The molecular mechanisms underlying cellular processes occur at the nanoscale. The advent of super-resolution microscopy (SRI) in the past ten years (2014 Nobel Prize in chemistry) has enabled biologists to study these mechanisms with nanoscale precision in living systems, bridging the gap between electron microscopy and classical fluorescent microscopy, which resolution is inherently limited by the diffraction of light to ~250 nm. These methods have led to the discovery of major biological structures and cellular organizations, and new concepts are emerging from these studies, revealing discreet organizational patterns of biomolecules that are essential for their functions. In the particular context of brain transmission where neuronal contacts are established in a highly confined, dense, and restricted area of ~20 nm*250-500 nm, SRI opens new avenues to study these nano-environments, to ultimately understand how they are established and modulated throughout life. However, like all developing techniques in their early days, SRI has revealed important limitations. Whereas optical setups have been increasingly ameliorated to optimize imaging resolution, down to a few nanometers only (~ 10 nm), biologists still rely on traditional imaging tools such as large divalent antibodies, which sizes of ~15 nm * 2 (primary/secondary), defeat the purpose of increasing optical resolution down to 10 nm. Thus, the development of proper imaging tools that enable full benefit from these nanoscopic resolutions is still lacking, and efforts towards the generation of novel molecular tools to probe protein organization at the nanoscale is critical. In this project, we will: - Develop versatile 3-nm monomeric ligands, called “nanoprobes”, that will enable full benefit from the available optical resolutions, in order to gain novel information on the dynamics and nano-architecture of biomolecules in living cells and tissues - Fully functionalize and characterize these nanoprobes for 3D and quantitative super-resolution imaging and tissue labeling - Use the newly generated nanoprobes to decipher the dynamic nanoscale organization of synaptic proteins underlying the formation of brain connectivity. For probe development, libraries of genetically diversified small recombinant protein domains (~10 kDa) will be screened against specific epitopes and endogenous adhesion receptors involved in severe neurodevelopmental disease (Autistic spectrum disorders, schizophrenia, epilepsy). This will enable the generation of highly versatile nanoprobes as well as probes against endogenous cell receptors involved in synapse establishment and remodeling, to understand brain connectivity. The newly generated nanoprobes will be conjugated to state of the art fluorophores for in-depth 3D super-resolution imaging and to DNA oligonucleotides for use in a novel multi-protein imaging scheme, allowing to visualize multiple proteins within the same structure, with nanoscale precision (DNA-PAINT) and extract quantitative information (qPAINT). Using these efficient nanoprobes to target adhesion receptors and the diverse components of synaptic adhesions within neuronal cells and brain tissue, we will be able to decipher for the first time the internal organization of brain connections with maximal accuracy, providing 3D cartographies of their ultrastructures, and understand the impact of specific genetic mutations on this organization. Thus, this project is expected to lift two major barriers in the fields of i) fundamental neuroscience, by allowing to study the molecular nano-architecture and remodeling of brain connectivity in neuronal cultures and inside living tissues, an objective that has long been hampered by the lack of suitable tools for protein targeting in confined environments and ii) bio-imaging, by enabling scientists to benefit from optimized molecular tools that will allow versatile protein labeling and full accessible nanoscale resolution.