the local RISEst network (Réseau d’Imagerie Strasbourg grand Est), is in the process of obtaining CORTECS and IBISA (STrasbourg Centre) accreditation and works closely with the other platforms in the network.

Microscopy systems available @PIC-STRA

InMagiC (INMED iMAGing Center) is the microscopy platform of the Institute of Neurobiology of the Mediterranean: INMED in Marseille on the Luminy campus.
The platform is under the scientific direction of Rosa COSSART and under the responsibility of the engineer François MICHEL rosa.cossart@inserm.fr / francois.michel@inserm.fr

INMAGIC gathers in one place the multi-scale study tools necessary to study the development and the physio-pathological functioning of the central nervous system.
These tools cover the scales of observation from the whole animal (binocular loupes) to the dynamic morphology of neuronal contacts (synapses) thanks to widefield, confocal and multiphoton microscopes.
In addition, the platform provides a large number of training courses at all levels, both theoretical and practical, for all audiences (students, engineers, researchers, etc.).

The strong and original points of our structure are:

  • A space dedicated to the techniques of tissue clearing and the acquisition of these samples by an ultramicroscope (light sheet) in platform access, as well as the expertise of the engineer in charge (active member of the GT transparencies of the RTMFM);
  • The use of the multiphoton microscopy for the fast measurement (10-20 Hz) of the neuronal activity in vitro and in vivo on large populations of cells (up to more than 500 simultaneous cells).

Two high performance analysis stations are also available to all users. They include free and commercial software dedicated to analysis such as FIJI, ICY, IMARIS, Neurolucida360, Matlab…

Finally, the platform is a laureate of the PIA Equipex + 2020 and in this context the availability of tools beyond the state of the art in minimally invasive in vivo imaging will soon be open to the community.
These new tools will be commercial with industrial co-developments: a 3-photon excitation microscope, an ultra-fast AOD scanning microscope, a 2-photon endoscope or in academic development with the collaboration of the Fresnel Institute such as an ultra-thin endoscope and a deep recording station by photo-acoustics.

NeurImag is a service and R&D Cell and Tissue imaging core facility of the Institute of Psychiatry and Neuroscience of Paris (IPNP, INSERM U1266 – Université de Paris) since October 2017.

It is divided in :

  • Imaging services using photonic microscopy,
  • Image analysis service providing « home made analysis solutions » through programming using either Icy, iLastik, ImageJ and/or MatLab.
  • Primary cell culture services that provide neuronal cells, organoïds and tissue to solve biological questions through live and super-resolution imaging.

To groundwork our scientific operation, the daily management is divided into three interconnected services (Sample preparation, Imagingand  Analysis) under the single management of a scientific director, Lydia Danglot (Inserm, “Chargée de Recherche Hors Classe”, Ph.D. in Neurosciences). Engineers performing technological operations are Sylvain Jeannin for Advanced Imaging, Philippe Bun for Data Analysis service,  Laurianne Beynac for in vivo functional imaging and David Boulet for the Sample preparation and Primary Cell Culture service.

NeurImag manages 9 light microscopy systems for (A) the observation of biological macromolecular structures (full field) with very high spatial (3 confocal, 1 Airyscan confocal,  1 super-resolution SIM and 1 3D-STED and STED-FCS) and temporal (spinning-disk, 2 photons imaging, Light-sheet imaging) resolution or high molecular localization precision (2 super-resolution microscope for PALM, SPT and 3DSTORM) correlated respectively with high speed Airyscan or swept-field confocal (SFC), (B) monitoring macromolecular dynamics and interaction within cells and/or living organism with minimal phototoxicity (full field, spinning-disk and fast Airyscan confocal) and (C) spectroscopy approaches for monitoring intracellular and molecular dynamics using light manipulation (spinning-disk with laser ablation and FRAP addons) and fluorescent lifetime decay measurements (fast-FLIM confocal) respectively. NeurImag also provides 3 workstations directly connected to microscope computers for image processing and data analysis using free- and commercial software such as ICY, ImageJ, Huygens, iLastik, Imaris, Neurolucida, Volocity, Zen Blue (Zeiss), SRX (Bruker) and MatLab.
The NeurImag cell and tissue culture services offers a unique service for biological sample preparation ranging from cells (primary culture of hippocampal/cortical neurons or glial cells, organoïdes, cells lines and tissues) with the optimization of immunofluorescence labeling or transfecting protocols to the management of human biological samples from the Pathology department of GHU-Paris/Sainte-Anne Medical Center, located on the same campus. The service is equipped with 6 biosafety level2-culture rooms including 15 Class II A2 biosafety cabinet, 18 CO2 incubators, 6 bench top centrifuge, 5 light microscope and one fluorescent microscope for a quick cell culture/transfection check. As an example, internal and external teams can buy weekly, plates of neurons seeded on glass coverslips to directly proceed for imaging or transfection. Light-sheet microscope is in L2 environment and can welcome human and IPS samples.
Since Neuroscience is a major research topic at IPNP, the NeurImag Imaging Facility addresses imaging techniques related to issues faced in this field. Imaging techniques currently available cover a wide range of spatiotemporal resolution, ranging from video-microscopy, super-resolution imaging (PALM, STORM, STED 3D and SIM), sample preparation and quantitative image analysis. If you wish to discuss about the potential and decisive contribution of imaging techniques to your research project, do not hesitate to contact us.

List of system and services available

  • Deconvolution widefield microscopy
  • Videomicroscopy and slide scanner system
  • Laser scanning confocal microscopy (LSCM) + Gated detectors + White Light Laser
  • Spinning disc confocal system +Fast 2 colors GEMINI module
    Swept-field confocal system
  • Total internal reflection fluorescence microscopy (TIRF)
  • Fluorescence Correlation Spectroscopy (FCS)
  • Fluorescence Cross-Correlation Spectroscopy (FCCS)
  • Fluorescence lifetime Imaging microscopy (FLIM)
  • Fluorescence resonance energy transfer (FRET)
  • Fluorescence recovery after photobleaching (FRAP)
  • 2 photon in vivo imaging
  • Light sheet microscopy for live (organoids, Zebrafish) or fixed samples
  • Laser ablation and Photon uncaging
  • Structured Illumination Microscopy (SIM)
  • Photoactivated localization microscopy (PALM)
  • STimulated Emission Depletion microscopy (STED) +/- Fluorescence Correlation Spectroscopy module
  • Stochastical Optical Reconstruction Microscopy (STORM) with 3D biplane module
  • Primary cell culture preparation + optimization on request/demand
  • Sample preparation (transfection, Immunostaining, 3D cultures)
  • Image Data Processing and Analysis +custom made solution
  • Workstations for data analysis (commercial softwares)

The imaging and Cytometry facility is part of Généthon, which is a pharmaceutical organization specialized in understanding rare diseases and designing new approaches of therapy, mostly gene transfer. The imaging and Cytometry facility offers services in imaging and cytometry, acquisition and analysis. The facility organizes its activity around 3 main missions: services, training, Research and Development.
The facility interacts with different partners:

  • inner departments of Genethon (5 R&D teams, vector production unit, technical development unit ; preclinical evaluation department)
  • Genethon belongs itself to a public interest group named Genopole, which gathers Start-up, companies, facilities, institutes and public laboratories, and promote their activity and research. Consequently, the facility has strong interaction with Genopole partners.

The facility is open to any user outside Genopole, and is currently working with the CEA, the institute of myology, the “Institut de Recherche Biomédial des armées (IRBA)” etc.

Scope of activity: The facility concentrates on quantitative imaging of muscles and neuromuscular disease.

Microscopy systems available @ImCy