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Amplification de la fluorescence par diffusion multiple : une étude exploratoire vers des conditions biocompatibles

Abstract : The purpose of this PhD has been to develop a method of amplification of the fluorescence by using the multiple scattering (introduction of dielectric, elastic and passive nanoparticles) that will be applicable to biological samples. This required doing the link between the biological laser and random laser concept for a use in an amplified regime, which precedes the laser regime, applicable to cell and tissue labelling. The generation of an amplification passes by the use of scatterers (nanoparticles of titanium dioxide in this work) homogeneously distributed. It required developing conditions of the colloidal stabilization to avoid the aggregation of nanoparticles into biological media and buffers by the adsorption of proteins (BSA). An optical setup has been developed to excite the fluorescence, by accurately evaluating the pump energy and the pulse response of fluorescence, while optimizing the observation window to limit photobleaching of fluorophores and cell toxicity. A first stimulated amplification was shown and validated with a colloidal suspension in a homogeneous aqueous solution of FITC at a concentration of 200 µM. This experiment has shown that it is possible to achieve a fluorescence gain up to 40 associated with a decrease in spectral width up to 5 nm, and an overall reduction in lifetime. The presence of the stimulated emission process in amplification is confirmed by the correlation between fluorescence and nanoparticle concentration or pump energy. This first step was extended to concentrations of 2 and 20 µM for which a rapid saturation of the gain (respectively ~ 10 and 20) was observed. Finally, amplification has been shown and validated on CFSE-labeled or GFP-expressing suspension cells in their culture medium. In these conditions, a decreasing of the gain compared with the previous conditions has been observed (up to 6 – 10 times). The explanation of this lower gain has been explored by testing the changes of media on amplification, and the presence of a layer of BSA surrounding the nanoparticles seems to be the cause because the protein decreases probably the refractive index of the nanoparticle leading a weak scattering.
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Sylvain Bonnefond. Amplification de la fluorescence par diffusion multiple : une étude exploratoire vers des conditions biocompatibles. Physique [physics]. COMUE Université Côte d'Azur (2015 - 2019), 2019. Français. ⟨NNT : 2019AZUR4080⟩. ⟨tel-02864775⟩

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