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, Cependant, contrairement à l'ADE qui avait abouti à une relation très intéressante entre le signal de CD-SHG mesuré et l'angle hors-plan ? des fibrilles, il ne semble pas possible, selon cette nouvelle approche, d'avoir accès à l'organisation 3D des fibrilles par une simple mesure de CD-SHG. En effet, d'après la seule forme autorisée pour la courbe du CD-SHG en fonction de ?, un même signal de CD-SHG peut être mesuré pour deux angles hors-plan différents. De plus, la courbe étant différente d'un pixel à l'autre du fait de la variabilité des tenseurs, il n'est pas possible d'établir de lien quantitatif absolu entre la valeur du signal de CD-SHG et le degré de planéité de la structure dans le volume focal. En particulier, le signe du CD-SHG ne correspond pas au signe de l'angle hors-plan mais à une certaine structuration dans le volume focal, Grâce à ces considérations de phase ainsi qu'aux résultats de nos expériences Up-Down, nous sommes donc en mesure d'affirmer que la prise en compte des contributions dipolaires magnétiques est indispensable pour décrire correctement le signal de dichroïsme circulaire en microscopie SHG 17

, Il a consisté à mettre en place et à appliquer de nouvelles modalités de SHG résolues en polarisation, c'est-à-dire avec des états de polarisation incidents linéaires ou circulaires contrôlés, afin d'obtenir des informations sur la structure hiérarchique du collagène. Il a inclus une phase d'optimisation du microscope, des protocoles expérimentaux et des programmes de traitement de données, une phase d'application de la P-SHG puis une phase d'étude expérimentale et théorique du CD-SHG, Le travail présenté dans ce manuscrit est centré sur l'utilisation de la microscopie SHG pour sonder l'architecture 3D du collagène dans différents tissus ou matériaux

, Les organisations à ces deux échelles ne sont pas sondées indépendamment mais regroupées sous un paramètre de désordre local appelé le paramètre d'anisotropie ?. Le but de la première application de la P-SHG était de pouvoir suivre l'état de conservation de parchemins historiques précieux sans contact ni prélèvement. Par des analyses sur des parchemins contemporains présentant différents stades de dégradation, nous avons démontré que la P-SHG est un outil puissant pour répondre à ce besoin. En effet, une dégradation avancée du collagène dans les parchemins s'accompagne d'une perte du signal SHG ainsi que d'une augmentation du signal de 2-PEF, ce qui se traduit par l'augmentation du rapport des intensités. L'apport spécifique de la P-SHG vient de la détection possible des premiers stades de dégradation grâce au suivi quantitatif de l'évolution du paramètre d'anisotropie ?. Nos résultats sont en accord avec les analyses chimiques classiques pour évaluer la dégradation (DSC), mais contrairement aux techniques classiques qui sont destructives, ils ont été obtenus sans contact et sans prélèvement. Cela nous a donc permis d'étendre nos analyses à des parchemins médiévaux précieux issus du grand fonds des manuscrits de la médiathèque de Chartres. La seconde étude en P-SHG portait sur la caractérisation de la structure du collagène dans la cornée dans des cas sains et pathologiques. Nos travaux sur coupes de cornées humaines saines et présentant un kératocône ont abouti à des résultats prometteurs : nous avons en effet montré une différence importante dans l'entropie de la distribution d'orientations des fibrilles de collagène. Il reste toutefois à valider ce diagnostic de manière significative sur davantage d'échantillons et sur différents types de kératocônes, car nous avons constaté que cette pathologie présente une grande variabilité. De plus, ces travaux préliminaires ont été conduits sur des coupes fixées et non pas sur le tissu intact dans sa forme physiologique. Une thèse en cours (Clothilde Raoux) poursuit ces travaux, tant sur des coupes histologiques que sur des cornées humaines entières, saines ou pathologiques, et vise à reconstituer un atlas complet de la structure de ce tissu en le sondant par P-SHG couplée à des essais mécaniques. Ces deux applications ont illustré la pertinence d, Dans un premier temps, nous avons utilisé la modalité de P-SHG pour répondre à deux problématiques spécifiques dans les domaines des sciences du patrimoine d'une part et de l'ophtalmologie d'autre part. Dans ces deux domaines, il est nécessaire de développer des diagnostics quantitatifs non-invasifs offrant une résolution subcellulaire. La P-SHG a permis l'obtention de paramètres d'ordre multiéchelle, sondant l'organisation du collagène à l'échelle de l'image (entropie ou indice d'orientation) mais également à des échelles dépassant la résolution optique, les échelles fibrillaire et moléculaire

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