Rozprawy doktorskie na temat „Fluorescence”

La détection de molécules toxiques pour l’Homme et son environnement est d’une importance cruciale et fait partie des préoccupations majeures de la société actuelle. Les résidus de pesticides tels que l’atrazine ainsi que la mélamine font notamment partie de ces molécules dangereuses pour la santé humaine. Ces deux molécules sont principalement dosées par des techniques lourdes et coûteuses comme la spectrométrie de masse, la chromatographie ou l’électrochimie. De même, la détection des amines biogéniques représente un intérêt sociétal. Elles sont produites par des bactéries durant la décarboxylation des acides aminés dans les cellules. Leur détection permet ainsi d’évaluer la contamination microbiologique et la dégradation potentielle d’un aliment. Elles sont aujourd’hui dosées par chromatographie en phase liquide ou en en phase gaz, par électrochromatographie capillaire et par spectroscopie UV-visible. Quelques exemples de détection par fluorescence ont déjà été décrits dans la littérature mais la nécessité de développer de nouveaux récepteurs fluorescents efficaces est bien réelle.La fluorescence est une technique qui offre de multiples avantages tels que la sensibilité, la sélectivité et un faible coût. De nombreuses sondes fluorescentes capables de détecter des métaux lourds ont été développées au laboratoire PPSM. Cependant, la détection de molécules neutres par fluorescence représente un défi supplémentaire en raison de la nature plus faible de l’interaction, comparée à celle entre espèces chargées.La première étape de cette thèse a été de concevoir et de synthétiser un ensemble de sondes moléculaires fluorescentes, aussi bien pour la détection de l’atrazine, de ses produits de dégradation et des dérivés de la mélamine que pour la détection des amines biogéniques. Des fluorophores dérivés de la molécule de maléimide, de naphthalimide et de l’acide barbiturique ont ainsi été développés pour sonder les dérivés de triazine en exploitant leur système de trois liaisons hydrogène pour la reconnaissance moléculaire. De même, un calix[6]arène fluorescent a été conçu pour déceler la présence des amines biogéniques qui provoqueront une réponse fluorescente par encapsulation dans le calixarène.La deuxième étape a consisté à étudier les propriétés photophysiques de ces sondes. La sonde Naphth-AlcyneOMe possède un rendement quantique élevé, s’est révélée fortement solvatochrome. Elle est de plus sensible à la déprotonation de sa fonction imide. Des études RMN et de modélisation moléculaire ont également été menées afin de caractériser les sondes de manière plus approfondie et de comprendre plus précisément leur réactivité. La spectroscopie RMN a confirmé l’interaction par liaison hydrogène entre les sondes maléimide et naphthalimides et la molécule d’atrazine. Elle a aussi mis en évidence l’encapsulation de l’heptylamine dans le calix[6]arène. Pour sa part, la modélisation moléculaire nous a permis de mieux comprendre la photophysique de la sonde Naphth-TriazoleOMe.Enfin, la capacité des sondes à détecter les divers analytes cibles par fluorescence a été évaluée lors de la dernière étape de ce projet. La sonde TPA-BARB a présenté une forte exaltation de fluorescence en présence des dérivés de mélamine alors que le calix[6]arène-quinoléine Calix-Quino est capable de détecter les amines aliphatiques par fluorescence
The detection of molecules toxic for man and his environment is one of the major concerns of our society. Melamine and the pesticide residues such as atrazine are some of these dangerous molecules. These two molecules are usually measured with time-consuming and costly techniques like mass-spectrometry, chromatography or electrochemistry. In the same way, the detection of biogenic amines is of the greatest importance. They are produced by some bacteria during the decarboxylation of amino acids in the cells. So their detection allows to assess the microbiologic contamination and the potential degradation of a food. Today they are measured by chromatography in the liquid or gas phase, capillary electrochromatography and UV-visible spectroscopy. Some examples of detection by fluorescence have been described in scientific literature, but it is really necessary to develop some new efficient fluorescent receptors.Fluorescence is a technique which offers many advantages such as sensitivity, selectivity and a low cost. A lot of fluorescent probes able to detect heavy metals have been developed in PPSM laboratory. However the detection of neutral molecules by fluorescence represents an additional challenge as the interaction is weaker than with charged species.The first step of this thesis was to design and synthesize a set of fluorescent molecular probes designed to detect atrazine, the products of its degradation and melamine derivatives as well as biogenic amines. Some fluorophores based on maleimide, naphtalimide and barbituric acid moieties have been developed for the detection of the triazines derivatives by exploiting their three hydrogen bonds for molecular recognition. In order to detect the presence of biogenic amines, a fluorescent calix[6]arene which lead to a fluorescent change upon encapsulation in the calixarene cavity has been designed.The second step consisted in studying the photophysical properties of these probes. Naphth-AlcyneOMe probe which has a high quantum yield turned out to be highly solvatochromic. Moreover it is sensitive to the deprotonation of its imide function. NMR studies and molecular modeling were conducted in order to deepen the characteristics of the probes and better understand their reactivity. NMR spectroscopy confirmed the interaction through hydrogen bonding between maleimide and naphtalimide probes and the atrazine molecule.It highlighted the encapsulation of heptylamine in the calix[6]arene. Molecular modeling enabled us to better understand the photophysics of Naphth-TriazoleOMe probe.Finally the capacity of probes to detect the various analytes by fluorescence was assessed in our last part. TPA-BARB probe presented a high exaltation of fluorescence in presence of melamine derivatives whereas the calix[6]arène-quinoleine Calix-Quino is able to detect aliphatic amines by fluorescence

Fluorescence resonance energy transfer (FRET)-based biosensors have been designed to fluorometrically detect everything from proteolytic activity to receptor-ligand interactions and structural changes in proteins. While a wide variety of fluorophores have demonstrated effectiveness in FRET probes, several potential sensor components are particularly notable. Semiconductor quantum dots (QDs) are attractive FRET donors because they are rather bright, exhibit high quantum yields, and their nanoparticulate structure enables the attachment of multiple acceptor molecules. Fluorescent proteins (FPs) are also of particular interest for fluorescent biosensors because design elements necessary for signal transduction, probe assembly, and device delivery and localization for intracellular applications can all be genetically incorporated into the FP polypeptide. The studies described in this thesis elucidate the important parameters for concerted QD-FP FRET probe design. Experimental results clarify issues of FRET pair selection, probe assembly, and donor-acceptor distance for the multivalent systems. Various analysis approaches are compared and guidelines asserted based on the results. To demonstrate the effectiveness of the QD-FP FRET probe platform, a ratiometric pH sensor is presented. The sensor, which uses the intrinsic pH-sensitivity of the FP mOrange to modulate the FP/QD emission ratio, exhibits a 20-fold change in its ratiometric measurement over a physiologically interesting pH range, making it a prime candidate for intracellular imaging applications.

Thesis (Ph. D.)--University of Oregon, 2007.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 143-151). Also available for download via the World Wide Web; free to University of Oregon users.

Les VEGFr (Vascular Endothelium Growth Factor receptors, récepteurs des facteurs de croissance de l’endothélium vasculaire) sont des protéines responsables de l’angiogenèse, c’està-dire, la croissance des vaisseaux sanguins. De fait, ils sont impliqués dans les maladies liées à une vascularisation néfaste comme dans la croissance des tumeurs cancéreuses ou de néovascularisation rétinienne. Traiter ces vaisseaux sanguins sans atteindre les tissus sains est un enjeu qui nécessite de les imager spécifiquement et précisément, ces deux critères pouvant être atteints par imagerie de fluorescence. La spécificité en imagerie de fluorescence repose sur l’emploi d’une sonde, c’est-à-dire un fluorophore sélectif du phénomène à imager. Pour synthétiser des sondes sélectives, nous nous sommes inspirés d’un ligand connu du VEGFr : l’axitinib. La structure chimique de l’axitinib comporte un hétérocycle indazole avec deux rôles essentiels : (i) dans la fluorescence de l’axitinib, (ii) dans sa sélectivité pour le VEGFr. Cette structure a été modifiée de façon à introduire des substituants rendant la molécule plus fluorescente tout en conservant au mieux le squelette responsable de l’activité biologique. Une librairie d’une vingtaine de fluorophores a été synthétisée et étudiée pour des applications en imagerie de fluorescence
VEGFr (Vascular Endothelium Growth Factor receptors) are proteins responsible for the angiogenesis, meaning the growth of blood vessels. Consequently, they are involved in diseases due to harmful vascularization such as tumor growth or retinal neovascularization. Treating those blood vessels without harming healthy tissues is an issue. It requires specific and precise images of the blood vessels, both criteria being achievable thanks to fluorescent imaging. The specificity of fluorescent imaging relies on the use of a probe, meaning a selective fluorophore. To synthetize selective probes, we were inspired by a known ligand of the VEGFr: the axitinib. The chemical structure of the axitinib has an indazole heterocycle with two key roles: (i) in the fluorescence of the axitinib, (ii) in its selectivity for the VEGFr. Substituents were introduced to increase the overall fluorescence of the molecule while preserving the backbone responsible for the biological activity to the best of our ability. A library of about twenty fluorophores was synthetized and studied for applications in fluorescent imaging

La mesure de la fluorescence de la chlorophylle in vivo est une methode d'investigation largement utilisee pour le suivi de l'activite photosynthetique, sur algues, chlorophastes ou feuilles entieres. Sur un couvert vegetal, la mesure a distance de la duree de vie de fluorescence permet de determiner directement le rendement quantique et peut ainsi etre envisagee comme methode de teledetection de l'etat physiologique de la vegetation. Dans cette perspective, nous avons etudie les possibilites qu'offre la fluorescence resolue en temps. Nous avons etudie l'effet du quenching energetique sur les composantes du declin de fluorescence et nous interpretons ces variations en termes de taille d'antenne et de limitation de la diffusion des excitons. Nous avons examine la faisabilite des mesures de duree de vie sur un couvert vegetal et defini les techniques instrumentales a mettre en uvre. Nous avons egalement developpe une methode specifique d'analyse qui permet de calculer la valeur de la duree de vie moyenne de fluorescence a partir des signaux emis par la canopee apres une impulsion lumineuse. A cote de l'emission rouge de la chlorophylle, les feuilles des vegetaux superieurs presentent une emission de fluorescence dans la region bleue du spectre apres excitation par un rayonnement ultraviolet. Cette emission a ete remarquee pour sa sensibilite au stress, mais son origine reste encore inconnue. Nous avons etabli les spectres d'excitation et d'emission des differents composes qui en sont responsables chez l'epinard, ce qui nous permet de proposer un certain nombre d'especes chimiques pour l'origine de cette emission. Nous avons etudie la faisabilite de sa detection a grande distance, simultanement avec l'emission chlorophyllienne, par la technique des lignes de fraunhofer et nous evaluons les performances attendues dans ce cas

Surgery has been a popular method for the treatment of cancers, in particular solid tumours; but the surgical margins for cancerous tissues are often indistinct and in most cases, the poor identification of residual cancer tissues can result in re-excision. Therefore, near infrared (NIR) fluorescence-guided surgery (FGS) is being developed as a real time intra-operative imaging technique to assist surgeons by improving the accuracy and precision of the removal of tumours. However, current FDA approved fluorophores suffer from poor chemical stability, limited water-solubility, and lack selectivity toward neoplastic tissue, limiting their clinical application. These current challenges have led to the development of new and improved fluorophores capable of absorbing and emitting light at NIR wavelengths, negating autofluorescence and improving deeper light transmission. Throughout this project, a series of BODIPYs, aza-BODIPYs and bacteriochlorins were synthesised and developed for bioimaging applications. Despite many of them showing interesting fluorescence properties, the investigation suggested aza-BODIPYs were the most promising red / NIR fluorophores (λem 600-700 nm) due to their excellent photostability. Methods have been developed to incorporate functionalities suitable for bioconjugation. Different bioconjugation strategies have been explored to covalently conjugate the NIR fluorophores to a clinically relevant protein, peptide and antibody under mild conditions. The viability of aza-BODIPY conjugates against biological targets were investigated and a range of other novel targeted NIR fluorophores were successfully developed. In vitro fluorescence imaging was subsequently carried out to demonstrate the enhanced selectivity of the targeting NIR fluorophores toward overexpressed receptors on various cancer cells lines. This project has demonstrated the potential of aza-BODIPY in biological imaging and developed targeted NIR fluorophores. Further biological evaluation is progressing with the eventual aim of developing a pre-clinical model for NIR FGS in oncology.

The work presented here improves the level of quantification achievable with fluorescence microscopy by integrating novel technologies and developing new experimental and theoretical methodologies. Initial work focused on the use of fluorescence microscopy for the quantification of molecular interactions in living cells. This resulted in the development of an analysis routine for the quantification of Förster resonance energy transfer (FRET) by intensity-based sensitised acceptor emission measurements. The developed technique enabled quantification of the strength of interaction as well as the relative stoichiometry of free and bound fluorophores. The work culminated in the dynamic measurement of the cyclin – cyclin dependent kinase interaction through the course of the cell cycle. To improve the flexibility of microscopy techniques, a confocal microscopy system was designed and built which used novel fibre-based supercontinuum illumination technique and a prism-based spectrometer to provide wavelength resolved measurements. The multiparametric imaging approach which this system enabled was shown to aid in the quantification of complex systems. The remainder of this thesis considers the development of new frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) techniques. The advantages of lifetime imaging techniques were illustrated through their application to quantitative chemical analysis in microfluidic devices. Novel illumination technology was integrated into FD-FLIM systems; both in the form of inexpensive light emitting diodes and fibre-based supercontinuum technology. An in-depth theoretical analysis permitted the development of systems with much improved photon economy. Using extensions of the AB analysis technique, multicomponent lifetime data could be accurately quantified. finally, a new experimental technique was implemented, termed ø²FLIM, which enabled the rapid acquisition of alias-free fluorescence lifetime data.

Les travaux de cette thèse portent sur l'élaboration et l'étude de sondes fluorescentes construites autour d'un squelette iminophénol, permettant ainsi d'accéder à un panel de composés absorbants et émettant sur une large gamme spectrale {UV, visible, proche infrarouge) et présentant des propriétés optiques remarquables : des coefficients d'absorption importants,des rendements quantiques élevés et de grands déplacements de Stockes. Des voies synthétiques simples et efficaces ont permis la constitution d'un catalogue de fluorophores modulables sur une large gamme du spectre électromagnétique. En particulier, la fluorescence liée à un phénomène de transfert de proton intramoléculaire dans l'état excité {ESIPT) a fait l'objet d'une grande partie des travaux. Une autre partie conséquente de ce travail de thèse concerne la synthèse et l'étude de complexes de bore luminescents. Les ligands sont rigidifiés par un atome de bore trivalent de configuration tétraédrique, ce qui permet une modulation des propriétés optiques des composés ainsi qu'une augmentation du rendement quantique de fluorescence
Projects of this thesis focus on development and photophysical studies of new fluorescent probes built around an iminophenol skeleton, providing access to a panel of compounds absorbing and emitting over a broad spectral range {UV, visible, near infrared) and having excellent optical properties: significant absorption coefficients, high quantum yields and large Stokes shifts. Simple and efficient synthetic routes allowed the creation of a catalog of fluorophores emitting on a wide range of the electromagnetic spectrum. The fine tuning of the absorption and emission wavelength of the fluorescent dyes were achieved by the substitution of different electro-attracting or -donating groups. ln particular, fluorescence due to an excited state intramolecular proton transfer process {ESIPT) has been studied. Syntheses and studies of boron complexes have also been achieved. Ligands are coordinated to a trivalent boron fragment, allowing a modulation of the optical properties and leading to highly luminescent B{lll) complexes

Fluorescence correlation spectroscopy has become a standard technique for modern biophysics and single molecule spectroscopy research. Here is presented a novel widefield extension of the established single-point technique. Flow in microfluidic devices was used as a model system for microscopic motion and through widefield fluorescence correlation spectroscopy flow profiles were mapped in three dimensions. The technique presented is shown to be more tolerant to low signal strength, allowing image data with signal-to-noise values as low as 1.4 to produce accurate flow maps as well as utilizing dye-labeled single antibodies as flow tracers. With proper instrumentation flows along the axial direction can also be measured. Widefield fluorescence correlation spectroscopy has also been utilized to produce super-resolution confocal microscopic images relying on the single-molecule microsecond blinking dynamics of fluorescent silver clusters. A method for fluorescence modulation signal extraction as well as synthesis of several novel noble metal fluorophores is also presented.

Fluorimetry in the very near infrared region ca. 600-1000nm is a new approach to photochemical analysis. The advantages include greatly reduced background fluorescence signals from important sample matrices (such as blood serum), reduced scattering, and reduced probability of sample decomposition. Also, the availability of low cost, efficient, stable and robust optical components (e.g. laser diodes and light emitting diodes), solid state detectors (e.g. single silicon photodiodes and diode arrays) and fibre optics, allows the construction of an inexpensive fluorimeter. In the near infrared region, there are some very bright fluorophores that can be adapted for use as fluorescent probes, labels for immunoassay, and as ion-pair agents. The advantageous performance of most types of fluorimetric analysis now undertaken In the ultraviolet and visible region of the spectrum may therefore be extended into the longer wavelength region. Excellent limits of detection are attainable, and some near infrared fluorophores show invaluable fluorescence probe properties, such as Nile Red. The most useful of the dye groups investigated were the phenoxazines and thiazines. Reactive derivatives of these dyes show great potential as fluorescent labels for Immunoassay. These dyes have also been used as probes due to their solvatochromism and sensitivity to pH.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Ocean Engineering, 1999.
Includes bibliographical references (p. 248-251).
Fluorescence can be a powerful tool for probing biological systems. Prior measurements from Caribbean corals identified five fluorescing pigments in reef corals. In this thesis I study coral fluorescence spectra. I wanted to learn if fluorescence could be useful for large scale mapping and monitoring of the reef as a part of an effort to stop the recently reported global decline in coral reefs condition. 3D excitation I emission spectra, average wavelength locations and shape variability studies of each of the pigments is presented. I also present an in situ corrununity study of the species Montastraea cavernosa and investigate the variability of fluorescence emission among colonies of one species at one location. Coral's fluorescence emission spectrum can result from the excitation of one or more fluorescing pigments. A mathematical algorithm was developed to separate coral fluorescence spectra into individual components. The un-mixing algorithm was combined with a prediction model whose purpose was to predict the response that will be produced by any excitation light source given knowledge of the response produced by a different light source. Energy coupling between two of the pigments was discovered. An empirical coupling efficiency factor was defined and calculated to account for this energy transfer. The energy coupling between these pigments may have important consequences in future investigation of coral's evolution. A new experimental method to separate the reflectance and fluorescence spectral components of fluorescing corals was developed for in vivo and in situ data. Two experimental methods are proposed to measure and calculate a newly defined quantity, "practical fluorescence efficiency". This efficiency factor is essential for correct prediction of coral spectra under different illumination conditions. This part of my work will benefit optical models that calculate light interaction with the bottom of the ocean in shallow waters. Lastly I present a prototype Fluorescence Imaging Laser Line Scanner system and discuss its potential use as a remote sensing system for reef mapping and monitoring. Recommendations are made to better tune the system to the fluorescence characteristics of reef corals.
by Eran Fuchs.
Ph.D.

Dans de nombreux cas la structure atomique des solides ne peut etre resolue par les techniques traditionnelles de la cristallographie. Cela peut etre le cas, par exemple, pour les etudes locales d'impuretes diluees, les interfaces enterrees et plus generalement les systemes non periodiques. En 1996 une nouvelle methode structurale, l'holographie x a resolution atomique est apparue. Elle a pour origine la technique d'imagerie holographique, inventee par gabor il y a 50 ans. Dans ce travail, nous presentons d'abord le principe et le cadre theorique de l'holographie par fluorescence, fondes sur le concept de source / detecteur interne. Puis nous decrivons les developpements techniques que nous avons progressivement obtenus, afin de transposer cette methode des rayons x de laboratoire vers la source synchrotron esrf ; ceci sous le double point vue du montage experimental et de l'analyse des donnees. Des resultats intermediaires interessants sont l'imagerie des configurations des lignes de kossel et des ondes stationnaires, a partir desquelles des informations structurales - parametre de reseau, symetrie et orientation cristallines - peuvent etre deduites. Puis l'hologramme et la reconstruction atomique de monocristaux modeles tels que coo(111) sont presentes, avec - pour la premiere fois, une resolution isotrope de 0,5 a et une qualite d'image qui n'avait pas ete obtenue jusqu'a present. Enfin, la premiere application de l'holographie par fluorescence aux films epitaxiques est donnee. Des differences significatives entre des films d'alliages fept chimiquement ordonne et desordonne ont ete obtenus, ouvrant la voie a l'etude de l'ordre a courte distance directionnel dans de tels systemes, au-dela des possibilites de la spectroscopie xafs. De nouvelles perspectives sont offertes en conclusion, concernant l'holographie atomique resolue en temps, ainsi que - sur la base d'une etude preliminaire d'holographie nucleaire - le potentiel de cette technique pour le magnetisme local et la selectivite en site.

Thesis (M.S.) -- University of Maryland, College Park, 2007.
Thesis research directed by: Dept. of Electrical and Computer Engineering. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Dental caries is a bacteria-infected disease. After being illuminated by an excitation light, the by-products of oral bacteria, mainly porphyrins, can emit visible auto-fluorescence, which can be used as a marker for bacteria-infected dentin. Thus the auto-fluorescence signals have been employed as an aid for the differentiation between infected and affected dentin and monitoring caries excavation. In 2002 fluorescence-aided caries excavation (FACE) was invented as an excavation device to improve the efficiency and accuracy of caries removal.This study has investigated a recent version of FACE.

Coral reefs are highly biodiverse ecosystems and provide vital resources for the human population. Due to increasing pressure by climate change and local stressors, these ecosystems currently face a global crisis. The development of tools to monitor how coral reefs respond to environmental change is a key aspect of the conservation of their biodiversity and resources. A number of reef organisms produce fluorescent molecules, including photosynthetic pigments and green fluorescent protein (GFP)-like pigments found in Anthozoa. These pigments are responsive to environmental conditions and can be optically monitored in vivo, making them a promising tool to investigate organism- and community-level processes on coral reefs. In this thesis, the fundamental principles and technological developments necessary for the application of fluorescence as a biomarker are explored. First, the mechanisms regulating coral fluorescence are considered for two functionally and biochemically distinct groups of GFP-like proteins. In mesophotic and depth-generalist symbiotic corals, incomplete light-driven maturation of the red GFP-like protein pool is shown to determine the spectrum of fluorescence emission. A role of this mechanism in adaptation to the reduced mesophotic light spectrum is discussed. In corals from shallow water environments, enhancement of internal light fluxes due to reduced absorption by symbiont pigments during bleaching is shown to induce expression of GFPlike proteins. High-level expression of these pigments in bleached tissue is shown to promote recovery of the symbiotic algae complement after a stress event. Second, a novel approach to fluorescence imaging for coral reef surveys is presented. The method enables automatic classification of reef benthic organisms based on the intensity of fluorescent signal in different excitation and emission bands. These findings demonstrate the potential of fluorescence as an in vivo marker for physiological and ecological studies of coral reef organisms, contributing to ongoing efforts to monitor and preserve the health of these ecosystems.

L'objectiu d'aquesta tesi és l'aplicació d'indicadors nadius de fluorescència per a la quantificació ràpida dels canvis generats per la calor en diversos marcadors de dany tèrmic, com ara: hidroximetilfurfural (HMF), grups sulfhidril (-SH), àcid ascòrbic (AA ) i riboflavina (RBF) en llet desnatada mitjançant espectroscòpia de fluorescència "front-face". Es van dur a terme quatre experiments; en els dos primers es va estudiar la cinètica de desaparició i / o aparició dels marcadors indicats i, es van construir models cinètics a partir de la informació generada, així com de predicció mitjançant marcadors de fluorescència. En el tercer experiment, amb la utilització de dos fluorímetres diferents (sobretaula i fibra òptica), es van validar i recalibrar els models de predicció obtinguts anteriorment, emprant mostres generades en planta pilot en condicions industrials. En el cas dels models cinètics, aquests van ser validats i recalibrats combinant dades dels tres experiments per tal d'ampliar el nombre de mostres i obtenir models millorats. L'aparició i / o degradació dels marcadors químics estudiats es va ajustar a cinètiques de primer ordre. Es van estimar alguns paràmetres cinètics com ara l'energia d'activació, constant preexpronencial d'Arrhenius i coeficient tèrmic. Durant el procés de validació dels models matemàtics de predicció dels marcadors, indicadors estadístics com ara CV (coeficient de variació) i SEP (error estàndard de predicció) van resultar elevats; més encara amb les dades de fibra òptica, a causa de diferències de configuració òptica entre els espectròmetres de sobretaula i fibra òptica. Per això es va realitzar per una banda un recalibrat dels models de sobretaula amb les dades dels tres experiments, si bé en el cas de l'equip de fibra òptica es va procedir al calibratge emprant exclusivament dades de planta pilot generats amb el tercer experiment i consegüent validació mitjançant el mètode de validació creuada "leave one out". La fluorescència del triptòfan, compostos intermedis de Maillard i riboflavina excitada a 370 nm van ser els predictors més importants en el calibratge dels models. El millor model seleccionat tant per a l'equip de sobretaula com per al de fibra òptica va ser el corresponent a la predicció de l'àcid ascòrbic amb CV <12% mentre que els models amb major variabilitat d'error van ser els del hidroximetilfurfural amb CV <45%. En un quart experiment es va desenvolupar un mètode de quantificació ràpida de riboflavina mitjançant la mesura de fluorescència "front-face". Els valors obtinguts amb el model desenvolupat per quantificar la riboflavina en llets comercials no van ser significativament diferents dels obtinguts amb els mètodes convencionals (HPLC). La fluorescència "front-face" és un mètode ràpid, senzill i sense manipulació de mostra que permet calibrar models de predicció dels marcadors de dany tèrmic en llet. L'aplicació en línia dels models desenvolupats presenta potencial per a la millora del control dels tractaments tèrmics en llet, si bé els resultats obtinguts fins al moment indiquen que la implementació dels mateixos requeriria una etapa prèvia de calibratge en planta.
El objetivo de esta tesis es la aplicación de indicadores nativos de fluorescencia para la cuantificación rápida de los cambios generados por el calor en varios marcadores de daño térmico, tales como: hidroximetilfurfural (HMF), grupos sulfhidrilo (-SH), ácido ascórbico (AA) y riboflavina (Rbf) en leche desnatada mediante espectroscopía de fluorescencia “front-face”. Se llevaron a cabo cuatro experimentos: en los dos primeros se estudió la cinética de desaparición y/o aparición de los marcadores indicados y, a partir de la información generada, se construyeron modelos cinéticos, así como de predicción mediante marcadores de fluorescencia. En el tercer experimento, con la utilización de dos fluorímetros diferentes (sobremesa y fibra óptica), se validaron y recalibraron los modelos de predicción obtenidos anteriormente, empleando muestras generadas en planta piloto en condiciones industriales. En el caso de los modelos cinéticos, éstos fueron validados y recalibrados combinando datos de los tres experimentos a fin de ampliar el número de muestras y obtener modelos mejorados. La aparición y/o degradación de los marcadores químicos estudiados se ajustó a cinéticas de primer orden. Se estimaron algunos parámetros cinéticos tales como la energía de activación, la constante preexponencial de Arrhenius y el coeficiente térmico. Durante el proceso de validación de los modelos matemáticos de predicción de los marcadores, los indicadores estadísticos tales como CV (coeficiente de variación) y SEP (error estándar de predicción) resultaron elevados; más aún con los datos de fibra óptica, debido a diferencias de configuración óptica entre los espectrómetros de sobremesa y de fibra óptica. Por ello se realizó por una parte un recalibrado de los modelos de sobremesa con los datos de los tres experimentos, si bien en el caso del equipo de fibra óptica se procedió a la calibración empleando exclusivamente datos de planta piloto generados con el tercer experimento y consiguiente validación mediante el método de validación cruzada “leave one out”. La fluorescencia del triptófano, los compuestos intermedios de Maillard y la riboflavina excitada a 370 fueron los predictores más importantes en la calibración de los modelos. El mejor modelo seleccionado tanto para el equipo de sobremesa como para el de fibra óptica fue el correspondiente a la predicción del ácido ascórbico con CV<12% mientras que los modelos con mayor variabilidad de error fueron los del hidroximetilfurfural con CV<45%. En un cuarto experimento se desarrolló un método de cuantificación rápida de riboflavina mediante la medida de fluorescencia “front-face”. Los valores obtenidos con el modelo desarrollado para cuantificar la riboflavina en leches comerciales no fueron significativamente diferentes de los obtenidos con los métodos convencionales (HPLC). La fluorescencia “front-face” es un método rápido, sencillo y sin manipulación de muestra que permite calibrar modelos de predicción de los marcadores de daño térmico en leche. La aplicación en línea de los modelos desarrollados presenta un gran potencial para la mejora del control de los tratamientos térmicos en leche, si bien los resultados obtenidos hasta el momento indican que la implementación de los mismos requeriría una etapa previa de calibración en planta.
The objective of this thesis is the application of native indicators of fluorescence for the rapid quantification of heat-induced changes in several thermal damage markers, such as hydroxymethylfurfural (HMF), sulfhydryl groups(-SH), ascorbic acid (AA) and riboflavin (Rbf) in skim milk by front-face fluorescence spectroscopy. Four experiments were carried out: in the first two, the kinetics of disappearance and / or appearance of the indicated markers were studied and kinetic models were constructed from the information generated as well as prediction models by fluorescence markers. In the third experiment, with the use of two different fluorimeters (benchtop and optic fiber), the prediction models previously obtained were validated and recalibrated using samples generated in a pilot plant under industrial conditions. In the case of kinetic models, these were validated and recalibrated by combining data from the three experiments in order to increase the number of samples and obtain improved models. The appearance and / or degradation of the chemical markers studied was adjusted to first order kinetic equations. Some kinetic parameters such as activation energy, Arrhenius pre-exponential constant and thermal coefficient were estimated. During the validation process of the markers prediction models, statistical indicators such as CV (coefficient of variation) and SEP (standard error of prediction) were high; even higher with optic fiber data, due to differences in optical configuration between benchtop and optic fiber spectrometers. For this reason, a recalibration of the benchtop models with the data of the three experiments was carried out, although in the case of optic fiber equipment, calibration was carried out exclusively using the pilot plant data generated in the third experiment and validation using the leave one out cross validation method. The fluorescence of tryptophan, intermediates compounds of Maillard reaction and riboflavin excited at 370 nm were the most important predictors in the calibration of the models. The best model selected for both the benchtop and optic fiber equipment was the prediction of ascorbic acid with CV <12%, while the models with the highest error variability were for hydroxymethylfurfural with CV <45%. In a fourth experiment, a rapid quantification method of riboflavin was developed by the measurement of front-face fluorescence. The values obtained with the model developed to quantify riboflavin in commercial milks were not significantly different from those obtained with conventional methods (HPLC). Front-face fluorescence is a fast, simple method. Without the need of sample manipulation, allows calibration of thermal damage predictive models. The inline application of the developed models presents potential for the improvement of the control of thermal treatments in milk, although the results obtained so far indicate that the implementation of the models would require a previous stage of calibration in plant.

Le stockage optique de l’information et l’imagerie à super-résolution sont des champs d’application dont les besoins en matériaux et en molécules photocommutables sont grandissants. Une des approches consiste à associer au sein d’une même structure moléculaire des photochromes et des fluorophores au sein de laquelle des transferts d’énergie résonants sont possibles. La combinaison des propriétés photophysiques des deux types d’entités conduit à la photocommutation de fluorescence recherchée. Pour concevoir de tels systèmes, nous avons basé notre approche sur le concept de click chemistry qui permet d’avoir une grande flexibilité du point de vue synthétique. Ainsi, en utilisant des plateformes moléculaires comme les dérivés de sucres et la β-cyclodextrine, nous avons synthétisé de nombreuses architectures multichromophoriques. En variant le ratio entre le nombre de photochromes (DAE) et de fluorophores (DCM) au sein d’une même molécule, nous avons pu progresser dans la compréhension des relations entre les structures et les propriétés photophysiques de ces systèmes, impliquant des transferts d’énergie multiples entre les différentes entités. Cette démarche nous a permis, d’une part, d’appréhender les effets d’extinction non-linéaire de fluorescence, et d’autre part, de découvrir l’effet d’hystérèse photocontrôlable résultant de la compétition entre les transferts d’énergie et les réactions photochromes
The fields of optical data storage and super-resolution imaging are in expansion and attract an increasing demand on photoswitchable materials and molecules. One approach consists in associating photochromic and fluorophores units in the same molecular structure, allowing resonant energy transfer processes. The combination of the photophysical properties of the two units leads to the expected fluorescence photoswitching. To design such systems, we based our approach on the click chemistry concept which offers a great flexibility in terms of synthesis pathways. Thus, using molecular platforms such as sugar derivatives and β-cyclodextrin, we have synthesized many multichromophoric architectures. By varying the ratio between photochromic (DAE) and fluorophore (DCM) units in the same molecule, we improved our comprehension of the structure-properties relationships, involving multiple energy transfers between the different entities. This allowed us, first, to understand the effects of non-linear fluorescence quenching, and secondly, to discover the light-controlled hysteresis effect resulting from the competition between energy transfers and photochromic reactions

Les maladies neurodégénératives comme la maladie d'Alzheimer, la maladie de Parkinson ou celle d'Huntington sont liées à un déséquilibre des neurotransmetteurs. Néanmoins, les sources de ces troubles de communication neuronale ne sont pas bien compris à ce jour, en partie à cause du manque d'outils permettant de suivre en temps réel et dans l'espace les neurotransmetteurs, dans les systèmes biologiques. Ainsi, il est extrêmement important de développer des outils tels que des sondes supramoléculaires fluorescentes pour l'imagerie des neurotransmetteurs.Dans une étude précédente réalisée dans notre groupe, des sondes fluorescentes basées sur un squelette cyclotriveratrylene, capables de reconnaître la choline ou l'acétylcholine (ACh) en solution aqueuse tampon (pH 7.4), ont été synthétisées. Cependant, ces molécules ne respectent pas tous les critères nécessaires à l'imagerie des espèces dans des conditions biologiques. En effet, leurs constantes de complexation sont insuffisantes. Pour améliorer l'affinité de liaison entre les sondes et les neurotransmetteurs, nous avons tourné notre attention vers des capsules construites à partir du noyau de cyclotriveratrylene, à savoir les hémicryptophanes (HC). Les hémicryptophanes sont connus pour reconnaître les invités ammoniums grâce à leur cavité préorganisée. De nouveaux hemicryptophanes fluorescents ont été synthétisés. La voie synthétique que nous avons développée nous permet de modifier facilement le motif fluorescent de l'HC. Certains d'entre eux montrent de bonnes affinités pour différents neurotransmetteurs comme l'acétylcholine, dopamine, sérotonine, ainsi qu'une bonne sensibilité et sont solubles dans la solution aqueuse tampon. Leur synthèse ainsi que leurs propriétés de fluorescence et de reconnaissance seront présentées
Neurodegenerative diseases like Alzheimer, Parkinson or Huntington are related to an imbalance of neurotransmitters. Nevertheless, the sources of this neuronal communication disorder are not well-understood to date, partly because of the lack of tools allowing real-time and real-space monitoring of neurotransmitters, in biological systems. Thus, it is extremely important to develop tools such as fluorescent supramolecular probes for the imaging of neurotransmitters.In a previous study carried out in our groups, fluorescent probes based on a cyclotriveratrylene skeleton, able to recognize either choline or acetylcholine (ACh) in buffer aqueous solution (pH 7.4), have been synthesized. However, these molecules do not respect all the criteria needed for the imaging of species in biological conditions. Indeed, their complexation constants are insufficient. To improve the binding affinity between probes and neurotransmitters we turned our attention to capsules built from cyclotriveratrylene core, namely hemicryptophanes (HC). HC are known to bind ammoniums guests thanks to their preorganized cavity. New fluorescent hemicryptophanes were synthesized. The synthetic pathway we developed allow us to easily modify the HC’s fluorescent moiety. Some of them show good affinities for different neurotransmitters like acetylcholine, dopamine, serotonin, good sensitivities and are soluble in buffer aqueous solution. Their synthesis as well as their fluorescence and recognition properties will be presented

Les molécules fluorescentes multiréactives peuvent ajuster leurs propriétés de fluorescence en réponse à des stimuli externes tels que des changements de température, de pression et d'environnement chimique. Cette capacité d'adaptation pourrait orienter efficacement le développement des technologies de capteurs, d'affichage et d'imagerie, offrant ainsi de nombreuses applications. Le mécanofluorochromisme (MFC) est une propriété fascinante des systèmes multiréactifs, où les matériaux subissent des changements fluorescents sous l'effet de contraintes mécaniques telles que la compression, le cisaillement et la friction. Les complexes difluorure de bore à ligand dicétone (DFB) attirent l'attention pour leurs caractéristiques photophysiques uniques, présentant non seulement un MFC mais aussi un polymorphisme et une fluorescence intense à l'état solide et en solution. Ce travail vise à explorer des méthodologies innovantes pour concevoir et synthétiser des matériaux DFB multi-réactifs et leurs composés précurseurs. L'accent est mis sur l'étude des caractéristiques photophysiques de ces matériaux et de leur réactivité à divers stimuli. En outre, les mécanismes sous-jacents du MFC sont étudiés. Tout d'abord, un nouveau DFB multiréactif et multicolore est discuté. La synthèse de l'amino-méthoxy-DFB (DFB-NH₂), impliquant l'introduction d'une amine primaire dans le cycle phényle par réarrangement de Curtius, a été entreprise. Grâce au groupe NH₂, la molécule présente un transfert de charge intramoléculaire (ICT) en solution et à l'état cristallin. Une structure de type quinoïde et une structure typique de dimère de type H tête-queue sont observées à l'état cristallin. Le cristal à faible émission rouge foncé présente une émission décalée vers le bleu après un frottement mécanique, ce qui constitue un comportement MFC original. Le composé déposé sur une feuille de papier présente également un MFC significatif. En outre, une réactivité acide/base caractéristique est observée en solution, dans les films polymères dopés et les échantillons de poudre. Deuxièmement, le système multiréactif est inséré dans une molécule symétrique en C₃. Un nouveau composé β-dicétone C₃-symétrique, BTA-D3, et son homologue monomère, D, ont été synthétisés avec succès. Notamment, l'émission induite par l'agrégation (AIE) est observée dans BTA-D3 contrairement à D. En outre, BTA-D3 présente des caractéristiques de fluorescence dépendantes du polymorphe, formant des fibres 1D avec une émission jaune dans le système THF/eau, tout en formant des feuilles 2D avec une émission bleue. En outre, les propriétés de transfert d'énergie intramoléculaire sont démontrées par BTA-D3, ce qui le distingue de D. Troisièmement, la migration d'énergie dans le gel, la formation d'assemblages, les effets mécaniques et la complexation du bore du BTA-D3 ont été explorés. Ces expériences ont permis de caractériser les propriétés de gélification et la fluorescence, révélant leur dépendance à la morphologie moléculaire. L'analyse de l'anisotropie dans le gel donne un aperçu de la migration de l'énergie à l'intérieur des molécules et entre elles, mettant en évidence des structures cruciales pour un auto-assemblage efficace. La structure unique contribue à diverses propriétés stimuli-réceptives, telles que l'induction chirale par des solvants chiraux et le MFC. Notamment, la boration de BTA-D3 donne une molécule très luminescente avec un déplacement marqué de la fluorescente vers le bleu par MFC. Ces résultats contribuent à une meilleure compréhension des molécules avec une symétrie C₃ et donnent un aperçu des stratégies de contrôle de l'assemblage moléculaire afin d'obtenir diverses colorations de fluorescence dans les matériaux moléculaires. L'ensemble de la thèse vise à fournir des lignes directrices pratiques et des idées pour le développement de nouveaux matériaux luminescents, contribuant ainsi à des avancées dans le domaine avec des applications potentielles
Multi-responsive fluorescent molecules can adjust their fluorescence properties in response to external stimuli such as changes in temperature, pressure, and chemical environment. This adaptability could efficiently direct the development of sensors, displays, and imaging technologies, providing various applications in the future. Mechanofluorochromism (MFC) is a fascinating property in multi-responsive systems, where materials undergo fluorescent changes under mechanical stress like compression, shear force, and friction. Difluoroboron β-diketonate (DFB) compounds draw attention for unique photophysical traits, featuring not only MFC but also polymorphism and intense fluorescence in both solid and solution. This research aims to explore innovative methodologies for designing and synthesizing multi-responsive DFB materials and their precursor compounds. Emphasis is placed on investigating these materials' photophysical characteristics and responsiveness to diverse stimuli. Additionally, the underlying mechanisms of MFC are studied. To achieve these objectives, a comprehensive approach is employed, integrating fluorescence spectroscopy, various microscopic techniques, anisotropic experiments, theoretical calculations, and other relevant methodologies.In the first topic of this thesis, a novel multi-responsive and multicolor DFB is discussed. The synthesis of amino-methoxy-DFB (DFB-NH₂), involving the introduction of a primary amine into the phenyl ring through Curtius rearrangement, was undertaken. Thanks to the NH₂ group, the molecule exhibits intramolecular charge transfer (ICT) in solution and in the crystalline phase. A quinoid-like structure and a typical head-to-tail H-type dimer structure are observed in the crystal state. The single crystal with dark-red weak emission demonstrates a blue-shifted emission after mechanical smearing, which constitutes an original MFC behavior. The drop-casted sample on a paper sheet also demonstrates significant MFC. Additionally, characteristic acid-/base-responsivity is observed in the solution phase, polymer-dispersed films, and powder samples.In the second topic of this thesis, the multi-responsive system is delved into a C₃-symmetrical molecule. A novel C₃-symmetrical β-diketone compound, BTA-D3, and its monomeric counterpart, D, are successfully synthesized. Notably, Aggregation-induced emission (AIE) is observed in BTA-D3 contrary to D. Additionally, BTA-D3 displays polymorph-dependent fluorescence characteristics, forming 1D fibers with yellow emission in the THF/water system, while forming 2D sheets with blue emission. In addition, intramolecular energy transfer properties are demonstrated by BTA-D3, distinguishing it from D.In the third topic of this thesis, energy migration in gel, assembly formations, mechanical effects, and boron complexation of BTA-D3 were explored. Through these experiments, the gelation properties and fluorescence are characterized, revealing their dependence on molecular morphology. Anisotropy analysis in gel offers insights into energy migration within and between molecules, highlighting crucial structures for efficient self-assembly. The unique structure contributes to diverse stimuli-responsive properties, such as chiral induction by chiral solvents and MFC. Notably, boronation of BTA-D3 results in a highly luminescent molecule with a distinctive blue-shift in MFC. These findings contribute to an enriched comprehension of C₃-symmetrical molecules and offer insights into strategies for controlling molecular alignment to achieve diverse fluorescence coloration in molecular materials. The whole thesis seeks to provide practical guidelines and insights for developing new luminescent materials, contributing to advancements in the field with potential applications

Les protéines fluorescentes sont très largement utilisées dans les études de biologie moléculaire depuis maintenant une vingtaine d’année. Pour autant, l’origine de leurs propriétés photophysiques n’est pas totalement élucidée. Dans cette thèse, nous avons essayé d’améliorer la compréhension de la photophysique de deux protéines fluorescentes particulières : Padron et EosFP.Dans la protéine Padron, nous avons étudié l’isomérisation du chromophore et cherché à déterminer si la protonation et l’isomérisation sont simultanées ou successives. Pendant l’isomérisation, le donneur de proton potentiel est le résidu Tyr159. Nous avons d’abord montré que dans le vide, le transfert de proton est peu probable quelle que soit la géométrie du chromophore. Dans la protéine (où l’effet de l’environnement n’est pas négligeable) nous avons mis en évidence par dynamique moléculaire que, durant l’isomérisation, le transfert de proton n’est presque jamais favorable et reste donc un marginal.Par ailleurs, ces mêmes dynamiques ont montré que, à la fin de l’isomérisation, il apparaît de nombreux chemins de molécules d’eau reliant le chromophore au solvant et pouvant permettre un transfert de proton. On conclut doncque l’isomérisation et la protonation ne sont pas simultanées mais successives.Dans le cas de la protéine EosFP, nous avons analysé l’effet d’une molécule d’eau présente dans une partie des structures cristallines. Les dynamiques avec le chromophore à l’état fondamental ont montré que cette molécule ne joue pas de rôle, que ce soit sur le réseau de liaison hydrogène ou sur le spectre d’absorption. Par contre, à l’état excité, les dynamiques ont montré que l’extinction de fluorescence est beaucoup plus rapide sans la molécule d’eau qu’en sa présence.Par ailleurs, ces dynamiques ont mis en évidence que la protéine bloque souvent le chromophore dans des géométries où il ne peut pas retourner à l’état fondamental ni par fluorescence, ni par conversion interne. Ces géométries « noires» jouent un rôle important dans la photophysique.Pour tenir compte de ces géométries, nous avons calculé le rendement quantique et le temps de vie de fluorescence par intégration directe le long des trajectoires et par cinétique chimique. Dans les deux cas, nous avons obtenu un accord qualitatif avec l’expérience
Fluorescent proteins are widely used in biology studies since 20 years. Yet, the origin of their photophysical properties aren’t totally explained. Here, we try to improve the understanding of two particular fluorescent proteins: Padron and EosFP.In the protein Padron, we work on the isomerization of chromophore and try to determine whether isomerization and protonation are simultaneous or successive processes. During the isomerization, the potential donor is Tyr159.First, we show that, in vacuum, the proton transfer is quite unlikely whatever the chromophore geometry.In the protein (where the environment effect isn’t negligible) we evidence with molecular dynamics that, during isomerization, proton transfer stays marginal.In addition, these dynamics shown the appearance, at the end of isomerization, of a lot of water molecules channel between the chromophore and the solvent allowing a proton transfer. We conclude that isomerization and protonation are successive processes.In the case of the protein EosFP, we first analyze the effect of a water molecule which is found only in some of the crystallographic structures.Molecular dynamics of the protein with the chromophore in the ground state show that the water molecule doesn’t play any role neither in the hydrogen bond network nor in the absorption spectra.On the contrary, in the excited state, dynamics without this water show a significant faster decay of fluorescence that those with the molecule.In addition, those dynamics have demonstrate that during long period, the protein retains the chromophore in geometries in which it is unable to convert to the ground state, neither by fluorescence nor by internal conversion. Those “dark” geometries play a crucial role in the photophysics.To take them into account, we calculate the quantum yield and the fluorescence lifetime by direct integration along trajectories and by a kinetic scheme. We obtain a good qualitative agreement with the two methods

L’encapsulation dans des nanomatériaux de polymères de colorants ioniques à l’aide de contre-ions hydrophobes volumineux apparaît être une méthode très efficace pour générer des nanoparticules (NPs) fluorescentes ultra-brillantes pour la bioimagerie. Nous avons d’abord étendu cette approche par contre-ions aux colorants cyanine opérant dans la gamme du bleu au proche infra-rouge. A partir de NPs chargés en cyanines, une methode de code-barre multicolore pour le traçage cellulaire à long terme a été développé. Ensuite, le rôle des contre-ions hydrophobes volumineux dans l’auto-assemblage des colorants cationiques à l’intérieur des NPs de polymères a été étudié en testant une large collection d’anions. Nous avons montré qu’une forte hydrophobicité du contre-ion augmente l’encapsulation du colorant, régule son clustering et empêche l’agrégation de nanoparticules, alors qu’une grande taille empêche l’auto-inhibition de fluorescence. Enfin, nous avons introduit les contre-ions à base d’aluminates et de barbiturates, qui sur-performent les tetraphénylborates fluorés. Ce travail procure une base solide au concept d’émission et d’encapsulation augmentées par contre-ions pour la préparation de NPs chargés en colorants fluorescents
Encapsulation of ionic dyes with help of bulky hydrophobic counterions into polymer nanomaterials emerged as powerful method for generating ultrabright fluorescent nanoparticles (NPs) for bioimaging. Here, this counterion-based approach is extended to cyanine dyes, operating from blue to near-infrared range. Based on cyanine-loaded NPs, a multicolour cell barcoding method for long-term cell tracking is developed. Second, the role of bulky hydrophobic counterion in self-assembly of cationic dyes inside polymeric NPs is studied by testing a large library of anions. We show that high hydrophobicity of a counterion enhances dye encapsulation, prevents particle aggregation and tunes dye clustering, while large size prevents dyes from self-quenching. Third, counterions based on aluminates and barbiturates are shown to outperform fluorinated tetraphenylborates. This work provides a solid basis for counterion-enhanced encapsulation and emission concept in preparation of dye-loaded fluorescent NPs

Le cadre général de cette thèse est une étude théorique par chimie quantique et dynamique moléculaire de la relation entre la structure et la fluorescence des protéines fluorescentes, en particulier, de la protéine fluorescente jaune (YFP). Dans cette protéine l'énergie de transition électronique est réduite par rapport à celle de la protéine fluorescente verte (GFP) en raison de l'empilement π entre le chromophore (la partie qui peut absorber et émetre de la lumiere visible au cœur de la protéine) et une tyrosine. Cet effet constitue la base de son utilité au laboratoire (transfert d'énergie par résonance «FRET» avec d’autres protéines). Ce travail comporte deux parties. D’une part, nous avons cherché à déterminer si un champ de force classique (ff99 de la suite AMBER) permet de représenter l’effet de π -stacking sur la dynamique à l’état excité. Pour cela nous avons effectué une série de calculs CASPT2 sur une grille de points. La conclusion est que la différence entre les surfaces d’énergie d’interaction résultant du champ de force et des calculs de chimie quantique CASPT2 ne semblent pas déterminante pour les propriétés de fluorescences.D’autre part, nous avons utilisé un modèle développé dans le groupe ThéoSim pour décrire le déclin à partir d’une série de dynamique (300ns) utilisant un champ de force classique. Cette méthode conduit à déterminer des paramètres en principe transférables d’une protéine fluorescente à une autre. Nous avons comparé la GFP et l’YFP. Cette approche ouvre la voie à une méthode rapide pour des propriétés de fluorescences pour de nouvelles protéines fluorescentes. Une prochaine étape serait d'améliorée la description du déclin radiatif utilisée dans ce modèle
The general framework of this PhD is a theoretical study by quantum chemistry and molecular dynamics of the relationship between the structure and the fluorescence properties of fluorescent proteins, particulary, of the yellow fluorescent protein (YFP). In this protein, the electron transition energy is reduced with respect to that of the green fluorescent protein (GFP) as a result of a π stacking between the chromophore (the part that absorbs and emits visible light in the protein) and a tyrosine . This effect is the basis of the usefulness of YFP in the laboratory (resonance energy transfer "FRET" with other proteins).This study has two parts. First, we have tried to determine if a classical force field (ff99 of the AMBER suite) can represent the effect of π stacking on the dynamics in the excited state. For this goal, we performed a series of CASPT2 calculations on a grid of points. The conclusion is that the difference between the interaction energy surfaces resulting from the force field and the CASPT2 calculations does not seem decisive for the fluorescence properties. Second, we used a model developed in the ThéoSim group to extract the fluorescence decay time from a series of dynamics (300ns) using a classical force field. This method leads to the determination of parameters in principle transferable across fluorescent protein. We compared GFP and YFP. This approach opens the way to a fast method for determining fluorescence properties for new fluorescent proteins. A next step would be to improve the description of radiative decay used in this model

Le travail présenté dans cette thèse est une contribution pour progresser vers des mesures thermiques plus quantitatives. Il s'agit de mesurer la température par la technique RIF de l'émission verte. Les travaux réalisés dans ce mémoire s'articulent en trois étapes. Au départ nous avons mesuré la température d'échauffement d'un cristal massif Sr0.3Cd0.7F2 codopés Er3+/Yb3+ d'épaisseur 0.3 mm. L'échauffement induit par l'excitation des ions Yb3+ à 974.4 nm a été mesurée à une distance (d) au bord de cristal, par l'émission verte des ions Er3+ excité par le laser rouge (652 nm) au bord du cristal. La seconde étape a eu pour but la mesure de la température d'échauffement du même cristal précédent, mais en dimension microscopique. Ces microparticules fluorescentes ont été fixées à l'extrémité d'une sonde thermique de Wollaston. L'échauffement des microparticules se fait par une excitation laser rouge à 652 nm ou par effet Joule en parcourant un courant électrique dans la sonde thermorésistive. La troisième étape a eu pour principal objectif la mesure de la température à l'échelle micrométrique en utilisant un microscope à force atomique (AFM) sur lequel est montée une sonde thermorésistive munie à son extrémité d'une microparticule fluorescente de Sr0.3Cd0.7F2 codopée Er3+/Yb3+ de 15 µm utilisée comme capteur de température. La technique est basée sur la variation de l'intensité de la fluorescence de la microparticule en contact avec une surface chaude. Cette nouvelle technique nous a permis d'obtenir une image cartographique de la température d'un microsystème, composé de lignes chauffantes submicroniques, chauffé par effet Joule
The work presented in this thesis is a contribution to progress towards more quantitative thermal measurements. This is to measure the temperature by RIF technique green emission. The work in this thesis is divided into three stages. Initially we measured the temperature rise of a massive crystal Sr0.3Cd0.7F2 codoped Er3 + / Yb3 + 0.3 mm thick. The heat induced by the excitation of Yb3 + ions to 974.4 nm was measured at a distance (d) at the edge of crystal, the green emission of the Er3 + ions excited by red laser (652 nm) at the edge of the crystal.The second step was designed to measure the temperature of the heating of the same previous crystal, but in microscopic dimensions. These fluorescent microparticles were attached to the end of a thermal probe Wollaston. The temperature rise of the microparticles is by a red laser excitation at 652 nm or by Joule effect through an electric current in the probe thermorésistive.The third step was the main aim of measuring the temperature using a micrometric scale atomic force microscope (AFM) on which is mounted at its end provided with one of a fluorescent microparticle thermorésistive probe Sr0.3Cd0.7F2 codoped Er 3 + / Yb 3 + 15 microns used as a temperature sensor. The technique is based on the change in fluorescence intensity of the microparticle in contact with a hot surface. This new technique allowed us to obtain a map image of the temperature of a microsystem consisting of submicron heating lines, heated by Joule effect

Chemical sensors capable of detecting a specific atom or molecule under various conditions have been utilized in biological and environmental analyses. Fluorescence based sensors are particularly advantageous in these studies because of their high sensitivity, relative ease in handling, and low technical costs. This dissertation focuses on the detection of two analytes, H+ and hypochlorous acid, which are of interest in biology because the presence of abnormal quantities of these analytes may be indicative of disease. We have established a new platform for which sensitive changes in various regions of pH can be detected using fluorescence. The aminomethylrhodamine (AMR) scaffold is highly versatile, i.e. the pH range in which the sensor is active can be tuned by introducing different substituents on the amine moiety. Overall this systematic approach to the design of pH sensitive fluorophores has allowed for a library of compounds that are responsive over a broad range of pH (pH 3 - 10) by simply changing the substituent on the amino group. We report the synthesis and characterization of a silicon analog of rhodamine for the fluorescence based detection of hypochlorous acid. This fluorophore exhibits a 90 nm bathochromic shift in its absorption and emission, relative to its oxygen counterpart. Hypochlorous acid is a biological agent linked to certain diseases. Therefore, the longer wavelength properties of the this far-red fluorescent sensor will be of significant benefit to imaging experiments of this analyte in biological media and tissue due to its spectral proximity of the so called NIR optical window. Furthermore, the novel synthetic methodology of this sensor possesses a key intermediate, which could potentially lead to future fluorescence based sensors. The characterization of a fluorescent probe designed for the detection of hypochlorous acid (HOCl) using a silicon analog of fluorescein (SiF) was also reported. Over a range of pHs, the probe reacts with a stoichiometric amount of HOCl resulting in a mixture of two pH dependent fluorescent species, a SiF disulfide product and a SiF sulfonate product. The unique colorometric properties of the individual SiF fluorophores were utilized to perform simultaneous detection of HOCl and pH. When an excess of HOCl is present, the SiF fluorophores become chlorinated, via an intermediate halohydrin, resulting in a more pH independent and red-shifted fluorophore. Finally, an attempt was made at developing a pH responsive photodynamic therapy agent. This system was designed to target the relatively low extracellular pH found around tumors. A di-bromohydroxymethylrhodamine system was synthesized and the photophysical properties were characterized. This system absorbs weakly under acidic conditions (ca. pH 3), however was shown to be a moderate photosensitizer under acidic conditions.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O objetivo do presente estudo foi avaliar in vitro o desempenho dos métodos baseados na medição da fluorescência induzida pela luz para detecção de lesões de cárie oclusal em dentes permanentes. Para tanto, foram realizadas três pesquisas: (1) O objetivo dessa pesquisa foi avaliar a influência da subtração do valor de fluorescência zero no desempenho de dois aparelhos que induzem fluorescência na detecção de cárie. Desse modo, foram utilizados 119 molares permanentes, nos quais três áreas da superfície vestibular (cúspide, central e cervical) das porções mesial e distal foram selecionadas. Além disso, uma lesão de cárie oclusal por dente foi escolhida como “sítio teste”. Dois examinadores mensuraram tanto as áreas nas superfícies vestibulares quanto as lesões oclusais utilizando o DIAGNOdent 2095 (DD, KaVo, Alemanha) e o DIAGNOdent 2190 (DDpen). Foi observada influência da subtração do valor de fluorescência zero no desempenho do DD, com diminuição da sensibilidade. Pode-se concluir que as mensurações com o DD podem ser realizadas sem a subtração do valor de fluorescência zero. No entanto, para o DDpen esse procedimento não pode ser eliminado. (2) Foram selecionados 119 dentes com o objetivo de comparar o desempenho na detecção de lesões de cárie oclusal de três aparelhos que induzem fluorescência, exame radiográfico convencional e sistema ICDAS II. Uma lesão de cárie por dente foi escolhida como “sítio teste”. Dois examinadores realizaram duas medidas independentes utilizando o DD, DDpen, câmera VistaProof (VP, Dürr Dental, Alemanha), além de exame radiográfico convencional e exame visual através do sistema ICDAS II. Conclui-se que cada aparelho variou seu desempenho de acordo com os valores de sensibilidade e especificidade.
The aim of this in vitro study was to asses the performance of light-induced fluorescence as auxiliary to conventional methods is detecting occlusal caries lesions in permanent teeth. For this reason, three studies were carried out: (1) The aim of this study was to evaluate the influence of zero value subtraction on the performance of two laser fluorescence devices for detecting occlusal caries. 119 permanent molars were selected. Three areas (cuspal, middle and cervical) of both mesial and distal portions and one occlusal site were assessed by two examiners using the DIAGNOdent 2095 (LF, KaVo, Germany) and DIAGNOdent 2190 (LFpen) devices. It was observed an influence of the zero value subtraction in the LF performance: the sensitivity decreased. However, because of the trend, it could be concluded that the LF readings could be performed without the zero value subtraction. Despite of that, it does not imply in wrong results clinically. For the LF pen, the zero value subtraction influences the performance though and should however not be eliminated. (2) This in vitro study compared the performance of fluorescence-based methods, radiographic examination, and ICDAS II system on occlusal surfaces. 119 molars were independently assessed twice by two experienced dentists using the laser fluorescence (LF and LFpen) and fluorescence camera (FC, Dürr Dental, Germany) devices, ICDAS II system and bitewing radiographs (BW). It can be concluded that the performance of each method changes according to the sensitivity and specificity.

Les travaux réalisés ont eu comme objectif de développer les méthodes optiques pour des études en biologie avec comme point de départ la synthèse chimique de sondes fluorescentes pour deux applications visant d'une part, les interactions biopolymères - cellules et d'autre part, la thérapie photo dynamique (PDT). La première partie est consacrée à la synthèse et à l'étude de polymères bioactifs de type polysaccharide, marqués par des rotors moléculaires fluorescents. Le travail a porté sur la synthèse et l'étude photophysique des dérivés coumarine associés à des dextranes bioactifs (CMDB) et à l'héparine. Les tests biologiques confirment la potentialité de la méthode: les CMDB fluorescents conservent l'action proliférative sur les cellules endothéliales; l'internalisation des CMDB et de l'héparine fluorescents, respectivement dans des cellules endothéliales et musculaires lisses a pu être visualisée par microscopie de fluorescence. La deuxième partie est consacrée au développement de nouveaux photosensibilisants pour la PDT. La PDT est un traitement médical de lutte contre certains types de cancer basé sur l'utilisation combinée d'un photosensibilisant (souvent des dérivés de porphyrine), de l'oxygène et de la lumière. Les effets secondaires et les domaines d'absorption des composés existants, de même qu'une fonctionnalisation spécifique permettant de cibler préférentiellement les cellules tumorales, justifient les travaux qui se poursuivent dans la recherche de nouvelles molécules. Avec l'objectif de vectorisation, nous avons développé la synthèse de porphyrines portant un nombre variable de cycles glucosamine. Différentes approches méthodologiques ont été réalisées, les protocoles de synthèse sont maintenant bien maîtrisés. L'étude photophysique met en évidence la formation d'agrégats, même à faibles concentrations. Les tests biologiques, en cours, devraient orienter les modifications à apporter
Researchs are done with the aim to develop optical methods for biological studies, with the starting point, the fluorescent trac ers chemical synthesis for two biological applications, namely for biopolymer - cells interactions studies and in photodynamic therapy, respectively. In the first part are described the synthesis and the study of bioactive polysaccharides polymers, labeled with fluorescent molecular rotors. The aim was to synthesise and to study the photophysical properties of coumarin derivatives associated with bioactive dextran (carboxymethyldextranbenzylamide (CMDB)) and heparin. The results of the biological tests prove the interest of the study : fluorescent CMBD polymers keep their stimulation effect on endothelial ceUs growth ; moreover, labeling of endothelial and smooth muscle cells with derivatized fluorescent dextran and heparin respectively, has been demonstrated by fluorescence microscopy. The second part is devoted to the development of new photosensitizers for photodynamic therapy (PDT). PDT is a medical treatment against sorne cancers, based on the combined use of a photosensitizer (often porphyrin compound), oxygen and light. Nowadays, compounds used suffer from sorne drawbacks, especially secondary effects and no specificity. This explains the extend of research in this field, to discover more efficient molecules. Our main objective being the targeting, we developed sorne new porphyrins linked with a variable number of glucosamine rings. Different synthetic routes have been explored and now, the synthese protocols are well known. The photophysical properties of compounds have been studied and aggregate formation, even at low concentration has been observed. Biological tests in course, will orientated the further chemical modifications for a good selectivity

While super-resolution microscopy has brought a significant improvement in nano-scale imaging of molecular assemblies in biological media, its extension to imaging molecular orientation using fluorescence anisotropy has not yet been fully explored. Providing orientational order information at the nano-scale would be of considerable interest for the understanding of biological functions since they are intrinsically related to structural fundamental processes such as in protein clustering in cell membranes, supra-molecular polymerization or aggregation. In this thesis, we propose a super-resolution polarization-resolved microscopy technique able to image molecular orientation behaviors in static and dynamic environments, in order to report structural information at the single molecule level and at nanometric spatial scale. Using direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with polarized detection, fluorescence anisotropy images are reconstructed at a spatial resolution of a few tens of nanometers. We analyze numerically the principle of the method in combination with models for orientational order mechanisms, and provide conditions for which this information can be retrieved with high precision in biological samples based on fibrillar structures. Finally, we propose an alternative technique based on stochastic fluctuations of single molecules: polarized super-resolution optical fluctuation imaging (polar-SOFI), and compare this approach with the previous one in terms of information gained and spatial resolution. We illustrate both techniques on molecular order imaging in actin stress fibers and tubulin fibers in fixed cells, DNA fibers and insulin amyloid fibrils.
La microscopía de súper resolución ha aportado una mejora significativa en la imagen, a escala nanométrica, de ensambles moleculares en medios biológicos. Sin embargo, su extensión, mediante la utilización de la anisotropía de fluorescencia para la obtención de imágenes de orientación molecular, aún no ha sido explorada a fondo. El proporcionar información sobre la orientación molecular a escala nanométrica es de gran interés para la comprensión de las funciones biológicas. Esta información está intrínsecamente relacionada con la estructura de los ensamblajes de proteínas en las membranas celulares, la polimerización y la agregación supra molecular, entre otros. En esta tesis, proponemos una técnica de microscopía de luz polarizada de súper resolución, la cual permite visualizar el comportamiento de la orientación molecular en ambientes dinámicos y estáticos. El objetivo final es el de poder reportar información estructural a nivel de molécula única y escala espacial nanométrica. Utilizando microscopía de reconstrucción óptica estocástica (dSTORM) en combinación con detección polarizada, las imágenes de anisotropía de fluorescencia son reconstruidas con una resolución espacial de varias decenas de nanómetros. Además, el principio del método ha sido validado numéricamente en combinación con modelos de mecanismos de orientación molecular y delimitando las condiciones en que esta información se puede obtener con una precisión alta en muestras biológicas, principalmente en estructuras fibrilares. Así también, se propone una técnica alternativa basada en la emisión de fluctuaciones estocásticas de moléculas individuales: imagen de polarización con súper resolución de fluctuaciones (polar-SOFI). Además comparamos esta técnica con la anterior, en términos de la información obtenida y la resolución espacial. Finalmente, ilustramos ambas técnicas para la obtención de imágenes del orden molecular de fibras de estrés de actina y tubulina en células fijas, fibras de ADN y fibrillas de insulina amiloide.
Alors que la microscopie super-résolue a apporté une amélioration considérable en imagerie des assemblages moléculaires dans les milieux biologiques à l'échelle nanométrique, son extension à l'imagerie de l'orientation moléculaire, utilisant l'anisotropie de fluorescence, n'a pas encore été complètement explorée. Apporter une information sur l'orientation moléculaire à l'échelle nanométrique aurait un intérêt considérable pour la compréhension des functions biologiques, puisque celles-ci sont fortement reliée à la structure des assemblages de prot éines dans les membranes cellulaires, la polymérisation ou l'aggrégation supramol éculaire par exemple. Dans cette thèse, nous proposons une technique de microscopie super-résolution résolue en polarisation, capable d'imager les comportements d'orientation moléculaire dans des environnements statiques et dynamiques, dans le but de rapporter une information structurale à l'échelle de la molécule unique et à des échelles spatiales nanométriques. En utilisant la microscopie par reconstruction stochastique (dSTORM) en combinaison avec une détection polarisée, des images d'anisotropie de fluorescence sont reconstruites avec une résolution spatiale de quelques dizaines de nanomètres. Nous analysons numériquement le principe de la méthode en combinaison avec des modèles des mécanismes d'orientation moléculaire, et donnons les conditions auxquelles cette information peut être obtenue avec une grande précision dans des échantillons biologiques basés sur des structures fibrillaires. Enfin, nous proposons une technique alternative basée sur l'émission de molécules uniques en fluctuations stochastiques: l'imagerie super-résolue polarisée par fluctuations (polar-SOFI), et comparons cette approche avec la précédente en terme d'information gagnée et de résolution spatiale. Nous illustrons les deux techniques pour l'imagerie de l'ordre moléculaire dans des fibres de stress d'actin et de tubuline dans des cellules fixées, des fibres d'ADN et des fibrilles d'amyloid à base d'insuline.

Fluorescence microscopy offers the opportunity to image noninvasively biological samples in real time. However, the phenomenon of diffraction limits the resolution of conventional fluorescence microscopes to submicrometer dimensions in both the horizontal and vertical directions. This limitation can be overcome by photoswitchable fluorescent probes able to undergo reversible saturable optically linear fluorescence transitions (RESOLFT). In this study, firstly, a photoswitchable fluorescent probe based on BODIPY fluorophore and Spiropyran photochrome were designed and its photophysical and photochemical properties were investigated in organic and aqueous environments. Also, its imaging with patterned illumination was showed by trapping them in PMMA matrix. Secondly, photochromic [1,3]oxazines with different substituents as well as polymers incorporating them were synthesized and their photochemical and photophysical properties were investigated. Thirdly, to improve the switching speeds and fatigue resistance of the BODIPY-Spiropyran conjugate, the photochromic part was replaced by [1,3]oxazines and dyads incorporating BODIPY fluorophore and [1,3]oxazine photochromes were synthesized. Lastly, a new strategy was designed to switch the fluorescence of fluorophores by a modular approach. It is based on photoinduced elongation of the absorption wavelength of a fluorescent chromophore with the aid of an appended photochromic auxochrome.

Corrected steady-state fluorescence emission spectra of various mechanical pulps and celluloses were measured using an excitation wavelength of 320 nm. Regardless of its source, cellulose displayed a relatively high characteristic emission. The emission spectra of mechanical pulps were red-shifted with respect to that of cellulose, and peak shapes were influenced by the pulping process, by hydrogen peroxide bleaching and by monochromatic UV irradiation of the sheets. Changes in emission spectra due to bleaching or UV-irradiation occurred principally in the 370-440 and 500-550 nm regions, and were similar to changes in reflectance spectra reported in the literature. Fluorescence emission spectra of commercial lignins and lignin model compounds were blue-shifted with respect to mechanical pulps, even upon absorption of the fluorophores on solid substrates. Concentration and inner filter effects were evident in emission spectra of commercial lignins, at concentrations as low as 60 ppm. These results suggest that, to a first approximation, emission spectra of mechanical pulps may be interpreted as a decrease in emission of cellulose caused by light absorption by lignin; changes due to bleaching and irradiation may be attributed to changes in lignin absorption.

The processes involved in the enhancement of the fluorescence profile of dipicolinic acid (DPA) were measured and analysed, with particular emphasis on their potential application to the rapid identification of suspicious powders. The research was conducted in contribution to the anthrax detector currently under development at this department. Using the enhancement of fluorescence as a method of determining whether a sample contains spores shows great potential because DPA is not found in most powders that do not contain spores. Thus, its detection is a good indication of the presence of spores. The research presented in this thesis primarily focuses on the optimisation of measurement and enhancement techniques. Both DPA and milk powder (containing spores) were used as anthrax simulants. We found that 210 nm light was the optimal wavelength for the enhancement of DPA; however, as most light sources have a higher intensity at longer wavelengths, the use of 270 nm light may be more effective. At low concentrations, there is a linear relationship between detected fluorescence intensity and the quantity of DPA present. A linear response was also found to the enhancement-light exposure time.

Fluorescence tomography (FT) is an emerging non-invasive in vivo molecular imaging modality that aims at quantification and three-dimensional (3D) localization of fluorescent tagged inclusions, such as cancer lesions and drug molecules, buried deep in human and animal subjects. Depth-resolved 3D reconstruction of fluorescent inclusions distributed over the volume of optically turbid biological tissue using the diffuse fluorescent photons detected on the skin poses a highly ill-conditioned problem, as depth information must be extracted from boundary data. Due to this ill-posed nature of FT reconstructions, noise and errors in the data can severely impair the accuracy of the 3D reconstructions. Consequently, improvements in the signal-to-noise ratio (SNR) of the data significantly enhance the quality of the FT reconstructions. Furthermore, enhancing the SNR of the FT data can greatly contribute to the speed of FT scans. The pivotal factor in the SNR of the FT data is the power of the radiation illuminating the subject and exciting the administered fluorescent agents. In existing single-point illumination FT systems, the illumination power level is limited by the skin maximum radiation exposure levels. In this research, a multiplexed architecture governed by the Hadamard transform was conceptualized, developed, and experimentally implemented for orders-of-magnitude enhancement of the SNR and the robustness of FT reconstructions. The multiplexed FT system allows for Hadamard-coded multi-point illumination of the subject while maintaining the maximal information content of the FT data. The significant improvements offered by the multiplexed FT system were validated by numerical and experimental studies carried out using a custom-built multiplexed FT system developed exclusively in this work. The studies indicate that Hadamard multiplexing offers significantly enhanced robustness in reconstructing deep fluorescent inclusions from low-SNR FT data.

Inkjet printing technology has been developing rapidly during recent years, pressing the ink and paper manufacturers to develop a better understanding of the mechanism of fixation of inkjet dye into the substrate. The aim of the work described in this thesis was to investigate the three-dimensional distribution of inkjet dye in paper and the interaction between dye and paper using advanced fluorescence microscopy techniques, Confocal Laser Scanning Microscopy (CLSM), and Two-photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM). It has been shown that CLSM is a valuable, non-destructive, rapid technique for threedimensional imaging of printed samples and evaluation of print quality. The intrinsic fluorescence of both the inkjet dye and the paper substrate can be used to determine the spread and penetration of ink droplets in different inkjet papers. The optical sectioning capability of CLSM enables the position of the ink layer relative to the paper surface and the penetration depth of the ink to be quantified. It was observed that while in the microporous type of inkjet paper the penetration depends on the quantity of ink in the printed sample, in the swellable type of inkjet paper the penetration is almost the same for different amounts of ink. 2P-FLIM has been employed to spatially map, in three-dimensions, fluorescence lifetimes by measuring the lifetime at each pixel in the image. Fluorescent molecules in both the ink and paper were analysed. Because the fluorescence lifetime is affected by the local molecular environment, the fluorescence lifetime maps provide information on the interaction between inkjet dye and paper. Analysis of fluorescence lifetime maps reveals the interaction between dye molecules and silica or alumina particles in the paper, variations of the molecular environment within a single ink dot and that interaction between dye and paper is affected by pH.

COORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIOR
O tema da dissertação, Caracterização de Áreas Contaminadas Através de Ensaios in Situ, aponta o estado-da-arte dos sensores que estão sendo adaptados ao conepenetrômetro, permitindo a detecção e o registro do poluente no subsolo e lençol freático.Talvez como uma conseqüência da atual conscientização da sociedade para o caráter emergencial no enfrentamento dos problemas relacionados à contaminação do subsolo,tem-se exigido uma significativa demanda de serviços especializados na caracterização dos agentes e processos de contaminação e nos procedimentos de remediação. A pesquisa apresenta técnicas de ensaio in situ para a caracterização de áreas contaminadas em função do contaminante que se quer detectar. O primeiro grande grupo abrange as tecnologias utilizadas na detecção dos hidrocarbonetos, em seguida estão agrupadas as técnicas de detecção dos compostos orgânicos voláteis e, finalmente, o terceiro grande grupo enfoca as técnicas utilizadas na detecção de metais pesados.Neste trabalho ensaios foram feitos em duas etapas: com o LIDAR-PUC e com o Fluorímetro, e são apresentados seus resultados, bem como as conclusões da pesquisa e algumas sugestões para estudos futuros, destacando-se as potencialidades e limitações de cada ensaio realizado.
The subject of the research, Characterization of Contaminated Areas by in Situ Tests, shows the state-of-art of the sensors that are being adapted to the conepenetrometer system, allowing for the detection and register of the polluent in the subsurface and water table.Perhaps as a consequence of the new conscientization of the society about the emergencial character in facing problems related to subsurface contamination, it has been claimed such a significative demand forward specialized services in characterization of agents and contamination processes, besides remediation procedures.In situ sampling techniques for the characterization of contaminated areas related to the targeted polluents are presented in this study. The first group covers technologies used in the detection of hydrocarbons, furthermore there are some techniques for the detection of volatiles organic compounds and, finally, the third group focuses the techniques used in the detection of heavy metals. In this work tests were done in two parts: with the LIDAR- PUC and with the Fluorimeter, and their results are presented as well the conclusions of the research and some suggestions for future works, principally the potentialities and limitations of each test done.