Polarization-resolved Second Harmonic Microscopy in collagenous tissues: theoretical and experimental developments, and application to microstructural imaging during biomechanical assays in tendon.

Abstract : Collagen is a major structural protein that forms various macromolecular organizations in tissues and is responsible for the biomechanical properties of most organs. Second harmonic generation (SHG) microscopy is a valuable imaging technique to probe collagen fibrillar organization. This work is aimed at implementing and characterizing polarization-resolved SHG (P-SHG) and coupling this technique to biomechanical assays to provide multiscale structural information on collagen tissues. We first studied the linear propagation effects that affect P-SHG imaging of thick anisotropic tissues such as collagen tissues. We developed a theoretical model that accounts for birefringence, diattenuation, and polarization scrambling, and obtained an excellent agreement with our P-SHG measurements in rattail tendon. Moreover we performed numerical simulations of light propagation and harmonic generation in tendon and cornea, which confirmed the crucial role of birefringence for P-SHG signal formation. We then implemented a new experimental device that combined mechanical testing with SHG imaging. It enabled visualization of the tendon crimp morphology at the micrometer scale during macroscopic strain-stress measurements. Our results proved that continuous SHG imaging allows for elucidating the link between macroscopic response and microscopic structure of tissues. Finally, we developed a theoretical model, which relates the P-SHG signal anisotropy to the orientational order of its SHG-capable constituents at submicrometer scale. We tested our model by performing P-SHG measurements in increasingly stretched tendon, and successfully characterized variations of fibril disorder within fascicle with strain.
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Ivan Gusachenko. Polarization-resolved Second Harmonic Microscopy in collagenous tissues: theoretical and experimental developments, and application to microstructural imaging during biomechanical assays in tendon.. Optics [physics.optics]. Ecole Polytechnique X, 2012. English. ⟨pastel-00910146⟩

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