L’un des enjeux de la bioingénierie consiste à synthétiser des biomatériaux visant à régénérer, remplacer ou suppléer des organes déficients. Afin de visualiser des structures dans des conditions proches de la réalité physiologique, et de manière non invasive, de nouvelles techniques d’imagerie ne cessent de se développer. En particulier, la microscopie associant une excitation multiphoton et la détection de signaux de fluorescence et de signaux de SHG (Second Harmonic Generation) permet l’observation en profondeur de composants de matrices extracellulaires sans marqueur fluorescent exogène. Ce travail basé sur ces techniques a permis dans un premier temps d’apprécier les probabilités d’absorption multiphoton en fonction de la modulation de l’excitation avec deux systèmes différents (Cavity Dumper et EOM). Une seconde partie de ce projet a été consacrée à la mise en place et l’optimisation de la détection de signaux SHG provenant du collagène dans les tissus biologiques. Nous avons utilisé ces méthodes d’imagerie pour mettre en évidence les modifications intervenant au niveau des réseaux de collagène de la MEC de cartilage suite à l’application d’une contrainte mécanique (compression) ou biochimique (enzymatique). Puis, nous nous sommes intéressés au domaine vasculaire, en montrant la possibilité d’imager sans marquage fluorescent les réseaux d’élastine de la média (en mettant à profit son autofluorescence) et le réseau de collagène de l’adventice (générant un fort signal SHG). Nous avons ainsi pu apprécier l’état des structures en fonction de différentes conditions de préservation (congélation, fixation) et le remodelage de substituts artériels implantés chez le lapin. Enfin, une dernière application biologique, basée sur l’étude de tumeurs, nous montre la complémentarité et l’intérêt d’une imagerie de type macroscopique avec les diverses modalités de détection en microscopie. / To repair, supply or regenerate deficient organs, the bioengineering field consists of synthesising functionalised biomaterials. To visualise the synthesised structures in a non invasive way and in physiological conditions, new imaging techniques tend to be developed. Among them, microscopy associating multiphoton excitation with fluorescence detection or Second Harmonic Generation enables a visualisation in depth of extracellular matrix structures, without any exogenous dye. The first part of this work was to characterise multiphoton absorption probability in function of different excitation conditions, that means a modulation of the excitation beam through two different systems (Cavity Dumper and EOM). In a second part, we implemented and optimised the detection of the SHG signal coming from collagen in biological tissues. Through SHG measurements, we showed modifications occurring on the collagen network of the extracellular matrix of cartilage, when sample were submitted to mechanical (compression) or biochemical (enzymatic) constraint. We also were interested by the vascular research field and showed the ability of multiphoton microscopy to image without any fluorescent dye, the elastin network of the media and the collagen network of the adventice. We could appreciate qualitatively the effect of cryopreservation or fixation on the arterial wall, and the remodelling of a substitute implemented in a rabbit to supply its carotid. A last biological application concerned study of tumors, and showed us the complementarities between a macroscopic study with information obtained by microscopy.

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Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references. Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI/Dissertation Abstracts International.

As one of the few 3D microfabrication techniques available to researchers, multiphoton lithography (MPL) has generated considerable interest in the scientific community. By allowing researchers to localize photochemistry to a femtoliter volume, MPL has permitted the fabrication of intricate, 3D microstructures from a range of materials, including protein hydrogels. MPL can be used to fabricate functional hydrogels on the scale of 100 μm, with features on the order of 1 μm. This dissertation examines existing MPL techniques to discover ways in which current processes can be modified to produce hydrogel products that are more useful for biomedical applications like tissue engineering. A new material is introduced that enables the fabrication of fully unconstrained hydrogel microstructures. In this context, A structure can be classified as “unconstrained” when it is free to translate and rotate without hindrance in three dimensions, and is not attached to the substrate or any other structure. New processes are demonstrated that permit the fabrication of larger MPL hydrogels without sacrificing feature resolution. This allows the fabrication of millimeter-scale, high aspect ratio structures with features smaller than 10 μm. Methods are described for tuning and measuring the mechanical properties of MPL-fabricated hydrogels, and ways of tuning the functional properties of the hydrogels are also examined. / text

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