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Life is made of molecular interactions, therefore probing molecules’ interactions at their native size, and in their natural environment, is the Holy Grail in biology. Yet, reaching such a high-level spatial resolution in living biological systems is extremely difficult. Towards that end, a large repertoire of super-resolution techniques have been developed in the last decades, however mostly focused on fluorescence microscopy: itself invasive, potentially perturbative, and, most importantly, leaving open many questions related to the larger population of molecules that are not labelled. Coherent Raman Scattering (CRS) microscopies have emerged as an alternative solution due to their high-speed character combined with label-free molecular-level chemical imaging capabilities. Nevertheless, CRS techniques are still far from reaching far-field super-resolution that is truly label-free, in particular for scattering specimens. Recently, we have developed a computational super-resolution CRS microscopy framework that is general, in the sense that it could be applicable to any CRS method. In COCOhRICO, we aim at extending this framework to enable fast far-field Raman nanoscopy, that is, to go beyond the 100 nm spatial resolution and for imaging within tissues and inside single cells. Our method relies on exploiting high nonlinearity of the structured illumination patterns, in order to readout high spatial frequency information of the specimen spectrum, typically lost in a conventional CRS microscope. For that, we exploit random structured illuminations, i.e. speckle patterns, that are robust against scattering and aberrations, and are furthermore easy to implement. While our current laser source only allowed for microscopy-only experiments, the new optical layout proposed in COCOhRICO will allow for extending the recent framework to microspectroscopy regimes. Such a setup, with improved chemical selectivity and sensitivity, will require also novel algorithm developments, and we plan to exploit speckle correlations and chemical sparsity priors for that. These tools are developed to tackle open-questions in challenging phytoplankton, in particular to unravel the internal chemical composition of diatoms. Because of diatoms’s small sizes, and inherent scattering exoskeleton (frustule), high-speed and scattering-resistant super-resolution methods are needed. We aim at exploiting the developed framework to address emerging questions in diatoms: what is the internal chemical composition of the organelles and how do they change with physical and chemical environmental changes. The answers provided COCOhRICO’s methods will have a wide impact in industrial use of phytoplankton and on Earth’s biogeochemical behavior. Finally, the outcomes of COCOhRICO paves the way to reach far-field super-resolution nanoscopy compatible with scattering specimens, therefore enabling answers to questions in systems where exogeneous labelling is challenging, or even prohibitive.
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