Nonlinear coherent scattering phenomena from single nanoparticles have been recently proposed as alternative processes for fluorescence in multiphoton microscopy staining. Commonly applied nanoscale materials, however, have reached a certain limit in size dependent detection efficiency of weak nonlinear optical signals. None of the recent efforts in detection of second-harmonic generation (SHG), the lowest order nonlinear process, have been able to cross a ~40 nm size barrier for nanoparticles (NPs), thus remaining at the level of "large" nanoscatterers, even when resorting to the most sensitive detection techniques such as single-photon counting technology. As we realize now, this size limitation can be significantly lowered when replacing dielectric insulators or wide gap semiconductors by direct-gap semiconducting quantum dots (QDs). Herein, a new type of highly nonlinear nanoprobes is engineered in order to surpass above mentioned size barrier at the single nanoparticle scale. We consider two-photon resonant excitation in individual zinc-blende CdTe QDs of about 12.5 nm diameter, which provide efficient coherent SHG radiation, as high as 105 Hz, furthermore exhibiting remarkable sensitivity to spatial orientation of their octupolar crystalline lattice. Moreover, quantum confinement effects have been found to strongly contribute to the second-order nonlinear optical susceptibility χ(2) features. Quantitative characterization of the χ(2) of QDs by way of their spectral dispersion and size dependence is therefore undertaken by single particle spectroscopy and ensemble Hyper-Rayleigh Scattering (HRS) studies. We prove that under appropriate conditions, χ(2) of quantum confined semiconducting structures can significantly exceed that of bulk. Furthermore, a novel type of semiconducting hybrid rod-on-dot (RD) QDs is developed by building up on crystalline moieties of different symmetries, in order to increase their effective quadratic nonlinearity while maintaining their size close to a strong quantum confinement regime. The new complex hybrid χ(2) tensor is analyzed by interfering the susceptibilities from each component, considering different shape and point group symmetries associated to octupolar and dipolar crystalline structures. Significant SHG enhancement is consequently observed, exceeding that of mono-compound QDs, due to a coupling between two nonlinear materials and slower decoherence, which we attribute to the induced spatial charge separation upon photoexcitation.

Lower urinary tract disorders are significant prognostic indicators of institutionalisation and lower quality of life in the elderly and their incidence increases with age. Urodynamics, the gold standard in diagnosis, replicates symptoms to assess functionality through controlled filling and voiding of the bladder but its interpretation is subjective and may be inconclusive; often requiring further testing or leading to inappropriate treatment. Normal filling and voiding biomechanics of the bladder relate directly to the structural composition of the bladder wall. Alterations to tissue composition in aging and pathology have significant impacts on biomechanics but are yet to be fully described. The aim of this thesis was to gain insight into the individual microstructural components of the bladder wall and how they relate to the gross mechanical response. Additionally, representation of these observations in a mathematical model that can be used to improve our understanding of urodynamic data. This aim was achieved through a combination of in situ mechanical testing and the development of a microstructural constitutive model, which was then included within an overall micturition framework to simulate filling and voiding functions, and evaluated with clinical data. Coupled systems of multiphoton microscopy and uniaxial, biaxial and inflation testing were used to correlate extra cellular matrix interactions with the mechanical response of young and aged murine bladder. Wall-layer specific collagen fibre orientation, dispersion and recruitment were quantified and implemented into a novel microstructural constitutive model. The bladder was modelled as a nonlinear elastic, constrainedmixture planar membrane with contribution from smooth muscle and collagen fibres in the detrusor. Collagen recruitment in the detrusor was observed to occur at a finite stretch; correlated with a steep increase in stiffness of the tissue, while collagen of the lamina propria plays a capacitance role. Collagen recruitment was modelled using a triangular probability density function; quantified from sequential microscopy images and fitted to mechanical data. Increased collagen area fraction and changes in dominant fibre orientation were attributed to reduced compliance in aged bladder. This behaviour was captured by the model. The microstructural model was modified to an isotropic thin-walled spherical membrane for the filling phase of a micturition model framework, consisting of a bladder outlet relation and urethral resistance relation. A contractile smooth muscle element was included in the active response. In the first steps towards clinical application the model was applied to male and female 'normal' patient urodynamic data to observe quality of model fit and estimate baseline parameter values. The model simulated key filling and voiding features seen in normal male and female clinical data. Mechanobiological modelling combined with clinically relevant micturition modelling has the potential to quantify bladder dysfunction. Moreover, improved understanding of how the microstructure influences macroscopic mechanics will yield improved understanding of how changes to the bladder impair its functionality. We predict that modelling will become a clinically relevant tool in urodynamics; leading to new options for diagnosis and management of patients with bladder dysfunction.

Les propriétés photoniques des tissus durs dentaires nous ont permis d’étudier l’email et la dentine a un niveau moléculaire (in vitro) en utilisant des techniques de microscopie optique non linéaires. La microscopie confocale Raman est technique d’imagine de haute résolution permettant d’analyse d’échantillon sans préparation spécifique ni marquage. Cette méthode nous a permis de reconstituer une cartographie de la réticulation du collagène et de la cristallinité au niveau de la jonction émail-dentine et cela avec une résolution spatiale non atteinte jusque-là. Cette analyse chimique de la jonction émail-dentine a permis de redéfinir la largeur de cette zone de transition. Cette largeur est nettement supérieure à celles proposées par les études précédentes. Par ailleurs, l’étude portant sur les changements de fluorescence intrinsèque entre les tissues dentaires sains et cariés suggèrent l’implication de la protoporphyrin IX et de la pentosidine dans l’expression de la fluorescence rouge des tissus cariés. La microscopie multiphotonique quant à elle nous a permis de détecter la lésion carieuse et de suivre son développement en utilisant la génération de seconde harmonique (SHG) et la fluorescence par excitation à deux photons (2PEF). Nos études ont démontré la validité du ratio SHG/2PEF comme paramètre fiable pour la détection de la lésion carieuse. Les études proposées par cette thèse montrent le potentiel des propriétés photoniques de l’émail et de la dentine en utilisant les microscopies Raman et multiphotoniques dans l’étude de ces tissus au niveau moléculaire. Cela offre de nouvelles perspectives en recherche et en applications cliniques. / Photonic properties of dental hard tissues allowed us to proceed to in vitro analysis of enamel and dentin on a molecular level. Confocal Raman microscopy has been used to produce a mapping of collagen cross-link and crystallinity of human dentin–enamel junction (DEJ) with a spatial resolution not achieved up to now. The method is a non-invasive, label-free and a high spatial resolution imaging technique. This chemical analysis of DEJ led us to redefine a wider width of this transition zone and advance our understanding of dental histology. A study on the intrinsic fluorescence changes of sound and carious tissues using conventional fluorescence microscopy suggests the involvement of protoporphyrin IX and pentosidine in the fluorescence red-shift observed in carious tissues. Multiphoton microscopy allowed to detect nonlinear optical signal changes during caries process using second harmonic generation (SHG) and two-photon excitation fluorescence (2PEF). Our studies led us to propose the ratio SHG/2PEF as valuable parameter to monitor caries lesion. Collectively, advances described in this thesis show the potential of photonic properties of enamel and dentin using Raman and multiphoton microcopies for molecular investigations on sound as much as on carious tissues. It opens new perspective in dental research and clinical applications.

La production de vanille (Vanilla planifolia, Orchidaceae) est limitée par la pourriture des racines et des tiges (PRT) provoquée par des champignons telluriques du genre Fusarium contre lesquels la lutte chimique ou prophylactique est inefficace. Ce travail décrit finement l'interaction entre les vanilliers et les Fusarium dans le but de développer la lutte génétique. L'analyse moléculaire (gène EF1α et IGS) et la détermination du pouvoir pathogène de 365 souches de Fusarium isolées de vanilliers à La Réunion et à Madagascar ont démontré que les souches pathogènes sur vanillier appartiennent presque toutes au complexe d'espèce F. oxysporum et ont une origine polyphylétique. Les observations en microscopie à champ large et multi-photons démontrent que la colonisation des racines débute au niveau des poils absorbants puis gagne le cortex, mais que le système vasculaire est épargné. Cela conduit à renommer l'agent pathogène en F. oxysporum f. sp. radicis-vanillae (Forv). Dans les interactions incompatibles (souche pathogène et vanillier résistant), l'épaississement et la lignification des parois de l'hypoderme, constitutive et induite par le champignon, joue un rôle significatif dans le blocage de l'invasion mycélienne. Le rôle important des composés phénoliques dans la résistance des vanilliers a été confirmé par les analyses d'expression différentielle de transcrits obtenus de novo par RNA-seq Illumina. Enfin, un test robuste de détermination in-vitro de la résistance à Forv a été validé et de nouvelles sources de résistance génétique à la PRT ont été identifiées. Nos résultats ouvrent des perspectives prometteuses pour la sélection de variétés améliorées de vanilliers. / Vanilla is a high value cash crop that is continuously demanded by the agri-food and cosmetics industries for its incomparable flavor. Most of vanilla comes from the cured fruits of V. planifolia G. Jackson, a hemi-epiphytic climbing orchid cultivated in the humid tropics. In all the countries were it is cultivated, the vanilla vines suffer from a root and stem rot (RSR) caused by the soil borne fungus Fusarium oxysporum which dramatically reduces plant production and the durability of plantations. No efficient control method is currently available for this disease. Sources of genetic resistance to RSR exist in few vanilla relatives, but so far no commercial resistant variety has been produced. The purpose of this thesis was to better describe the diversity and histopathology of the causal agent of RSR and to evaluate the potential sources of genetic resistance that could be used in breeding programs. In a first step, a collection of 377 single-spored Fusarium isolates recovered from rotten roots and stems during surveys conducted in 52 vanilla plots from Reunion Island, Madagascar and French Polynesia were characterised. Representative subsets of isolates were genotyped using the Elongation Factor 1α and Intergenic Spacer gene sequences. Their pathogenicity was assayed by root dip inoculation on the susceptible V. planifolia accession pla0001. Results showed that F. oxysporum was the principal species responsible for the disease in the field, although a few F. solani isolates showing slight pathogenicity were also isolated. Fusarium oxysporum isolates were highly polyphyletic regardless of geographic origin or pathogenicity. Remarkably, their pathogenicity varied in gradient between non- pathogenic (about 42% of isolates) to highly pathogenic (14%). In a second step, 254 vanilla accessions comprising 18 species and six types of hybrids were assessed for resistance to RSR in the field (natural inoculum) and in the lab (in-vitro plants inoculated with Fo072). The strong resistance to RSR of all V. pompona accessions and hybrids of V. planifolia X V. pompona or V. phaeantha, were confirmed, and novel sources of resistance to RSR were added including, V. bahiana, V. costariciensis and V. crenulata. Most of the V. planifolia accessions, V. ×tahitensis and V. odorata were susceptible to RSR. However, three inbreeds of V. planifolia showed a high level of resistance to Forv. To our knowledge this is the first report of resistance to RSR in V. planifolia accessions. For the 26 accessions evaluated in both conditions, a strong correlation was observed between long term (9 years) evaluation in the field and ratings on in-vitro plants at 15dpi. Thirdly, we monitored by wide field and multiphoton microscopy the root infection process and the responses of one susceptible accession (V. planifolia pla0001) and two resistant accessions (V. planifolia pla0020 and V. pompona pom0018) to challenge inoculation with the severe isolate Fo072. In the compatible interaction (Fo072 – pla0001) invasion started from penetration of hyphae emitted from germinated conidia in the hairy region of root rapidly colonizing the cortex but never expanded to the vascular bundles up to the 9th dpi. It was therefore suggested to prefix the forma specialis name of the causal agent of RSR with radicis to point out its non-vascular pathogenicity in vanilla. In the two incompatible interactions, the important role played by hypodermis cells for impeding the invasion of the cortex by Fo072 was demonstrated by specific staining and spectral analysis of lignin precursors. Both constitutive and pathogen induced defense mechanism were described in pla0020 and pom0018. The mechanisms included the deposition of lignin in the hypodermal cell wall, entrapment of hyphae in specific hypodemal cells and polyphenolics secretion in intercellular spaces. Further, a de novo transcriptome analysis was experimentedon 8 pooled samples.

La microscopie non linéaire a transformé le domaine de la neurobiologie depuis les années 1990, en permettant d'acquérir des images tridimensionnelles de tissus épais avec une résolution subcellulaire. Cependant, les profondeurs d'imagerie accessibles sont limitées à quelques centaines de micromètres dans des tissus diffusants tels que le tissu cérébral. Au cours des dernières années, plusieurs stratégies ont été développées pour dépasser cette limitation de profondeur et accéder à de plus grands volumes de tissu. Ces avancées récentes ont jusqu'à présent été limitées en terme de modes de contrastes accessibles, et ont souvent été réduites à des approches monochromes. Ce travail de thèse vise à développer des techniques d'imagerie non linéaires de grands volumes et de grande profondeur dotées de diverses possibilités de contrastes, indispensables pour l'étude de tissus complexes tels que le tissu cérébral. Dans un premier chapitre, nous présentons les difficultés associées à l'imagerie de grand volume de tissu cérébral, avec une emphase particulière sur les puissantes stratégies de marquages génétiques dont l'usage à jusqu'à présent été limité à des faibles étendues. Ensuite, nous introduisons la microscopie Chrom-SMP (chromatic serial multiphoton), une méthode développée au cours de cette thèse et consistant à combiner l’excitation deux-photon multicouleurs par mélange de fréquences avec une technique d'histologie automatisée (i.e découpe sériée) pour accéder à plusieurs contrastes non linéaires à travers de grands volumes de tissus ex vivo, allant de plusieurs mm3 à des cerveaux entiers, avec une résolution micrométrique et un coalignement intrinsèque des canaux spectraux. Dans un troisième chapitre, nous explorons le potentiel de cette nouvelle approche pour la neurobiologie. En particulier, nous démontrons l'histologie multicouleur de plusieurs mm3 de tissu "Brainbow" avec une résolution constante dans l’ensemble du volume imagé. Nous illustrons le potentiel de notre approche à travers l'analyse de la morphologie, des interactions et du lignage des astrocytes du cortex cérébral de souris. Nous explorons également l’apport du Chrom-SMP pour le suivi multiplexé de projections neuronales marquées par des traceurs de couleurs distinctes sur de grandes distances. Enfin, nous présentons dans un quatrième chapitre le développement de la microscopie à trois photons multimodale, approche permettant d’augmenter la profondeur d’imagerie sur tissus vivants. / Multiphoton microscopy has transformed neurobiology since the 1990s by enabling 3D imaging of thick tissues at subcellular resolution. However the depths provided by multiphoton microscopy are limited to a few hundreds of micrometers inside scattering tissues such as the brain. In the recent years, several strategies have emerged to overcome this depth limitation and to access larger volumes of tissue. Although these novel approaches are transforming brain imaging, they currently lack efficient multicolor and multicontrast modalities. This work aims at developing large-scale and deep-tissue multiphoton imaging modalities with augmented contrast capabilities. In a first chapter, we present the challenges of high-content large-volume brain imaging, with a particular emphasis on powerful multicolor labeling strategies which have so far been restricted to limited scales. We then introduce chromatic serial multiphoton (Chrom-SMP) microscopy, a method which combines automated histology with multicolor two-photon excitation through wavelength-mixing to access multiple nonlinear contrasts across large volumes, from several mm3 to whole brains, with submicron resolution and intrinsic channel registration. In a third chapter, we explore the potential of this novel approach to open novel experimental paradigms in neurobiological studies. In particular, we demonstrate multicolor volumetric histology of several mm3 of Brainbow-labeled tissues with preserved diffraction-limited resolution and illustrate the strengths of this method through color-based tridimensional analysis of astrocyte morphology, interactions and lineage in the mouse cerebral cortex. We further illustrate the potential of the method through multiplexed whole-brain mapping of axonal projections labeled with distinct tracers. Finally, we develop multimodal three-photon microscopy as a method to access larger depths in live settings.

This project considers the co-rotating twin screw extrusion of a confectionery paste comprising powdered proteins, sugars, water and fats. As is the case with many food industry products, this process has been developed experimentally with little quantitative understanding of how variations in processing conditions influence the formation of the extrudate. A variety of techniques have therefore been developed to characterise and quantify the dispersive mixing, distributive mixing and rheological flow properties of this complex, multiphase, viscoelastic, unstable material. These techniques have then been utilised in a pilot plant extruder study of the mechanics of mixing and paste formation during extrusion, considering the influence of both processing conditions and screw profile. The internal evolution of paste microstructure has been successfully tracked along the length of screw profile using dead-stop extractions of the screws. A rigorous off-line assessment of shear yield strength behaviour using cone penetrometry has shown the use of conventional off-line rheometers to be unviable due to rapid post extrusion hardening. This highlighted the need for an in-line rheological measurement technique for continuous extrusion analysis where the extruded material is severely time dependent and not extractable. In pursuit of this, a novel arrangement of bender elements is proposed and trialled, to rapidly characterise material parameters of viscoelastic pastes. A second technique looking to extend the application of shear wave interface reflection to multiphase pastes is also trialled. A novel analysis of thermogravimetric data (TGA) has generated a viable index of distributive mixing, suitable for use on complex multi-component materials where thermal decomposition temperatures of the components are not well defined. Quantitative image analysis of pastes using scanning electron microscopy (SEM), optical microscopy protein staining and a novel application of multiphoton microscopy (MPM) have been used to visualise paste microstructure and quantify dispersive mixing. From the pilot plant extruder study, the application of these techniques was successful in mapping the evolution of paste mixing and the resulting microstructure, as well as identifying key differences between pastes mixed by twin screw extrusion and batch mixing.

Les modèles numériques personnalisables d'organes du corps humain offrent un formidable potentiel pour évaluer le risque lésionnel dans les domaines de la sécurité des transports, du médical ou du sport. Suivant les applications, différents niveaux de détails peuvent être nécessaires. En particulier, lorsque le comportement mécanique des tissus biologiques doit être finement reproduit, les modèles de comportement doivent intégrer des considérations sur la structure du tissu, et simuler les mécanismes suivant lesquels il réagit à un chargement mécanique. Le travail de thèse présenté ici s'est focalisé sur la capsule de foie, notamment sur ses propriétés microstructurales et mécaniques, afin d'identifier les hypothèses importantes à intégrer dans la construction d'un modèle constitutif de tissu fibreux basé sur la microstructure. La méthodologie expérimentale a été mise en place afin de caractériser le comportement mécanique de ce tissu, en lien avec l'organisation de sa microstructure. Des essais de traction uniaxiale et de gonflement sous microscope confocal biphotonique ont été développés, pour observer l'évolution de la microstructure sous chargement. Des déformations macroscopiques ont été mesurées, et une méthode de mesure de champs de déformations locaux a été développée pour quantifier l'état de déformation du réseau de fibres. La réorganisation du réseau de fibre de collagène a également été quantifiée. L'analyse des liens existant entre les grandeurs mesurées à l'échelle macroscopique et ces phénomènes microscopiques est proposée, pour préciser les hypothèses à adopter dans les modèles permettant de passer de l'échelle des fibres au comportement global du tissu / Customized human body models offer a great potential to assess the injury risks in the fields of transport safety, surgery or sport. Various detail levels can then be needed, according to the targeted application. In particular, when the mechanical behavior of biological tissues needs to be accurately reproduced, numerical models have to include information about the structure of the tissue, and model the mechanisms of the response to mechanical loading. The work presented here focuses on the microstructural and mechanical characterization of the human liver capsule, in order to identify the important hypotheses that need to be included in a fibrous tissue constitutive model, based on microstructure. Thus, an experimental methodology has been developed to identify the mechanical behavior of this particular tissue, related with its microstructural organization. Uniaxial tensile tests, as well as bulge tests under a multiphoton confocal microscope have been performed, to observe the microstructure evolution during loading. Macroscopic strain has been assessed, and a method to measure local strain fields has been developed, to quantify the strain state of the fibrous network. The reorganization of the collagen fibers network has also been quantified. An analysis of the links between the measured macroscopic parameters and the microscopic phenomena is given. Therefore, the hypotheses that need to be included in constitutive models are highlighted, with particular consideration given to the affine transformation hypothesis which allows to link the fibers behavior to the global response of the tissue

No description available.