La technique de spectroscopie basée sur la diffusion Raman Stokes spontanée est un procédé standard employé dans de nombreux domaines allant de la thermodynamique à la médecine, en passant par la science des matériaux. À la faveur d'un échange d'énergie inélastique, elle permet de déterminer les fréquences des vibrations moléculaires présentes dans un objet. On peut ainsi remonter à l'identification des molécules et ainsi caractériser l'objet d'étude sans utiliser de marqueur spécifique. Cette méthode est néanmoins affligée de défauts. Outre la présence d'un signal de fluorescence qui peut submerger la réponse Raman, le désavantage majeur est le long temps d'exposition que requière cette technique. Dans le cas d'étude d'échantillon biologique, cela proscris son usage pour des mesures de microspectroscopie : la cartographie spectrale d'objet microscopique. Afin de pallier ce problème, de nouvelles techniques ont été développées. C'est le cas de la spectroscopie employant la diffusion Raman anti-Stokes Cohérente (ou CARS pour Coherent Anti-Stokes Raman Scattering). Du fait de sa cohérence et de sa directivité le signal anti-Stokes affiche une intensité 10^5 to 10^6 fois plus importante que dans le cas de la diffusion Raman spontanée, ce qui permet alors d'abaisser le temps d'exposition à un niveau tolérable pour les objets biologiques lors d'une mesure de microspectroscopie. De plus, le caractère anti-Stokes du signal l'épargne de la contribution de la fluorescence. Pourtant, un défaut majeur limite encore l'utilisation de cette technique : le bruit de fond non résonant. Ce phénomène peut diminuer, voir noyer la contribution résonante qui porte l'information. Cette thèse a permis le développement de techniques CARS autorisant une réduction du bruit de fond non résonant. Pour ce faire un dispositif de spectroscopie CARS multiplex (M-CARS) en configuration copropagative a été construit. Ses capacités sont illustrées par des mesures spectrales d'échantillons minéral, végétal et biologique. À partir de ce système, il a été établi une méthode innovante permettant de discriminer le signal résonant du bruit non résonant en utilisant un champ électrique continu. Il est aussi démontré la mise en place d'un procédé qui a permis de mener la première mesure de microspectroscopie M-CARS en configuration contrapropagative sur un échantillon biologique. Cette configuration limite la collecte du signal à l'objet d'étude, empêchant ainsi l'acquisition du signal résonant et non résonant issu du solvant, principal responsable du bruit de fond non résonant lors d'une mesure CARS en configuration copropagative. / The spectroscopy technique based on spontanée Raman Stokes scattering is a standard process used in many fields spanning from thermodynamic and medicine, to materials sciences. An inelastic energy exchange permits to determinate the frequency of the molecular vibrations in an object. One can identify the molecules and thus, can characterize the object of study in a label-free way. Nevertheless, this method is afflicted with faults. Beside the presence of fluorecence that can drown the Raman answer, the main drawback is the long exposition time required. In the case of biological sample, this can prohibit the use of spontaneous Raman scattering for microspectroscopy measures: the spectral mapping of microscopic objects. To avoid this problem, new techniques have been developed. It is the case of Coherent anti-Stokes Raman scattering (CARS) spectroscopy. Due to its coherence and its directivity, the anti-Stokes signal has an intensity 105 to 106 times greater than the spontaneous Raman scattering one. The exposition time is then reduced to a tolerable level for biological objects during microspectroscopy measures. Moreover, the anti-Stokes characteristic of the signal prevents the fluorescence contribution. However, a major fault still limits the use of this technique: the nonresonant background. This phenomenon can diminish, even overwhelm the resonant contribution carrying the information. This thesis permitted the development of CARS approaches that allow the reduction of the nonresonant background. To do so, a multiplex CARS (M-CARS) spectroscopy apparatus in a forward configuration has been built. Its abilities are illustrated with spectral measures of mineral, vegetal and biological samples. Based on this system, it has been established an innovative method that can discriminate the resonant signal from the nonresonant one thanks to a static electric field. It has been also been demonstrated the development of a process that has allowed the first M-CARS microspectroscopy measure of a biological sample in a contrapropagative configuration. This setup limits the collect of the signal to the object of study, avoiding the acquisition of the resonant and resonant signals coming from the solvent, responsible for the major part of non resonant background during a CARS measure in a forward configuration.

Doctor of Philosophy / Department of Biochemistry / Lawrence C. Davis / The fate and bioavailability of organic contaminants in plants is a major ecological and human health concern. Current wet chemistry techniques that employ strong chemical treatments and extractions with volatile solvents, such as GC-MS, HPLC, and radiolabeling, although helpful, degrade plant tissue resulting in the loss of spatial distribution and the production of artifacts. Synchrotron radiation infrared microspectroscopy (SR-IMS) permits direct analysis of plant cell wall architecture at the cellular level in situ, combining spatially localized information and chemical information from the IR absorbances to produce a chemical map that can be linked to a particular morphology or functional group. This study demonstrated the use of SR-IMS to probe biopolymers such as cellulose, lignin, and proteins in the root tissue of hydroponically grown sunflower and maize plants as well as to determine the fate and effect of several organic contaminants in those root tissues. Principal components analysis (PCA), a data compression technique, was employed to reveal the major spectral variances between untreated and organic contaminant treated root tissues. Treatment with 1H-benzotriazole (BT) caused alterations to the lignin component in the root tissue of plants. The BT was found in xylem and epidermal tissue of sunflower plants but not associated with any particular tissue in maize roots. 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) altered the pectin and polysaccharide structure in both maize and sunflower. SR-IMS revealed the reduction of DNTs to their aromatic amine form in the vascular and epidermal tissues at low concentration. At high concentration, DNTs appeared to be associated with all the plant tissues in maize and sunflower. Exposure of sunflower and maize to 2,6-dichlorophenol (2,6-DCP) caused alterations to the polysaccharide and protein component of the root tissue. In some cases, phenolic compounds were observed in the epidermal tissue of maize and sunflower roots. The results of this research indicate that SR-IMS has the potential to become an important analytical tool for determining the fate and effect of organic contaminants in plants.

Over the past decades, Fourier Transform Infrared (FTIR) microspectroscopy has emerged as a potential candidate to complement Histopathology in the study and diagnosis of tissue diseases. Contrary to the histological examination, which relies on the morphological tissue alterations assessed by visual inspection of stained samples, FTIR chemical imaging is a rapid and label-free tool that provide simultaneously information about histological structures as well as the localisation and magnitude of basic molecular units that compose tissue sections (proteins, nucleic acids, lipids, and carbohydrates). Despite the many proof-of-concept studies demonstrating the effectiveness of FTIR spectroscopy in detecting biological disorders with high levels of sensitivity and specificity, translation into clinical practice has been relatively slow due to the substantial cost of infrared transparent substrates required to collect the images. Thus, the main objective of this research is to evaluate the diagnostic potential of infrared chemical images collected from samples placed on conventional histology glass slides as alternative substrates for FTIR spectroscopy. Swiss mice were submitted to a well-established chemical carcinogenesis protocol, in which cancerous and non-cancerous cutaneous lesions were obtained by varying the exposure time of the animals to carcinogenenic factors. FTIR hyperspectral images were acquired in transmission mode over the mid-infrared region from tissue specimens placed on conventional infrared substrates (calcium fluoride - CaF2) and glass slides. In the first phase of our study, spectral datasets were segmented using k-means (KMCA) and Hierarchical Cluster Analysis (HCA) as clustering algorithms to reconstruct the hyperspectral images aiming to evaluate the ability of the false-color maps in reproducing the histological structures of tissue specimens. The images were segmented by each clustering technique using several different combinations varying parameters including the substrate used to place the samples (CaF2 or conventional glass) and the methods employed to preprocess the datasets. Fingerprint (1000-1800 cm-1) and high wavenumber (3100-4000 cm-1) regions from images collected on CaF2 were separately used as input for image reconstruction and only the high wavenumber range was employed in the case of samples placed on glass. All pseudocolor maps were compared to standard histopathology in order to evaluate the quality and consistency of images after segmentation. KMCA presented slightly superior ability in correctly assigning the pixels of morphochemical maps to the histological structures of the specimen, nevertheless, our findings indicate that the choice of the substrate, input data, preprocessing methods, and sample preparation have more influence in the final results than the clustering algorithm used to reconstruct the images. In the second phase of our study, Principal Component Analysis (PCA) was employed to compare datasets from healthy group to animals exposed to chemicals for 8, 16, and 48 weeks in order to evaluate the biochemical changes induced by chemical carcinogenesis. The performance of classification in each pairwise comparison was calculated using a binary classification test based on Linear Discriminant Analysis associated to PCA (PC-LDA). The method achieved satisfactory discrimination (over 80%) comparing healthy tissue to samples that were classified as papilloma (16 weeks) and invasive squamous cell carcinoma (48 weeks) regardless of the substrate used to place the samples. Statistical measurements obtained comparing healthy skin to animals exposed to carcinogenic factors for 8 weeks (free of malignancy based on the morphological and clinical evidence) ranged from 35-78%, indicating that the ability of PC-LDA in correctly classifying spectral data from cancerous and pre-cancerous lesions vary with the stage of the disease during the tumorigenesis process. Thus, as a proof-of-concept, we demonstrate the feasibility of FTIR spectroscopy in evaluating the biological events triggered by cancer using a label-free methodology that do not rely on expensive substrates and do not disrupt the pathologist workflow. This is a major step forward towards clinical application, since the method can be used to complement the diagnostic process of cancer as a non-subjective alternative that do not require laborious and time-consuming procedures nor expensive probes as biomarkers. / Nas últimas décadas, a microespectroscopia de absorção no infravermelho por transformada de Fourier (FTIR) tem surgido como potencial ferramenta para complementar a Histopatologia no estudo e diagnóstico de doenças teciduais. Ao contrário do exame histológico, que se baseia na inspeção visual de amostras coradas visando avaliar as alterações morfológicas que as doenças ocasionam no tecido, o imageamento químico obtido pela técnica de FTIR baseia-se nas características bioquímicas da amostra sem o uso de colorações. Apesar da vasta literatura comprovando a eficácia da espectroscopia FTIR em detectar alterações biológicas causadas por doenças com altos níveis de sensibilidade e especificidade, a implementação do método na prática clínica tem sido relativamente lenta devido ao alto custo dos substratos transparentes no infravermelho que são necessários para aquisição de dados. Diante disso, o objetivo principal do presente trabalho é avaliar a capacidade diagnóstica de imagens hiperespectrais coletadas de amostras em lâminas de vidro como substratos alternativos para a espectroscopia FTIR. Camundongos Swiss foram submetidos a um protocolo de carcinogênese química, no qual lesões cutâneas cancerosas e não-cancerosas foram obtidas variando-se o tempo de exposição dos animais aos fatores carcinogênicos. Imagens hiperespectrais FTIR foram adquiridas no modo de transmissão na região do infravermelho médio a partir de amostras de tecido depositadas em substratos transparentes no infravermelho (fluoreto de cálcio - CaF2) e vidro convencional. Na primeira fase de nosso estudo, os dados espectrais foram segmentados usando as técnicas estatísticas k-means (KMCA) e Análise Hierárquica de Clusters (HCA) como algoritmos de agrupamento para reconstruir as imagens hiperespectrais com o objetivo de avaliar a capacidade dos mapas de cores falsas em reproduzir as estruturas histológicas das amostras de tecido. As imagens foram segmentadas por cada técnica de agrupamento variando-se o substrato usado para colocar as amostras (CaF2 ou vidro convencional) assim como os métodos de tratamento utilizados para pré-processamento dos dados. As regiões de impressão digital (1000-1800 cm-1) e de altos números de onda (3100-4000 cm-1) das imagens coletadas em CaF2 foram usadas separadamente como dados de entrada para a reconstrução das imagens, enquanto apenas a faixa de altos números de onda foi utilizada no caso de amostras colocadas em vidro. Ao fim do processo de segmentação os mapas de cores falsas obtidos foram comparados com a Histopatologia padrão a fim de avaliar a qualidade e consistência das imagens. Os resultados obtidos pela técnica de KMCA foram ligeiramente superiores com relação a HCA na identificação de pixels dos mapas morfo-quimicos correspondentes às estruturas histológicas da amostra. No entanto, nossos achados indicam que a escolha do substrato, dados de entrada, métodos de pré-processamento e preparação de amostras têm mais influência nos resultados finais do que o algoritmo de agrupamento usado para reconstruir as imagens. Na segunda fase do nosso estudo, a Análise de Componentes Principais (PCA) foi empregada para comparar os dados do grupo saudável aos animais expostos aos produtos carcinogênicos por 8, 16 e 48 semanas a fim de avaliar as alterações bioquímicas induzidas pela carcinogênese química. O desempenho da classificação em cada comparação pareada foi calculado usando um teste de classificação binária baseado na Análise de Discriminante Linear associada à técnica de PCA (PC-LDA). O método obteve discriminação satisfatória (acima de 80%) comparando tecido saudável com as amostras que foram classificadas como papiloma (16 semanas) e carcinoma espinocelular invasivo (48 semanas) independentemente do substrato usado para colocar as amostras. A comparação de pele saudável com animais expostos aos fatores carcinogênicos por 8 semanas (livres de malignidade de acordo com as evidências clínicas e morfológicas) apresentou figuras de performance cujos valores variaram entre 35-78%, indicando que a habilidade da técnica de PC-LDA em classificar corretamente dados espectrais de lesões cancerosas e pré-cancerosas variam com o estádio da doença durante o processo de tumorigênese. Diante disso, como uma prova de conceito, demonstramos a viabilidade da espectroscopia FTIR na avaliação dos eventos biológicos desencadeados pelo câncer usando uma metodologia que não requer colorações e substratos caros, assim como não interrompe/altera o fluxo de trabalho atual do patologista. Este é um passo importante na implementação da tecnologia no ambiente clínico, uma vez que o método pode ser usado para complementar o processo de diagnóstico do câncer como uma alternativa não-subjetiva e que não requer procedimentos trabalhosos e demorados, nem sondas caras como biomarcadores.

Les biofilms sont des communautés complexes de microorganismes, enchassées dans une matrice auto-secrétée de substances polymériques extracellulaires ou EPS. Les biofilms se forment à la surface de la plupart des matériaux, qu’ils soient de nature biologique ou non, et sont à l’origine de divers problèmes économiques et sanitaires. Les bactéries dans un biofilm, dites bactéries sessiles, présentent en effet des propriétés phénotypiques qui les distinguent de leurs homologues planctoniques, notamment par une résistance accrue aux antibiotiques et aux traitements de désinfection. D’où, la nécessité de prévenir leur formation et/ ou de leur élimination à partir de stratégies mieux adaptées à ce mode de vie en communauté. Le développement de telles stratégies passe entre autre par une meilleure connaissance des contributions physico-chimiques gouvernant les interactions de ces microorganismes avec leur environnement proche notamment lors des étapes initiales de la formation des biofilms. Deux grands objectifs ont été fixés au début de cette thèse, le premier visant à caractériser, in situ et en temps réel la formation de jeunes biofilms de deux modèles bactériens : une souche naturelle et ubiquitaire de Pseudomonas fluorescens et une souche modèle d’Escherichia coli obtenue par génie génétique pour surexprimer un seul type d’EPS. Le deuxième objectif de cette thèse, consiste à étudier leurs réponses à un stress environnemental ou chimique, notamment quand les biofilms doivent se développer dans des conditions extrêmes de pH. Pour atteindre ces objectifs, différentes techniques ont été combinées pour étudier de l’échelle moléculaire à l’échelle cellulaire le développement des biofilms. La spectroscopie Infrarouge à Transformée de Fourier en mode réflexion totale atténuée (FTIR-ATR) a été utilisée pour suivre en temps réel le développement des biofilms. Nous avons pu suivre l’évolution des empreintes spectrales IR-ATR au cours de la formation des biofilms, sous des conditions favorables ou non à leur croissance. De jeunes biofilms de 24 h ont été étudiés par microspectroscopie Raman confocale (MRC), celle ci permettant d’obtenir des informations localisées sur la composition chimique des biofilms. La structure générale des biofilms a été visualisée par la microscopie à épifluorescence. Finalement, les propriétés physico-chimiques des EPS ont été quantifiées par spectroscopie de force atomique à l’échelle de la molécule unique (SMFS pour Single Molecule Force Spectroscopy). / Biofilms are complex communities of microorganisms, embedded in an auto-produced matrix of extracellular polymeric substances or EPS. Biofilms form on the surface of most materials, whether or not they are of biological nature, and cause major economic problems as well as public health concerns. Bacteria within a biofilm also called sessile bacteria, have phenotypic characteristics that distinguish them from their planktonic counterparts, rendering them more resistant to antibiotics and to disinfection strategies. Hence, the prevention of their formation and/ or their elimination requires the use of strategies that are well suited to the sessile mode of life. The development of these strategies begins with a better understanding of the physicochemical contributions that govern the interaction between the sessile community and its environment especially during the first steps of biofilm formation. Two main objectives were defined at the beginning of this thesis, the first was to characterize, in situ, and in real time the development of nascent biofilms. Two bacterial models were studied: a natural and ubiquitous strain of Pseudomonas fluorescens and a model strain of Escherichia coli genetically modified to overexpress one type of EPS. The second objectif was to study their responses towards an environmental or chemical stress; particularly how their development would be affected under extreme conditions of pH. To gain these objectives, different techniques were combined to study from the molecular to the cellular scale the development of biofilms. Fourier Transform Infrared spectroscopy in attenuated total reflection mode was used to evaluate in real time the development of biofilms. We were able to detect changes in the IR-ATR spectral profile along biofilm formation under favorable and non favorable growth conditions. 24 h - old biofilms were characterized using confocal Raman microspectroscopy, which allowed us to gather localized information on their chemical composition. The structure of biofilms was visualized using epifluorescence microscocopy. Finally, physico-chemical properties of EPS were quantified using single molecule force spectroscopy

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No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Jandira Brandao.PDF: 3676451 bytes, checksum: f81e6261408c6c253ba5df2e649db174 (MD5) / CAPES, FAPES / Considerando o forte impacto social e financeiro causado pelas fraudes em documentos, principalmente o papel-moeda, e a diversidade de métodos empregados nas falsificações, sua crescente disseminação e sofisticação, é preciso desenvolver novas metodologias para análise de documentos que sejam sensíveis e não destrutivas, pois o material examinado precisa ser preservado na íntegra para continuidade dos processos judiciais. Assim, o objetivo deste trabalho foi desenvolver uma metodologia utilizando as técnicas de Microespectroscopia Raman e Microscopia de Força Atômica, que possibilite distinguir os documentos autênticos dos fraudados e determinar os meios empregados nas falsificações, de maneira confiável, com baixo tempo de análise e sem danos às amostras. Neste estudo foram utilizadas cédulas de R$100,00 e CNH’s autênticas e fraudadas, cédulas autênticas de dólar de diferentes valores (US $ 10,00 e US $ 20,00), e cédulas autênticas de € 5,00 de diferentes fabricantes; para cada tipo de amostra (autêntica e fraudada) foram utilizados três exemplares distintos. Todas as análises foram realizadas in situ, sem qualquer preparação das superfícies, utilizando microscópio confocal Alpha 300R WITEC do NCQP/UFES acoplado com microespectroscópio Raman e microscópio de força atômica, sendo selecionadas as mesmas regiões tanto nos documentos autênticos quanto nos fraudados. O trabalho foi dividido em duas partes. A primeira parte abordou a análise das amostras utilizando AFM, através da avaliação dos parâmetros de topografia, fase e rugosidade dos diferentes papéis utilizados na fabricação desses documentos. Os resultados obtidos por AFM permitiram diferenciar os documentos autênticos (com superfícies mais uniformes e regiões topográficas características para os elementos de segurança) dos fraudados (com superfícies mais irregulares e perfil topográfico semelhante em todas as regiões), discriminar o tipo de papel utilizado nas fraudes (propriedades físico-químicas semelhantes às do papel tipo Office), e ainda, distinguir entre cédulas autênticas produzidas por diferentes fabricantes (diferença nos valores de SSK e SKU). Na segunda parte foi abordada a análise das amostras utilizando RM, através da identificação dos pigmentos utilizados na confecção dos diversos documentos. Os resultados obtidos por RM permitiram distinguir os documentos autênticos (com predominância de bandas características para o carbon black, ftalocianina de cobre, diarileto e dióxido de titânio), dos fraudados (com predominância de fluorescência e bandas características do carbonato de cálcio utilizado no tratamento do papel comercial), além de identificar o tipo de impressão utilizada nas falsificações. A combinação das duas técnicas mostrou ser promissora para a análise forense de documentos, pois fornece resultados precisos e reprodutíveis, em pouco tempo e, principalmente, sem prejuízo ao material analisado. / Considering the strong social and financial impact caused by document forgery, especially banknote, and the diversity of methods used in the forgeries, their increasing spread and sophistication, it is necessary to develop new methods for document analysis that are sensitive and non-destructive, because the material examined must be preserved in its entirety for continuity of legal proceedings. The objective of this study was to develop a methodology using the techniques of Microspectroscopy Raman and Atomic Force Microscopy, which allows to distinguish the authentic documents of counterfeit, and to determine the means employed in the forgery, that is reliable, with low analysis time and undamaged the samples. This study used authentic and counterfeit banknotes of R$ 100.00 and CNH, authentic dollar banknotes of different values ($ 10.00 and $ 20.00), and authentic banknotes of € 5.00 from different manufacturers; for each sample type (authentic and counterfeit) were used three different copies. All analyzes were performed in situ without any surface preparation, using confocal microscope Alpha 300R WITEC of NCQP / UFES coupled with Raman microspectroscope and atomic force microscope, being selected the same regions in both the authentic and counterfeit documents. The study divided into two parts. The first part dealt with the analysis of samples using AFM, by assessing the topography parameters, phase and roughness of the different papers used in manufacturing these documents. The results obtained by AFM could differentiate the authentic documents (with smoother surfaces and topographical regions characteristics for the security features) of counterfeit (over uneven surfaces and similar topographic profile in all regions), discriminate the type of paper used for forgery (physicochemical properties similar to Office type paper), and also distinguish between authentic banknotes produced by different manufacturers (difference between SSK and SKU values). In the second part, we addressed the analysis of samples using RM, through the identification of pigments used in the preparation of various documents. The results obtained by RM possible to distinguish authentic documents (with a predominance of characteristic peaks for the carbon black, copper phthalocyanine, diarylide and titanium dioxide), of counterfeit (predominantly fluorescence and calcium carbonate bands characteristic of the commercial paper treatment), and identify the type of printing used in the forgeries. The combination of the two techniques has shown promise for the forensic analysis of documents because it provides accurate and reproducible results in a short time and, above all, without prejudice to the analyzed material.

L'imagerie a pris une importance croissante dans l'étude des processus biologiques. L'utilisation de la microscopie de fluorescence a notamment favorisé le développement de nombreuses molécules sondes pour le marquage et la visualisation de biomolécules en milieu biologique complexe. D'autre part, des techniques d'imagerie complémentaires émergent, comme la microspectroscopie infrarouge. Les complexes organométalliques ont un potentiel important comme outils pour l'imagerie biologique, car ils peuvent combiner sur un même cœur moléculaire des propriétés permettant leur détection par différents types d'imagerie. En particulier, des complexes rhénium tricarbonyl ont été utilisés comme sondes pour l'imagerie multimodale. Ces " SCoMPIs " (Single Core Multimodal Probes for the Imaging) sont luminescents et présentent des signaux infrarouge et de fluorescence X intenses et qui permettent leur détection spécifique en milieu biologique. Dans cette étude, nous nous sommes intéressés aux possibilités d'imagerie multimodale de peptides et de protéines exogènes ou endogènes. Différents SCoMPIs ont été préparés et caractérisés, permettant le marquage N-terminal de peptides, le marquage d'une protéine exogène (homéodomaine) via une réaction thiol-maléimide ou le marquage par affinité d'une protéine endogène (anhydrase carbonique). L'évolution des propriétés de luminescence de SCoMPIs en fonction de l'environnement a été étudiée. Les imageries de luminescence et infrarouge ont permis la détection des peptides en contexte cellulaire, mais pas des protéines marquées avec des SCoMPIs. En revanche, il a été possible de détecter ces faibles quantités par microspectroscopie de fluorescence X. / Bioimaging is now widely used for the study of biological processes. The expansion of fluorescence microscopy has led to the development of small chemical probes for the labelling and imaging of biomolecules in complex biological environments. Alternative imaging techniques, such as infrared microspectroscopy, are also emerging. Organometallic complexes have a great potential as tools for bioimaging, since they can combine on a single molecular core properties enabling their detection by various imaging techniques. In particular, rhenium tricarbonyl complexes have been used for multimodal imaging. These “SCoMPIs” (Single Core Multimodal Probes for the Imaging) are luminescent and exhibit intense, specific infrared and X-ray fluorescence signals, which make possible their multimodal detection in biological media. In this study, we focused on the possibility to perform the multimodal imaging of SCoMPI-labelled peptides and proteins in cells. Various SCoMPIs were prepared and characterized, that were suitable for N-terminal labelling of peptides, labelling of an exogenous protein (homeodomain) through thiol-maleimide labelling, or the affinity-guided labelling of endogenous proteins (carbonic anhydrases). Dependance on the luminescence properties of SCoMPIs with their environment was investigated. SCoMPI-labelled peptides could be easily detected by luminescence and infrared imaging, which was not the case for SCoMPI-labelled proteins. However, it was possible to detect these small amounts of proteins by X-ray fluorescence microspectroscopy.

Master of Science / Department of Grain Science and Industry / David L. Wetzel / During germination, biochemical changes occur in the wheat kernel by stimulation of enzymes and hormones, and the seed reserves are mobilized. Infrared microspectroscopy and imaging enables a localized chemical inventory, upon germination, to study the process. Frozen sections of germinated wheat mounted onto BaF[subscript]2 were mapped to produce functional group images for comparison with corresponding sections of ungerminated kernels. Relative functional group populations in the scutellum and embryonic axis were assessed before and after germination. An average 23% reduction in lipid to protein ratio was observed in the scutellum based on the comparison of 53,733 spectra. As a result of the early germination process, lipid in the scutellum was depleted to provide energy for the growing embryo. Germination of the kernels while in the field before harvest due to high humidity is known as preharvest sprouting. Preharvest sprouting has detrimental effects on the end use quality of the wheat (sprout damage) and cause economic loses. Tolerance to preharvest sprouting is highly desirable. To assist breeding program, a nondestructive near-IR chemical imaging method has been developed to test new lines for resistance to preharvest sprouting. The higher sensitivity of subsurface chemical imaging, compared with visual detection, alpha amylase determination, or viscosity testing, permits germination detection at early stages. A near-IR chemical imaging system with an InGaAs focal plane array (FPA) detector in the 1100 nm-1700 nm range was used. Kernels from six different cultivars, including HRW and HWW wheat, were exposed to moist conditions for 6, 12, 24, 36, and 48 hours. Images of each 90 kernel group were examined; kernels exposed to moisture for 36 hours were compared with kernels treated for 3 hours as a control. Each kernel was classified as sprouted or not sprouted with the criteria of log 1/R intensity at select wavelengths or select factors of principle component analysis (PCA) treatment of reflectance intensity data. Imaging wavelength range was expanded beyond 1700 nm to 2400 nm with the use of InSb FPA. Study for the potential for unsupervised determination in nondestructive near-IR imaging with detection wavelengths 1200-2400 is ongoing. Some preliminary results presented are encouraging.

Les microsphères d'embolisation, apparues dans les années 2000, sont des dispositifs médicaux dirigées contre les tumeurs hépatiques non opérables. Elles sont calibrés et peuvent être chargées en anticancéreux. Ces avancées majeures permettent de contrôler le niveau d'occlusion et la concentration en principe actif à injecter dans la tumeur. Cependant, le type, la taille des microsphères ou encore la concentration en anticancéreux varient d'un centre à un autre et d'un pays à un autre. Notre travail vise à comparer, sur des modèles de tumeurs hépatiques, les propriétés d'élution et l'efficacité antitumorale de différentes préparations de microsphères. La microspectroscopie infrarouge est utilisée pour mesurer la quantité de doxorubicine présente dans les microsphères à différents délais alors que la microspectrofluorimétrie permet d'évaluer la concentration et la distribution de la doxorubicine autour des billes. L'évaluation de l'activité antitumorale du traitement est mesurée sur les images spectrales infrarouge grâce à un modèle de prédiction et confirmée par un examen histopathologique. Les résultats ont montré que la vitesse d'élution dépend des propriétés physicochimiques de la microsphère, de sa taille et de la concentration de chargement. Les concentrations tissulaires de doxorubicine mesurées induisent une réduction significative de la viabilité tumorale. Le model de prédiction est un outil robuste et précis pour évaluer les modifications tissulaires. Ces résultats permettent de formuler des hypothèses mécanistiques sur l'activité antitumorale de différentes préparations de microsphères afin d'optimiser leur utilisation dans une stratégie thérapeutique clinique. / Transarterial chemoembolization is the most common treatment for patients with unresectable liver tumors. Calibrated drug eluting beads offer the advantages of controlling the level of occlusion, the amount of drug delivered, and the duration of drug delivery to the tumor. However, optimal procedure still remains unanswered and treatments differ through the use of various beads sizes or dose of loading. Our work is to compare, on experimental liver tumor model, the release properties and antitumor effects for different preparations of doxorubicin eluting beads. The amount of drug retained inside the beads, at different time point, is assessed by infrared microspectroscopy. Doxorubicin concentration and distribution in the tissue are determined by microspectrofluorimetry. Tissue modifications are quantified by a prediction model on infrared images and compared with the conventional pathological examination of stained tissue sections. Results show that elution rate of doxorubicin depend on the beads composition, the size and the loaded concentration. The doxorubicin tissue concentration induces a significant decrease of tumor viability. The prediction model established by infrared microspectroscopy is an accurate and robust tool to quantify tissue modifications. These results allow the formulation of mechanistic hypotheses on antitumor activity of different preparations of beads to optimize their use in a clinical therapeutic strategy.

Ce travail de recherche rapporte l'utilisation de techniques biophotoniques pour investiguer des questions biomédicales, de la recherche fondamentale (interaction champs électriques impulsionnels / cellules) aux études cliniques (toxicité cutanée induite chez les patients traités par des thérapies ciblées).La microspectroscopie confocale Raman et de la microscopie terahertz ont été utilisées pour étudier le processus d’électroperméabilisation cellulaire d'un point de vue moléculaire. Nos résultats démontrent l'implication des protéines. De plus, nous avons montré que la signature Raman des cellules peut être utilisée comme un biomarqueur précis des différents états des cellules exposées aux chocs électriques, correspondant à une électroperméabilisation non détectable, électroperméabilisation et irréversibleEn tant que projet parallèle de ce doctorat, une recherche clinique a été réalisée afin d'étudier la toxicité cutanée induite chez les patients traités par des thérapies anticancéreuses ciblées. Bien que l'efficacité de ces thérapies ne soit pas discutée, de nombreux effets cutanées secondaires graves sont associés. Dans cette étude, nous avons étudié l'opportunité de prédire l’apparition de la toxicité cutanée au moyen de la microspectroscopie Raman confocale réalisée sur la peau des patients. Nous avons réussi à déterminer un nouveau biomarqueur pharmacodynamique spécifique de la toxicité cutanée grâce aux signatures Raman de la peau des patients; alors que l'évaluation dermatologique ou histologique n'a détecté aucune modification. / This research work reports the use of various biophotonics techniques to investigate biomedical questions, from basic research (interaction between pulsed electric fields and cells) to clinical studies (skin toxicity induced in patients treated with targeted anticancer therapies).Confocal Raman microspectroscopy and terahertz microscopy have been used to investigate cell electropermeabilization process from a molecular point of view. Our results demonstrate the involvement of the proteins in cell electropermeabilization. Moreover, we have shown that the Raman signatures of the cells could be used as an accurate biomarker of the different states of the cells exposed to pulsed electric fields, corresponding to no detectable electropermeabilization, reversible and irreversible electropermeabilization.Finally, this doctorate research demonstrates the opportunity to predict skin toxicity induced by targeted anticancer therapies by means of confocal Raman microspectroscopy. We succedded to determine a novel and specific pharmacodynamic biomarker for skin toxicity based on the Raman signatures of the patient’s skin, whereas dermatological or histologicalevaluation did not detect any modifications.

La peau est un organe particulier de l'organisme dont la fonction principale est un rôle de protection vis-à-vis du milieu extérieur. Cette fonction est assurée grâce à la structure du tissu cutané en trois couches (épiderme, derme, hypoderme). Le derme est responsable de la résistance et de la souplesse de la peau. Le composant moléculaire majeur du derme est le collagène de type I, qui est fortement altéré au cours du vieillissement chronologique. Dans ce contexte, notre étude a pour objectif d'évaluer les modifications moléculaires du collagène dermique associées au vieillissement cutané par spectroscopies vibrationnelles (diffusion Raman et absorption infrarouge). Par déconvolution de la bande Amide I du signal Raman, nous avons mis en évidence, en fonction de l'âge de la peau, des modifications au niveau des interactions entre le collagène et les molécules d'eau ; ce qui reflète un espacement croissant des faisceaux de fibres de collagène au cours du vieillissement. En micro-imagerie infrarouge polarisée, le ratio des bandes Amide I/ Amide II permet d'évaluer l'orientation des fibres de collagène qui deviennent parallèles à la surface de la peau lors du vieillissement. Des expérimentations préliminaires ont également montré la possibilité de localiser sans marquage la jonction dermo-épidermique de la peau grâce aux caractéristiques spectrales du collagène de type IV. Une analyse ciblée de cette structure nécessite de développer de nouveaux instruments basés sur la spectroscopie en champ proche (Tip Enhanced Raman Scattering, NanoIR) ; ce qui devrait permettre de suivre les altérations du collagène de type IV au cours du vieillissement cutané. / Skin is a particular organ of the body whose the main function is a protective role towards the external environment. This function is provided by the structure of skin tissues in three layers (epidermis, dermis, hypodermis). The dermis is responsible for the strength and elasticity of the skin. The major molecular component of the dermis is the type I collagen, which is strongly altered during chronological aging. In this context, our study aims at evaluating the molecular modifications of the dermal collagen associated with skin aging by vibrational spectroscopy (Raman diffusion and infrared absorption). Using curve-fitting of Raman Amide I band, modifications in the interactions between collagen and water molecules were highlighted depending of the donor age. Such result reflects an increasing spacing of the collagen fiber bundles during aging. In addition, the collagen fibers orientation can be evaluated from the amide I/ amide II ratio calculated in polarized infrared micro-imaging. It appeared that the collagen fibers become orientated parallel to the skin surface with aging. Preliminary experiments showed also the ability to localize in a label-free manner the dermo-epidermal junction of the skin using the spectral characteristics of type IV collagen. A precise analysis of this structure requires the development of new instruments based on near-field spectroscopy (Tip Enhanced Raman Scattering, NanoIR); which could permit to follow the collagen IV alterations during skin aging.