Le travail de thèse est consacré aux interactions spatio-, temporo- et spectro- résolue de laser avec des tissus biologiques. L'objectif de cette thèse était d'étudier l'influence des nanoparticules colloïdales embarqués dans les tissus biologiques multicouches sur leurs propriétés optiques afin de fournir plus profond et / ou plus précise de sondage. Pour ce faire, les paramètres spectroscopiques intégrales et durée de vie de fluorophore dans les environs de nanoparticules métalliques ont été analysées théoriquement et expérimentalement. L’autre partie de l'étude était de proposer de nouvelles solutions algorithmiques pour l'amélioration de la performance du processus d'estimation des valeurs des propriétés optiques de la résolution spatiale des mesures spectroscopiques. La dernière partie de la thèse est la modélisation expérimentale et théorique de la cinétique de fluorophores en présence des nanoparticules d'or colloïdales. La composante ultra-courte pico-seconde (environ 100 ps) a été résolue et a été corrélée à champ dipolaire forte des nanoparticules qui compense le dipôle de la molécule / The thesis work is devoted to spatially-, temporally- and spectrally- resolved laser and biological tissue interactions. The aim of the present thesis was to investigate the influence of colloidal nanoparticles embedded into multilayered biological tissues on their optical properties in order to provide deeper and/or more precise probing. To do so, the integral spectroscopic parameters and lifetime of fluorophore in vicinity of metal nanoparticles were analyzed theoretically and experimentally. Another part of the study was to propose new algorithmic solutions for improving the performance of the estimation process of the optical properties values from spatially resolved spectroscopic measurements. The last part of the thesis was the experimental and theoretical modelling of fluorophore’s kinetics in presence of colloidal gold nanoparticles. The ultra-short pico-second component (around 100 ps) was resolved and correlated to strong nanoparticles dipole field which is compensating the molecule’s dipole

L'objectif de la thèse était de développer une méthode de prédiction de l’origine tissulaire de particules dans des poudres issues de plantes lignocellulosiques en exploitant les propriétés d’autofluorescence des parois. L’entrenœud sous épi de la tige de maïs a été utilisé comme modèle. Le macroscope à fluorescence a été validé comme outil pour acquérir rapidement des grandes images multispectrales avec des champs de vue représentatifs de l’ordre du cm². Une approche chimiométrique par analyse d’images multispectrales a été retenue pour construire le modèle de prédiction des tissus à partir de leurs propriétés d’autofluorescence observées dans des coupes d’entrenœuds. La collection étalon comprenait 40 coupes transversales et longitudinales prélevées à différentes positions de l’entrenœud pour 10 tiges d’une même variété. Nous avons validé par analyse supervisée que la variabilité de fluorescence des tissus était prépondérante devant la variabilité inter et intra tiges. Afin de prendre en compte sans a priori toute la variabilité de fluorescence des tissus dans le maïs, le modèle de prédiction a été construit à partir d’une classification non supervisée des pixels des 40 images. Le grand nombre de pixels a conduit à développer une méthode de classification multiéchelle basée sur la méthode des K-means en utilisant des pyramides d’images. Le modèle a permis d’identifier les principaux tissus constitutifs de la tige : parenchymes lignifiés et non lignifiés, gaines de sclérenchyme des faisceaux dans la moelle et dans l’écorce, épiderme, fibres de xylème. L’étape suivante sera de tester le modèle de prédiction sur des poudres / The objective of the thesis was to develop a method for predicting the tissue origin of particles in powders of lignocellulosic plants using the autofluorescence properties of cell walls. The internode under the ear of maize stems was used as a model. Fluorescence macroscopy has been validated for acquiring large multispectral images with representative fields of view about 1 cm². A chemometric approach by multispectral image analysis was applied to build the predictive model of tissues based on their autofluorescence properties observed in stem sections. The calibration set consisted of 40 transverse and longitudinal sections taken at different positions from 10 stems of the same variety. Variability of fluorescence in tissues was found preponderant compared to inter and intra-stems variability by supervised analysis. In order to take into account all the fluorescence variability of the tissues in maize stem without any a priori, the prediction model was built from an unsupervised clustering of all the pixels from the 40 images. The large number of pixels led to the development of a multi-scale clustering method based on K-means method using image pyramids. The model made it possible to identify the main tissues of the stem: lignified and non-lignified parenchyma, sclerenchyma sheaths in vascular bundles in the pith and in the rind, epidermis, fibers of xylem. The next step will be to test the prediction model on powders.

Dans un contexte de vieillissement de la population et d’accroissement des maladies chroniques liées à l’âge comme le diabète, de nouveaux biomarqueurs de l’état de santé à long terme doivent être étudiés. Les produits de glycation avancée (AGE) sont des molécules témoins de la charge métabolique accumulée au cours du temps, dénommée "mémoire métabolique". Les AGE jouent un rôle important dans les lésions à long terme dans le diabète et dans le déclin du métabolisme global lié au vieillissement. L’accumulation cutanée des AGE peut être mesurée par autofluorescence (AF) de manière instantanée et non invasive grâce à l’AGE-READER. Les objectifs de cette thèse étaient d’évaluer la valeur de l’AF cutanée des AGE en tant que marqueur de mémoire métabolique chez des personnes âgées de la cohorte des 3-Cités et parallèlement d’évaluer la valeur pronostique de l’AF pour les complications du diabète chez des patients porteurs de diabète de type 1. Chez les personnes âgées, nous avons montré que l’AF reflétait les statuts glycémique et rénal 10 ans avant la mesure. Chez les patients atteints de diabète de type 1, l’AF était associée à la présence d’une neuropathie 4 ans plus tard. De plus, dans cette même population, nous avons décrit l’évolution de l’AF sur 4 ans de suivi. Nous avons montré que les principaux déterminants de son évolution étaient la fonction rénale et le traitement par pompe à insuline. Enfin nous avons trouvé que l’augmentation de l’AF sur 4 ans de suivi était associée à la survenue de la maladie rénale. Ces travaux soulèvent de nouvelles perspectives de recherche quant à l’intérêt de l’AF à différents âges clés de la vie en tant que biomarqueur de pathologies qui évoluent sur des dizaines d’années. / In the context of the ageing of the population and the increase of age related diseases such as diabetes, new biomarquers of the long-term health status should be considered. Advanced glycation end products (AGE) are molecules indicators of the metabolic burden over time, called “metabolic memory”. AGE play an important role in long term diabetes injuries and in the global decline of the metabolism related to ageing. Skin accumulation of AGE can be measured by autofluorescence instantly and non-invasivly with a tool called AGE-READER. The objectives of my dissertation were to evaluate the value of the skin autofluorescence (sAF) of AGE as marker of metabolic memory in elderly people from the 3-City cohort and in parallel, in patients with type 1 diabetes, evaluate the prognostic value of sAF for diabetes complications. In the elderly population, we showed that sAF reflected glycemic and renal status of 10 years before. In patients with type 1 diabetes, sAF was associated to the presence of neuropathy 4 years later. Moreover, in this same population, we described the evolution of sAF in 4 years of follow-up and we showed that the principal determinants of the evolution of sAF were kidney function and insulin pump therapy. Finally, we also found that increase of sAF in 4 years was associated with the occurrence of kidney disease. This work rises new research opportunities about the interest of sAF at differents key ages as biomarker of pathologies which evolve in several decades.

L'imagerie optique de fluorescence permet de localiser des cibles biologiques comme des tumeurs, marquées par des fluorophores. Pour des applications au diagnostic chez l'Homme où l'épaisseur des tissus atteint plusieurs centimètres, la détection parasite de l'autofluorescence naturelle des tissus compromet la détection de la fluorescence d'intérêt et son élimination est la condition sine qua non d'une localisation correcte de la tumeur. L'objet de cette thèse a été l'étude spectrale de l'auto fluorescence des tissus et la mise au point d'une méthode de séparation de spectres aveugle permettant de supprimer sa contribution des mesures. La Factorisation en Matrices Non-négatives a été privilégiée, et de nouveaux algorithmes ont été proposés et testés sur données réelles. Nous avons démontré les performances de notre méthode dans l'amélioration de la détection des marqueurs et la reconstruction de la position de la tumeur en tomographie optique diffuse de fluorescence.

L'incidence des cancers cutanés est en constante progression. Leur diagnostic précoce et leur caractérisation in vivo constituent donc un enjeu important. Notre approche multi-modalités non invasive en spectroscopie fibrée résolue spatialement vise à coupler des mesures d'AutoFluorescence (AF) et de Réflectance Diffuse (RD). L'instrumentation développée permet des mesures co-localisées en multiple excitation d'autofluorescence (7 pics d'excitation centrés à 360, 368, 390, 400, 410, 420 et 430 nm) et en réflectance diffuse (390 à 720 nm) résolues spatialement à 5 distances inter-fibres (271, 536, 834, 1076, 1341 µm). Le protocole d'étude expérimental a porté sur les stades précoces de cancers cutanés UV-induits sur un modèle pré-clinique. L'analyse histopathologique a permis de définir 4 classes (états) de référence de tissus cutanés : Sain (S), Hyperplasie Compensatoire (HC), Hyperplasie Atypique (HA) et Dysplasie (D), menant à 6 combinaisons de paires histologiques à discriminer. Suite au prétraitement des spectres bruts acquis (suppression des artefacts, moyennage, filtrage, correction spectrale), puis à l'extraction, la sélection et la réduction de jeux de caractéristiques spectroscopiques les plus discriminantes, les performances de trois algorithmes de classification supervisée ont été comparées : k-Plus Proches Voisins (k-PPV), Analyse Discriminante Linéaire (ADL) et Machine à Vecteur de Support (MVS). Les contributions des différentes modalités ont également été évaluées : mono-excitation d'AF seule, Matrices d'Excitation-Emission en AF seules (EEMs), réflectance diffuse (RD) seule, couplage EEMs – RD et couplage EEMs – RD résolue spatialement. L'efficacité finale de notre méthode diagnostique a été évaluée en termes de sensibilité (Se) et de spécificité (Sp). Les meilleures résultats obtenus sont : Se et Sp ≈ 100% pour discriminer CH vs autres ; Sp ≈ 100% et Se > 95% pour discriminer S vs AH ou D ; Sp ≈ 74% et Se ≈ 63% pour discriminer AH vs D.

Microscopic imaging in the biomedical sciences allows for detailed study of the structure and function of normal and abnormal (i.e., diseased) states of cells and tissues. The expression patterns of proteins and/or physiological parameters within these specimens can be related to disease progression and prognosis, and are often heterogeneously spread throughout the entire specimen. With conventional microscopy, a large number of individual image ‘tiles’ must be captured and subsequently combined into a mosaic of the entire specimen. This has the potential to introduce artefacts at the image seams, as well as introducing non-uniform illumination of the entire specimen. A further limitation often encountered in biomedical fluorescence microscopy is the high background due to the autofluorescence (AF) of endogenous compounds within cells and tissues. Often, AF can prevent the detection and/or accurate quantification in fluorescently- labelled tissues and, in general, can reduce the reliability of results obtained from such specimens. AF spectra are relatively broad and so can be present across a large number of image spectral channels. The intensity of AF also increases as the excitation wavelength is decreased, causing increasing amounts of autofluorescence when exciting in the blue and near-UV range of the spectrum (400 - 500 nm). This thesis reports the development of hyperspectral, fluorescence and brightfield imaging of entire, paraffin-embedded, formalin-fixed (PEFF) tissue slides using a prototype confocal scanner with a large field of view (FOV). This technology addresses the challenges of imaging large tissue sections through the use of a telecentric f-theta laser scan lens thus allowing an entire microscope slide (22x70 mm) to be imaged in a single scan at resolution equivalent to a 10x microscope objective. The development and optimization of brightfield and single-channel fluorescence imaging modes are discussed in the first half of this thesis, while the second half and appendices concentrate on the spectral properties of the system and removal of AF from PEFF tissue sections. The hyperspectral imaging mode designed for this system allows the fluorescence emission spectrum of each image pixel to be sampled at 6.7 nm/channel over a spectral range of 500-700 nm. This results in the ability to separate distinct fluorescence signatures from each other, and enables quantification even in situations where the AF completely masks the signal from the applied labels.

The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and function of islet β-cells, the mechanisms are currently unknown. This thesis examines mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2) protein expression in mouse islets. Autofluorescence microscopy showed difference in NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets showed significant decreased mitochondrial energetics when acutely stimulated with high concentrations of glucose and palmitate. This decrease in energetics correlated with increased generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state. These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high fat loading and reduces insulin during nutrient stress.

The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and function of islet β-cells, the mechanisms are currently unknown. This thesis examines mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2) protein expression in mouse islets. Autofluorescence microscopy showed difference in NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets showed significant decreased mitochondrial energetics when acutely stimulated with high concentrations of glucose and palmitate. This decrease in energetics correlated with increased generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state. These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high fat loading and reduces insulin during nutrient stress.

Microscopic imaging in the biomedical sciences allows for detailed study of the structure and function of normal and abnormal (i.e., diseased) states of cells and tissues. The expression patterns of proteins and/or physiological parameters within these specimens can be related to disease progression and prognosis, and are often heterogeneously spread throughout the entire specimen. With conventional microscopy, a large number of individual image ‘tiles’ must be captured and subsequently combined into a mosaic of the entire specimen. This has the potential to introduce artefacts at the image seams, as well as introducing non-uniform illumination of the entire specimen. A further limitation often encountered in biomedical fluorescence microscopy is the high background due to the autofluorescence (AF) of endogenous compounds within cells and tissues. Often, AF can prevent the detection and/or accurate quantification in fluorescently- labelled tissues and, in general, can reduce the reliability of results obtained from such specimens. AF spectra are relatively broad and so can be present across a large number of image spectral channels. The intensity of AF also increases as the excitation wavelength is decreased, causing increasing amounts of autofluorescence when exciting in the blue and near-UV range of the spectrum (400 - 500 nm). This thesis reports the development of hyperspectral, fluorescence and brightfield imaging of entire, paraffin-embedded, formalin-fixed (PEFF) tissue slides using a prototype confocal scanner with a large field of view (FOV). This technology addresses the challenges of imaging large tissue sections through the use of a telecentric f-theta laser scan lens thus allowing an entire microscope slide (22x70 mm) to be imaged in a single scan at resolution equivalent to a 10x microscope objective. The development and optimization of brightfield and single-channel fluorescence imaging modes are discussed in the first half of this thesis, while the second half and appendices concentrate on the spectral properties of the system and removal of AF from PEFF tissue sections. The hyperspectral imaging mode designed for this system allows the fluorescence emission spectrum of each image pixel to be sampled at 6.7 nm/channel over a spectral range of 500-700 nm. This results in the ability to separate distinct fluorescence signatures from each other, and enables quantification even in situations where the AF completely masks the signal from the applied labels.

Antibiotic resistance has emerged as one of the World's leading public health challenges. The inexorable emergence of drug resistant pathogens, combined with a steep decline in antibacterial drug discovery, has led to a major crisis. One of the most common drug resistance mechanisms involves bacteria adapting to reduce intracellular drug accumulation. To understand these resistance mechanisms, one needs quantitative information about the membrane permeability of drugs. In this Thesis, we develop a novel optofluidic permeability assay that allows us to quantify the permeability coefficient of drugs crossing lipid membranes. Lipid vesicles are used as model systems and drug molecules are tracked directly using their autofluorescence in the ultraviolet. The permeability coefficient of the drug is inferred by studying the increase in drug autofluorescence intensity within vesicles as they traverse a microfluidic network while exposed to the drug for well defined times. This provides a novel platform from which we can develop membrane models for understanding drug permeability. We incorporate the Escherichia coli outer membrane protein OmpF in vesicles and quantify its role in the transport of fluoroquinolone antibiotics. We provide direct visualisation of OmpF mediated fluoroquinolone transport. We study the pH dependence of antibiotic transport both through pure phospholipid membranes and through OmpF, and present a physical mechanism to explain the pH dependence of E. coli fluoroquinolone susceptibility. We also show the importance of lipid composition on drug permeability - changing the lipid composition of the membrane is shown to change antibiotic permeability by over an order of magnitude. Finally, we report on the discovery of a novel signalling mechanism in E. coli that relies on the transport of small drug-like molecules, and discuss the role it plays in stress response in the microbial community.