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Génération de seconde harmonique par le collagène et application à l'étude de fibroses par microscopie multiphoton.

Abstract : Multiphoton microscopy is an optical imaging technique which has the unique ability of providing three-dimensional micrometer resolution within thick tissues. Another attractive feature is that it is possible to visualise the structure of an unstained cell or tissue using endogenous sources of contrast that allow for a less invasive imaging method. Moreover, different contrast modes like two-photon excited fluorescence (2PEF), second harmonic generation (SHG) or third harmonic generation (THG) can be easily combined, and all these features enable a wide range of applications of multiphoton microscopy in fields like neurosciences, oncology, embryology,... Meanwhile, progresses are still needed in order to completely characterize the endogenous sources of nonlinear optical contrast observed in biological media. Thus, the second harmonic generation from fibrillar collagen raised different questions in the beginning of this work, mainly on the role of the collagen type and the role of its triple helical structure (that means the chirality) in the SHG signal. The study of collagen is particularly interesting for this protein represents 30% of the total human body proteins. The collagen is the main constituent of the extracellular matrix of a wide variety of tissues and organs and hence it is involved in the remodelling of the extracellular matrix and numerous pathologies. But nowadays there's a lack of appropriate methodologies to visualise the 3D architecture of collagen at a micrometer scale. In this context, the SHG microscopy appears as a promising tool for the visualisation of the distribution of collagen within the tissues. This work started with polarisation-resolved surface second harmonic generation experiments on thin films of collagen I and IV molecules, which allowed us to demonstrate that the SHG microscopy is a sensitive probe of collagen macromolecular organization and not of the collagen type. We next applied these results to the study of pulmonary and renal fibrosis in animal models, a pathological accumulation of fibrillar collagen which can progress to a terminal renal insufficiency or to a respiratory insufficiency often lethal eventually. We demonstrated that multiphoton microscopy permit to visualise the morphology of these tissues, to evidence all the characteristic features of these pathologies and quantitatively evaluate the remodelling of the extracellular matrix during fibrosis. Finally, we have proposed fibrosis scores based on volume fractions of significant SHG signals which allowed us to appreciate the role of different factors (cells/assembling enzymes) in fibrosis. This methodology will help to develop new therapeutic approaches based on a better understanding of these fibrotic pathologies.
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Ana-Maria Pena. Génération de seconde harmonique par le collagène et application à l'étude de fibroses par microscopie multiphoton.. Biophysique [physics.bio-ph]. Ecole Polytechnique X, 2006. Français. ⟨pastel-00002033⟩

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