Imagerie Quantitative du Collagène par Génération de Seconde Harmonique

Abstract : Collagen is an ubiquitous protein which plays a central role in the architecture and the mechanical integrity of connective tissues. Synthesized as triple helices, collagen self-assembles into fibrils both in vivo and in vitro to form three-dimensional networks. It is essential to probe this fibrillar organization to characterize tissu remodeling involved in many diseases and guide tissue engineering. Multiphoton microscopy based on second harmonic generation (SHG) is a very specific technique to visualize unstained collagen deep into tissues, with sub-micron resolution. This thesis aims to develop quantitative approaches in SHG imaging of collagen, at both fibrillar and tissular scales. We first showed that SHG microscopy is a relevant tool to measure the dynamics of formation of collagen networks, even at single fibril scale. We also characterized the structure of collagen gels controlled by adding functionalized silica nanoparticles. We then performed correlative electron/SHG imaging on these fibrillar gels. This allowed us to measure the sensitivity of our set-up and to calibrate the response of an isolated fibril as a function of its diameter. In addition, we derived the hyperpolarizability of a triple helix, which validated the additive model used to calculate the nonlinear response of a fibril. Concurrently, we developed specific image analysis to phenomenogically quantify the organization of the fibrillar network at micrometer scale with the view investigate the function/structure relationship in biological tissues. This was validated by characterizing the changes of biomechanical properties in genetically modified mice model of Ehlers-Danlos syndrome.
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Stéphane Bancelin. Imagerie Quantitative du Collagène par Génération de Seconde Harmonique. Biophysique [physics.bio-ph]. Ecole Polytechnique X, 2013. Français. ⟨pastel-00958171⟩

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