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.

Over the last ten years, the joint effort of the platform PLATIM and the laboratory of Plant Development and Reproduction (RDP) has developed a strong and recognized expertise in the imaging and quantification of cell mechanical properties in living plant issues. Specific pipelines have been developed to map and quantify:

1. Wall mechanical properties through direct measurements of key parameters such as wall stiffness, adhesion, and resistance to piercing, using AFMs and nano-indenters 

2. Cell hydrostatic pressure through stiffness measurements made using AFMs or nano-indenters and the subsequent application of physical models but also through the development of a nano-pressure probe (in progress).

In addition of the originality of these technological approaches in mecanotransduction, the interest of our R&D teams is to develop and support their evolution directly into the environment of an IBiSA core facility. Thus, the R&D Team PLATIM/RDP is one of the rare facilities in France to provide different and integrated mechanical evaluations for both plant and animal multicellular organisms.

Publications:

  • Bauer, A., Ali, O., Bied, C., Boeuf, S., Bovio, S., Delattre, A., Ingram, G., Golz, J.F. & Landrein, B. Spatiotemporally distinct responses to mechanical forces shape the developing seed of Arabidopsis. (2024) EMBO J. https://doi.org/10.1038/s44318-024-00138-w
  • Creff, A., Ali, O., Bied, C., Bayle, V., Ingram, G. & Landrein, B. Evidence that endosperm turgor pressure both promotes and restricts seed growth and size. (2023) Nature Comm. https://www.nature.com/articles/s41467-022-35542-5

Since 2010, we have developped and implemented numerous methods in optogenetics, and even chemogenetics, in order to have access to dynamics and reversible perturbations of key biological functions such as cell adhesion, cell signaling, transcription factors, inflammation, functions of immune cells and even metabolism. Our lab proposes to share this expertise to the FBI users through access for consulting and even direct collaboration. In the Rhonalpin node, our lab is focused on the coupling between optogenetics, biosensors and metabolism imaging through FLIM imaging. Indeed, we have developed a TIRF microscope presenting a module of FastFLIM imaging. Through the use of dark acceptors, this technology allows the users to extend the possibilities in terms of combining optogenetics, biosensors, metabolic imaging through ratiometric probes and classical multicolors TIRF imaging. Moreover, TIRF imaging allows long term live imaging with low levels of photoxicity. This is essential for metabolic imaging and preserving photon budget for FLIM imaging. In the future, this system will be coupled with a module of evanescent field patterning (EFP) in order to have a specific TIRF-mode illumination of only  a region of interest (microm scale).

Publications

1- An optogenetic approach to control and monitor inflammasome activation. Julien Nadjar, Sylvain Monnier, Estelle Bastien, Anne-Laure Huber, Christiane Oddou, Léa Bardoulet, Gabriel Ichim, Christophe Vanbelle, Bénédicte Py, Olivier Destaing*, Virginie Petrilli*. Recently accepted in Science Signaling. bioRxiv 2023.07.25.550490; doi: https://doi.org/10.1101/2023.07.25.550490

2-Optogenetic control of YAP cellular localisation and function. Toh PJY, Lai JKH, Hermann A, Destaing O, Sheetz MP, Sudol M, Saunders TE. EMBO Rep. 2022 Sep 5;23(9):e54401. 

3-Control of SRC molecular dynamics encodes distinct cytoskeletal responses by specifying its signaling pathway usage. Kerjouan A, Boyault C, Oddou C, Hiriart-Bryant E, Pezet M, Balland M, Faurobert E, Bonnet I, Coute Y, Fourcade B, Albiges-Rizo C, Destaing O. J Cell Sci. 2021 Jan 25;134(2):jcs254599.

4-β1A integrin is a master regulator of invadosome organization and function. Destaing O, Planus E, Bouvard D, Oddou C, Badowski C, Bossy V, Raducanu A, Fourcade B, Albiges-Rizo C, Block MR. Mol Biol Cell. 2010 Dec;21(23):4108-19.

The translational Cardiovascular Research team develops fluorescence-based 3D imaging techniques and intravital microscopy approaches, in order to apply them for solving key questions related to the molecular and cellular functions, notably related to inflammatory responses in hearts and vessels. The researchers and technicians of the team, in collaboration with PRIMACEN, have implemented and developed set-ups for macroconfocal lymphangiography, vascular thrombosis evaluations as well as light sheet imaging, which are regularly optimized for new applications and projects. The team implemented in 2016 the first cardiac lymphangiography in rodents. Later, the team developed approaches for light sheet 3D imaging. These protocols are accessible to the community through their integration into the platform PRIMACEN (IBISA).

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.

Expertise in 3D imaging of live and fixed organoids. Drug screening tests using morphological characterization of organoid cultures as readouts. Organization of an open platform for Organoid development.

3D-electron microscopy approaches and correlative microscopies at different scales, from single molecules to tissues. Strong expertise in molecular imaging by cryo-EM and single particle analysis

Expertise of the team: Combination of cuttin-edge techniques in optical and electron imaging, material science, cell mechanics and intra-vital imaging to elucidate how phagocytes, in particular macrophages and osteoclasts, interact with the extracellular matrix, to decipher the mechanisms of macrophage 3D migration.

The team develops original systems to fluorescently label and track DNA (ANCHOR technology – NeoVirtech SAS) in real time at nanoscale resolution to understand physical principles underlying regulation of gene expression and DNA repair, in cellular plasticity and tumorigenesis.