Les techniques de microscopie non linéaire connaissent un essor considérable ensciences du vivant, du fait de leur capacité à imager les tissus biologiques en profondeur et àexploiter différents contrastes dont les plus connus sont la fluorescence excitée à deux photons(2PEF) et la génération de second harmonique (SHG).Ce travail de thèse ’articule autour de la métrologie de milieux diffusants et d’applicationsbiomédicales de la microscopie non linéaire. Après une présentation générale de la technique, nousdécrivons une méthode originale de mesure du coefficient de diffusion μs et du facteur d’anisotropieg de milieux turbides épais basée sur la comparaison des intensités de fluorescence épi-collectéesselon trois modalités de notre microscope non-linéaire. Notre méthode est alors appliquée à lacaractérisation de gels biomimétiques et d’échantillons d’intérêt biologique. Le manuscrit abordeensuite le problème de l’imagerie en profondeur d’explants de peau humaine ré-innervée par desneurones sensoriels de rats nouveau-nés. Le choix du marqueur neuronal fluorescent fait l’objetd’une mesure in situ de la section efficace d’absorption à deux photons de différents fluorophores.La faisabilité d’une imagerie bimodale exploitant la fluorescence de ce marqueur et la réponse SHGdu collagène fibrillaire du derme est démontrée. Le manuscrit s’achève par une étude faisant suiteà des travaux de thèse antérieurs relatifs à la quantification de la fibrose hépatique par microscopieSHG/2PEF couplée. Nous appliquons la méthode de scoring SHG développée précédemment àune cohorte de patients infectés par le virus de l’hépatite C et comparons nos résultats aux testsMETAVIR et Ishak. / Nonlinear microscopy techniques are experiencing a considerable growth in lifescience, thanks to their ability to image biological tissues at high depth with different contrastssuch as two-photon excitation fluorescence (2PEF) and second harmonic generation (SHG).This manuscript focuses on metrology of scattering media and on biomedical applications ofnonlinear microscopy. After an overview of the technique, we describe a novel method for measuringthe scattering coefficient μs and anisotropy factor g of thick turbid media based on the comparisonof fluorescence intensities epi-collected through the three modalities of our nonlinear microscope.Our method is then applied to biomimetic gels and to biological samples. The manuscript thentackles the problem of imaging deeply human skin explants re-innervated by sensory neurons fromneonatal rats. The choice of the fluorescence probe is the subject of an in situ measurement ofthe two-photon action cross-sections of various fluorophores. The feasibility of bimodal nonlinearimaging of re-innervated skin explants based on the 2PEF signal of the molecular probe and onthe SHG response of fibrillar collagen of the dermis is demonstrated. The manuscript ends witha study subsequent to the work of previous thesis regarding the quantification of liver fibrosisby SHG/2PEF microscopy. We apply the method of fibrillar collagen scoring by SHG previouslydeveloped to a new cohort of patients infected with hepatitis C virus and we analyze our resultsin terms of METAVIR and Ishak tests.

Background: The shoulder joint is one of the most freely movable joints in the human body and has therefore high importance for upper limb functionality. Several techniques have been developed to replace the glenohumeral joint including humeral hemiarthroplasty, anatomical total shoulder arthroplasty, and reverse total shoulder arthroplasty, depending on the underlying pathology. For the soft tissue reconstruction, the innervated latissimus dorsi musculocutaneous flap is a reliable solution flap in shoulder and arm reconstruction. - Case presentation: We present the case of a 16-year-old male patient with a complete destruction of the shoulder joint and soft tissues after ballistic trauma. We performed the reconstruction of the shoulder joint using a humeral hemiarthroplasty with a mesh fixation to the remaining glenoid. The soft tissue coverage and the restoration of the deltoid muscle function were insured with a pedicled innervated latissimus dorsi musculocutaneous flap. One year postoperatively, the patient showed a good function of the shoulder joint with an excellent aesthetical result and no pain. - Conclusion: The pedicled latissimus dorsi musculocutaneous flap can safely restore the shoulder function, while the humeral hemiarthroplasty with mesh fixation can be a reliable solution for the reconstruction of a completely destructed shoulder joint.