Label-free (N)on-(L)inear (O)ptical Imaging of (M)etabolism and (M)yelin distribution in (I)ntact (T)issues – NLOMMIT

Multiple Sclerosis (MS) induces destruction of intrinsically normal myelin (demyelination) and disrupts cellular metabolism leading to neurodegeneration. Myelin is an ubiquitous lipidic structure within the nervous system essential for the normal conduction of impulses along axons and the energetic support of neurons. Preventing demyelination and promoting myelin repair appear to be a crucial therapeutic challenge, not only as they allow restoration of normal conduction and functional recovery, but also as they prevent axonal and neuronal degeneration. MS diagnosis and evolution are usually followed by Magnetic Resonance Imaging (MRI), which allows rapid identification of demyelinating MS lesions but with a poor spatial resolution and specificity for single myelin fibers. The relation between the observed MRI contrast and microscopic myelin distribution is unknown and very few translational works have been conducted to assess the specificity of MRI toward myelin. Potent label-free and non-invasive optical methods to investigate myelin and metabolism pathology and repair at the sub-cellular scale are the key tools for the analysis of demyelinating lesions in MS in vivo. NLOMMIT proposes to develop a methodology and a platform based on nonlinear optical (NLO) microscopy for probing in a label-free manner and simultaneously cellular metabolism and myelin lipid distribution in intact mouse nervous tissue during myelination and demyelination processes. The project will rely on the association of fluorescence lifetime imaging and third-order coherent contrasts: two-photon Fluorescence Lifetime Microscopy (2p-FLIM) signals from cellular endogenous fluorophores (metabolic coenzyme NADH) will be used to non-invasively investigate the energetic and metabolic redox state of intact/living tissues at the cellular level; in parallel, Third Harmonic Generation (THG) and Coherent Anti-Stokes Raman Scattering (CARS) signals will allow the visualization of lipids and provide a label-free probe of molecular ordering at the sub-micron scale in intact tissues. Finally the project intend to correlate the optical description of myelin distribution obtained by THG and CARS microscopy with the high resolution MRI images within ex vivo brain slices. NLOMMIT aims to achieve a multi-parametric imaging of myelinated tissues on a "multiscale" level, providing an experimental and theoretical framework to relate the imaging data to the myelin organization at the macromolecular (sub-µm) to tissue (tens-to-hundreds of µm) scales. This will allow cutting edge progresses in the understanding of the biological consequences of cortical myelin pathology at the cellular and neuronal network levels, and for the development and evaluation of future promyelinating therapies.