5D-SURE

Super-resolution (SR) microscopy is a revolution that allows us to revisit biology at the nanoscale. Among SR strategies, Single Molecule Localization Microscopies (SMLM) offers the highest spatial resolution (~20-nm) for fixed cell imaging whereas Structured Illumination Microscopy methods (SIM and its variants) provide lower resolution (~140 nm) with a high temporal resolution (~1-s) compatible with live-cell imaging. Ideally, multi-scale spatial and temporal resolution would be observed over the same field of view (FOV). If SR microscopy is becoming a widely used tool, it still suffers from limitation inherited from classical microscopy, in particular inhomogeneous fluorescence excitation which limits the field of view and can compromise the final image resolution. SR microscopes will not only strongly benefit from dedicated optical design, but can now integrate smart, real-time analysis and feedback for optimal performances. To address these crucial needs, we propose to develop an integrated instrument offering a continuum of super-resolution modalities from live cell SR imaging with multifocal SIM (MSIM) to molecular resolution in fixed samples using SMLM. Furthermore, we will increase the throughput of this new instrument by increasing the FOV and improving its multi-color capabilities, as well as developing probes to interrogate the pH of living cell at the nanoscale (fifth dimension). Dedicated analysis solution will speed up and provide smart control over the whole imaging workflow. Overcoming these technological barriers requires a multidisciplinary approach that includes optical design, new probes and optimized photophysics, as well as optimization of the data management. We formed a transdisciplinary consortium and designed the 5D-SURE project to push the limits of the nanoscope. At the hardware level, the proposed instrument is based on a new excitation strategy offering MSIM illumination and smart control of the irradiance for SMLM, advances in adaptive data processing, and a microfluidic device fully controlled by an intelligent software platform. Several new targeted live-cell compatible and dual-color pH probes SMLM will also be designed and developed during this project. We will demonstrate the benefits on a biological application with high societal impact: the functional nano-architecture of recently discovered axonal actin structures (rings, hotpsots, trails). In addition to these technological and scientific advances, the project will also have a significant economic impact: by propelling Abbelight’s best-in-class offer, it will increase its competitivity and lead to the creation of new jobs.