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Raman spectroscopy is a powerful tool in the field of biomedicine for disease diagnosis owing to its potential to provide the molecular fingerprint of biological samples. However due to the inherent weak nature of the Raman process, there is a constant quest for enhancing the sensitivity of this technique for enhanced diagnostic efficiency. This thesis focuses on achieving this goal by integrating advanced methods with Raman spectroscopy. Firstly this thesis explores the applicability of a laser based fluorescence suppression technique – Wavelength Modulated Raman Spectroscopy (WMRS) - for suppressing the broad luminescence background which often obscure the Raman peaks. The WMRS technique was optimized for its applications in single cell studies and tissue studies for enhanced sensing without compromising the throughput. It has been demonstrated that the optimized parameter would help to chemically profile single cell within 6 s. A two fold enhancement in SNR of Raman bands was demonstrated when WMRS was implemented in fiber Raman based systems for tissue analysis. The suitability of WMRS on highly sensitive single molecule detection techniques such as Surface Enhanced Raman Spectroscopy (SERS) and Surface Enhanced Resonance Raman Spectroscopy (SERRS) was also explored. Further this optimized technique was successfully used to address an important biological problem in the field of immunology. This involved label-free identification of major immune cell subsets from human blood. Later part of this thesis explores a multimodal approach where Raman spectroscopy was combined with Optical Coherence Tomography (OCT) for enhanced diagnostic sensitivity (>10%). This approach was used to successfully discriminate between ex-vivo adenocarcinoma tissues and normal colon tissues. Finally this thesis explores the design and implementation of a specialized fiber Raman probe that is compatible with surgical environments. This probe was originally developed to be compatible with Magnetic Resonance Imaging (MRI) environment. It has the potential to be used for performing minimally invasive optical biopsy during interventional MRI procedures.

Στο συγκεκριμένο ερευνητικό πρωτόκολλο μελετήθηκαν οι πιθανές ένοχες βλάβες σε φλεβικά μοσχεύματα ασθενών με οξέα στεφανιαία σύνδρομα με τη μέθοδο της Οπτικής Συνεκτικής Τομογραφίας (OCT). • Οι αθηροσκληρωτικές βλάβες των φλεβικών μοσχευμάτων έχουν μελετηθεί in vivo με τη μέθοδο της αγγειοσκόπησης και της ενδαγγειακής υπερηχογραφίας (IVUS). Απεναντίας, η απεικόνιση των μοσχευμάτων με τη μέθοδο της OCT, η οποία χαρακτηρίζεται από σημαντικά μεγαλύτερη διακριτική ικανότητα σε σχέση με το IVUS και βελτιωμένη διεισδυτική ικανότητα συγκρινόμενη με την αγγειοσκόπηση, δεν έχει μελετηθεί συστηματικά. • Μέθοδος: Η απεικόνιση των ένοχων βλαβών των μοσχευμάτων πραγματοποιήθηκε, κατόπιν αγγειογραφίας τους, με τη μέθοδο χωρίς απόφραξη της OCT σε ασθενείς με ασταθή στηθάγχη (UA), έμφραγμα μυοκαρδίου με ανάσπαση του διαστήματος ST (STEMI) και έμφραγμα μυοκαρδίου χωρίς ανάσπαση του διαστήματος ST (non-STEMI). Ο ινώδης ιστός, ο λιπώδης ιστός, οι εναποθέσεις ασβεστίου, ο θρόμβος και η ρήξη της πλάκας ορίστηκαν σύμφωνα με τα κριτήρια απεικόνισης στοιχείων για την OCT, όπως περιγράφηκαν και στο γενικό μέρος. • Αποτελέσματα: Απεικονίστηκαν 28 φλεβικά μοσχεύματα (μέσης ηλικίας 14.6 ετών) σε 26 ασθενείς. Οι βλάβες χαρακτηρίστηκαν ως σύμπλοκες αγγειογραφικά σε ποσοστό 96.4%, ενώ εμφάνιζαν εξέλκωση σε ποσοστό 32.1% και θρόμβο σε ποσοστό 21.4%. Η OCT αποκάλυψε ινολιπώδη σύσταση σε όλες τις βλάβες, εναπόθεση ασβεστίου στο 32.1% των βλαβών, ρήξη πλάκας σε ποσοστό 60.7% και παρουσία θρόμβου σε ποσοστό 46.4%. Η παρουσία του θρόμβου ήταν προοδευτικά συχνότερη ανάμεσα στις ομάδες ανάλογα με το κλινικό τους σύνδρομο (UA έως STEMI, p=0.003, UA έναντι εμφράγματος μυοκαρδίου, p=0.006). Η λεπτή ινώδης κάψα καταγράφηκε οριακά συχνότερα στους ασθενείς με οξύ έμφραγμα μυοκαρδίου (UA έναντι εμφράγματος μυοκαρδίου, p=0.06, STEMI 100% έναντι non-STEMI 53.3% έναντι UA 20%, p=0.03). Βλάβες με στοιχεία ευθρυπτότητας, όπως απεικονίζονταν στην OCT παρουσιάζονταν σε ποσοστό 67.9%, χωρίς όμως συσχέτιση με την κλινική εικόνα. • Συμπέρασμα: Οι ένοχες βλάβες φλεβικών μοσχευμάτων μεγάλης ηλικίας ασθενών με οξέα στεφανιαία σύνδρομα, όπως αυτές απεικονίζονται στην OCT, εμφανίζουν ινολιπώδη σύσταση, σχετικά λεπτή ινώδη κάψα, ρήξη πλάκας και θρόμβο που συσχετίζονται με το κλινικό φάσμα των οξέων στεφανιαίων συνδρόμων. Αυτά οδηγούν στο συμπέρασμα ότι οι ίδιοι μηχανισμοί αθηροσκλήρωσης που οδηγούν στην εμφάνιση οξέων στεφανιαίων συνδρόμων στα γηγενή στεφανιαία αγγεία, είναι πιθανόν να ενέχονται και στην πρόκληση οξέων στεφανιαίων συνδρόμων λόγω αποτυχίας των φλεβικών μοσχευμάτων. / This study sought to assess, with optical coherence tomography (OCT), presumably culprit atherosclerotic lesions of saphenous vein grafts (SVGs) in patients with acute coronary syndromes (ACS). Background: Atherosclerotic lesions of SVGs have been studied in vivo with angioscopy and intravascular ultrasound. However, imaging with OCT, which has a higher resolution than intravascular ultrasound and better penetration than angioscopy, has not been conducted systematically. Methods Using a nonocclusive OCT technique, we performed angiography and OCT of culprit SVG lesions in patients with unstable angina (UA), ST-segment elevation myocardial infarction (STEMI), and non-STEMI. Fibrous and fatty tissue, calcification, thrombus, and plaque rupture were defined according to OCT objective criteria. Results: Twenty-eight SVGs (average age 14.6 years) in 26 patients were imaged. Lesions on angiography were complex (96.4%), with ulceration in 32.1% and thrombus in 21.4%. OCT disclosed a fibrofatty composition in all lesions, calcification in 32.1%, plaque rupture in 60.7%, and thrombus in 46.4%. Thrombus was progressively more frequent across groups (UA to STEMI, p=0.003; UA vs. myocardial infarction, p=0.006). A thin fibrous cap was marginally more frequent in myocardial infarction patients (UA vs. myocardial infarction, p=0.06; STEMI 100% vs. non-STEMI 53.3% vs. UA 20%, p=0.03). OCT features of friability were present in 67.9% of SVGs not correlating with clinical presentation. Conclusions: OCT of culprit lesions of old SVGs in patients with ACS demonstrates fibrofatty composition, relatively thin fibrous cap, plaque rupture, and thrombus, which correlate with the clinical spectrum of ACS. This suggests that similar mechanisms with native vessels’ atherosclerosis may be involved in SVG-related ACS.

La capacité de la tomographie par cohérence optique (OCT) à délivrer des images tomographiques de tissus biologiques in vivo, de manière non invasive et en temps réel, a suscité un intérêt croissant pour de nombreuses applications biomédicales, principalement en ophtalmologie pour l’imagerie de la rétine et du segment antérieur de l’œil. Toutefois, pour l’imagerie à haute résolution de tissus biologiques fortement diffusants, comme la sclérotique et la cornée œdémateuse, la technique nécessitait des améliorations technologiques. Dans cette thèse, un système d’OCT « Fourier-domain » (FD-OCT) à très haute résolution spatiale (< 4 µm), à la longueur d’onde de 1,3 µm, a été développé dans la Laboratoire Charles Fabry – Institute d’Optique Graduate School. Avec ce système original, nous avons réussi, pour la première fois, à visualiser correctement le canal de Schlemm de l’œil humain qui se trouve à une profondeur d’environ 0,8 mm dans le limbe de la cornée, milieu fortement diffusant. L’imagerie du canal de Schlemm est capitale afin d’envisager la chirurgie par laser du glaucome, qui consiste à inciser cette partie de l’œil afin d’améliorer l’écoulement de l’humeur aqueuse. Par ailleurs, en collaboration avec le Laboratoire d’Optique Appliquée de l’ENSTA ParisTech, nous avons démontré la possibilité de contrôler en temps réel par OCT des découpes par laser femtoseconde pratiquées dans la cornée humaine in vitro. Ces travaux ont montré que l’opération du Glaucome par laser femtoseconde, contrôlée par OCT, devrait être possible. / The ability of optical coherence tomography (OCT) to deliver tomographic images of biological tissues in vivo non-invasively and in real-time has been a growing interest in many biomedical applications, mainly in ophthalmology for imaging the retina and the anterior segment of the eye. However, developing high-resolution OCT for imaging strongly scattering biological tissues like sclera and edematous cornea has still been the main challenge. In this PhD work, an ultrahigh-resolution (< 4 µm) Fourier-domain OCT (FD-OCT) system optimized at 1.3 µm center wavelength was developed in Laboratoire Charles Fabry – Institut d’Optique Graduate School. Using this OCT system, we have, for the first time, properly visualized the Schlemm’s canal of the human eye that is located in the strongly scattering corneal limbus at depth of ~ 0.8 mm. Schlemm’s canal has been our target for OCT imaging because it plays an important role for the management of the aqueous humor that is responsible for causing glaucoma - an eye disease that can potentially lead to blindness. In collaboration with Laboratoire d’Optique Appliquée at ENSTA ParisTech, we have also demonstrated real-time OCT imaging of the femtosecond laser surgery in excised human cornea. These studies have thus shown that the surgery of glaucoma by femtosecond laser, monitored by OCT, would be possible.

OBJETIVO: Caracterizar morfologicamente e funcionalmente as alterações observadas no fundo de olho de pacientes com a doença de Vogt-Koyanagi- Harada (VKH) no estágio tardio. MÉTODOS: Estudo prospectivo, transversal, com inclusão de 36 pacientes com diagnóstico de doença de VKH no estágio tardio (definido como 6 meses ou mais após o início da doença) após obtenção do termo de consentimento livre e esclarecido. Fundo de olho (retinografia, RG) foi estratificado baseado em alterações difusas e focais assim como a função global da retina (eletroretinograma campo total, ERGct) segundo o método matemático de aglomeração (cluster). A concordância entre os dois métodos de estratificação foi estimada pelo teste kappa. Atividade clínica foi correlacionada com alterações indicativas de atividade de coróide pela angiografia com indocianina verde (AICV) (teste exato de Fisher). Alterações na integridade dos segmentos interno e externo de fotorreceptores (IS/OS) avaliada pela tomografia de coerência óptica espectral (OCT espectral) foram correlacionadas com a função macular (eletroretinograma multifocal, ERGmf) e acuidade visual (AV) (teste de Mann-Whitney). RESULTADOS: Houve concordância substancial entre os observadores em relação ao sistema analítico para estratificação fundoscópica proposto (kappa=0,78; intervalo de confiança 95%(IC95%)=0,63-0,93). Este sistema analítico também se correlacionou de maneira substancial com os achados do ERGct (kappa=0,68; IC 95% 0,52-1,07). Na avaliação com AICV, a proporção dos olhos com resultados compatíveis com atividade subclínica de coróide foi maior em pacientes com doença fundoscópica leve (12/13 olhos) se comparados exclusivamente com aqueles portadores de doença fundoscópica grave (11/19 olhos) (p=0,049). Os pacientes estratificados de acordo com as características fundoscópicas não diferiram de maneira substancial quanto aos aspectos clínicos. Atividade subclínica de coróide detectada pela AICV foi observada em 36 dos 51 olhos (72%). Atividade clínica (células na câmara anterior) foi observada em 21 dos 51 olhos (41%). Destes 76% (16/21) demonstravam sinais de atividade de coróide, ao passo que 5 de 21 olhos (24%) não os apresentavam. Dos pacientes com atividade clínica de doença e com AICV negativa, a maioria tinha doença grave (4/5 olhos) e nenhum tinha doença leve pela classificação fundoscópica. Quanto à função macular, as amplitudes e latências das ondas N1 e P1 diferiram dos controles (p<0,01). A junção IS/OS macular estava alterada em 17 de 42 olhos (40%) dos olhos. A AV foi diferente entre grupos estratificados de acordo com os resultados do ERGmf ( leve= 0,0 [20/20] e grave = 0,2 [20/32]; p<0,05) assim como entre os grupos estratificados pelo OCT espectral (IS/OS+ = 0,0 [20/20] e IS/OS-= 0,7 [20/100]; p<0,05). O grau de concordância entre estas estratégias foi baixo (K=0,17). CONCLUSÕES: O sistema analítico para achados fundoscópicos proposto teve alta reprodutibilidade e correlacionou-se com medidas objetivas de função retiniana (ERGct). A gravidade de doença estimada segundo a estratégia fundoscópica influencia a interpretação da AICV, sendo que pacientes com achados leves tendem a ter maior prevalência de atividade subclínica detectada pela AICV assim como pacientes graves apresentaram exame angiográfico negativo mesmo na vigência de atividade de segmento anterior. Os grupos estabelecidos com as estratégias funcional (ERGmf) e morfológica (OCT espectral) de análise macular diferiram significativamente em termos de acuidade visual, sendo que o ERGmf discerniu grupos com acuidades semelhantes. A concordância entre as duas técnicas de análise macular foi baixa devido à maior sensibilidade do ERGmf em detectar alterações. Estes dados sugerem que o dano funcional pode preceder e/ou ocorrer na ausência de alterações maculares detectáveis com o OCT espectral em pacientes no estágio tardio da doença de VKH / OBJECTIVES: To characterize the structural and functional derangements observed in eyes of patients with late-stage Vogt-Koyanagi-Harada (VKH) disease. METHODS: Cross sectional, prospective study including 36 patients diagnosed with late-stage VKH disease (defined as more than 6 months from disease`s onset) after obtainment of informed consent. Fundoscopy (retinography, RG) was stratified based on diffuse and focal findings as well as according to global retinal function (full-field electroretinogram, ffERG) applying cluster method of aggregation. The concordance between the two methods of stratification was estimated with the kappa test. Clinical disease activity was correlated with findings suggestive of subclinical choroidal activity on indocyanine green angiography (ICGA) (Fisher`s exact test). Disruption of photoreceptors inner and outer segments\' junction (IS/OS) evaluated using spectral optical coherence tomography (spectral OCT) was correlated with macular function (multifocal electroretinogram, mfERG) and best corrected visual acuity (BCVA)(Mann- Whitney test). RESULTS: Substantial interobserver concordance was detected with regard to the analytic system for fundus findings proposed (kappa=0.78; confidence interval 95%(CI95%); 0.63-0.93). This system also showed substantial correlation with ffERG findings (kappa=0.68; CI 95%; 0.52-1.07). Upon ICGA investigation, the proportion of eyes presenting signs of disease activity on ICGA was significantly greater among mild fundus cases (12/13) when compared exclusively with those presenting severe fundus disease (11/19) (p=0.049). The different severity categories based on fundoscopic evaluation did not differ in terms of clinical characteristics. Subclinical choroidal activity was observed in 36 of 51 eyes analyzed (72%). Clinical disease activity (cells in the anterior chamber) was observed in 21 of 51 eyes (41%). Most patients with clinical activity and negative ICGA examination (4/5) had severe disease and none had mild disease according to fundoscopic stratification. On macular evaluation, patients\' amplitudes and latencies of N1 and P1 waves differed from controls (p<0.01). Macular IS/OS junction was altered in 17 out of 42 eyes (40%). BCVA differed significantly between groups stratified according to both, mfERG (mild = 0.0 [20/20] and severe = 0.2 [20/32]; p<0.05) as well as spectral OCT (IS/OS+ = 0.0 [20/20] and IS/OS-= 0.7 [20/100]; p<0.05) assessment techniques. Concordance between these strategies of macular assessment was weak (K=0.17). CONCLUSIONS: The analytic system for fundus findings proposed herein demonstrated high reproducibility and substantial interobserver concordance with objective measures of retinal compromise (ffERG). The severity grading proposed influenced the interpretation of ICGA, as patients with mild disease had a greater proportion of positive findings while patients with severe disease presented negative ICGA in spite of anterior segment activity. Groups established according to both functional (mfERG) and morphological (spectral OCT) macular analysis strategies differed in terms of BCVA, although the mfERG distinguished groups with similar BCVA. The low concordance between the two strategies detected is attributable to the greater sensibility of the mfERG to detect alterations. These findings suggest that functional derangements may precede and/or occur in absence of alterations detectable with the spectral OCT in patients with late-stage VKH disease.

O presente trabalho experimental, tem como objetivo avaliar \"in vitro\" possíveis falhas na cimentação de fragmentos cerâmicos sobre substrato de esmalte dentário previamente submetido ao clareamento dental e analisados pelo sistema de tomografia por coerência ótica. O ensaio experimental foi realizado in vitro, fazendo uso de trinta pré-molares superiores humanos. Cada elemento dental teve sua porção de esmalte vestibular aplainada por meio de disco de sílica na granulação 120, foram cimentados fragmentos cerâmicos retangulares de 1.5mm de espessura. Os dentes foram distribuídos aleatoriamente em 3 grupos de 10 dentes cada (n=10), Grupo 1: Fragmentos cimentados sem serem submetidos ao tratamento clareador; Grupo 2: Fragmentos cimentados 24 hrs. Após serem submetidos ao clareamento dental; Grupo 3: Fragmentos cimentados 7 dias após serem submetidos ao clareamento dental. Os espécimes foram transferidos para o sistema de tomografia por coerência óptica para seus respectivos monitoramentos após exposição ao agente clareador e cimentação da restauração indireta e ao final dos ensaios de envelhecimento artificial por termociclagem. A variável de resposta, falhas na interface esmalte e cimento resinoso, foi avaliada por escores (0,1 e 2) que representam em ordem crescente as falhas entre o substrato dental e a restauração indireta. Os valores obtidos foram analisados por meio de testes não paramétricos (Kruskal-Wallis e Wilcoxon). Os resultados constituíram de 60 escores de fendas referentes aos 3 grupos (G1;G2;G3) analisados imediatamente após a cimentação e após a termociclagem. A comparação entre os 3 grupos após termociclagem demonstrou haver diferença estatisticamente significante (p<0,0001) pela comparação dois a dois pelo teste complementar de Dunn e detectou-se que o G2 cimentado após 24 horas do clareamento apresentou mais fendas que G1 e G3. Com base nestes resultados e na metodologia utilizada pode-se concluir que, dentes submetidos ao tratamento clareador, o protocolo clinico de cimentação de restaurações cerâmicas indiretas, quando realizado 7 dias após o clareamento gerou menor quantidade de fendas na interface, propondo assim melhor qualidade na adaptação dente/restauração. / The present experimental study has the objective of evaluating in vitro possible failures in the cementation of ceramic fragments over dental enamel substrate previously submitted to dental bleaching and analyzed by optical coherence tomography system. The experimental trial was performed in vitro using thirty human maxillary premolars. Each dental element had have it\'s portion of vestibular enamel planed by a silic disk in granulation 120, ceramic fragments with a diameter of 1.5mm thickness were cemented. The teeth were randomly distributed in 3 groups of 10 teeth each (n = 10), Group 1: Bonded Veneer without being subjected to bleaching treatment Group 2: Bonded Veneer 24 hours after being submitted to dental bleaching Group 3: Bonded Veneer 7 days after being submitted to dental bleaching. The specimens were transferred to the optical coherence tomography system for their respective monitoring after being exposured to the whitening agent and cementation of the indirect restoration and at the end of the artificial aging tests by thermocycling. The variable of response, at faults in the interface enamel and resin cement, was evaluated by scores (0,1 and 2) that represent in increasing order the failures between dental substrate and indirect restoration. The values obtained were analyzed by non-parametric tests (Kruskal-Wallis and Wilcoxon). The results consisted of 60 gaps scores for the three groups (G1, G2, G3) analyzed immediately after bonding and after thermocycling. The comparison between the 3 groups after thermocycling, showed a statistically significant difference (p <0.0001) by the two-to-two comparison by Dunn\'s complementary test and it was detected that G2 after 24 hours of bleaching, showed more gaps than G1 and G3. Based on these results and the methodology used, it was concluded that, when teeth were submitted to the bleaching treatment, the clinical cementation protocol of indirect ceramic restorations, when performed 7 days after bleaching, generated fewer gaps at the interface, thus proposing a better quality in the relation Tooth / restoration.

A terapia fotodinâmica (PDT) é uma alternativa promissora de tratamento para lesões pré-cancerosas e para câncer de pele não-melanoma, como o carcinoma espinocelular, agressivo e potencialmente metastático. Visando melhorar a eficiência da terapia fotodinâmica de carcinoma espinocelular de pele, este estudo otimizou o tempo para início da terapia, avaliou a eficácia dos fotossensibilizadores ácido aminolevulínico (ALA- 20%) e o metil-ester aminolevulínico (MEALA-10%) e verificou o coeficiente de atenuação relativo à pele normal dos grupos experimentais, por meio de Tomografia por Coerência Óptica. Para a indução do tumor foi realizada a carcinogênese química (DMBA/TPA) por um período de 28 semanas. A espectroscopia de fluorescência foi utilizada para monitoração da emissão da molécula de protoporfirina IX, induzida pelo ALA e MEALA. A aquisição de dados a cada 30 minutos totalizando um período de 360 minutos, permitiu verificar a máxima incorporação de ALA e MEALA em 300 e 330 minutos após a aplicação, respectivamente. Após a otimização foi realizada a PDT, avaliação clínica, histopatológica e análise por OCT dos grupos experimentais, por meio das quais verificou-se maior eficiência do grupo tratado com MEALA. No período de 20 dias, o percentual de lesões com redução de área maior que 50%, foi de 33% na PDT com ALA e 83% para o MEALA. Os coeficientes de atenuação ópticos para os grupos com neoplasia foram maiores do que os do grupo controle. Os grupos tratados com PDT apresentaram valores de coeficiente de atenuação que se aproximam dos valores obtidos para a pele sadia, evidenciando a resposta ao tratamento. Entretanto para ambos os fotossensibilizadores utilizados, a PDT mostrou-se eficaz quando iniciada nos tempos determinados neste estudo, e a técnica de OCT mostrou-se uma potencial ferramenta na avaliação deste tratamento. / Photodynamic therapy (PDT) is a promising alternative for pre-cancerous lesions and non-melanoma skin cancer treatment as squamous cells carcinoma, which is aggressive and potentially metastatic. Aiming the improvement of photodynamic therapy efficiency for skin squamous cells carcinoma, this study has optimized the time to start the therapy, it also evaluated the effectiveness of the aminolevulinic acid (ALA-20%) and methyl-ester aminolevulinic (MEALA-10%) as photosensitizers as well as verified the optical attenuation coefficient relative to normal skin of the experimental groups by optical coherence tomography (OCT). A chemical carcinogenesis was performed (DMBA/TPA), by a period of 28 days. Fluorescence Spectroscopy was used to monitor the emission of protoporphyrin IX induced by ALA and MEALA. Signal acquisition was performed every 30 minutes for a total period of 360 minutes, showing a maximum incorporation of ALA and MEALA, 300 and 330 minutes after the application. PDT was conducted after the optimization of the irradiation time. Clinical and histopathologic evaluation and OCT analysis of the experimental groups showed higher efficiency of the MEALA-treated group. After 20 days of treatment, the percentage of lesions with area reduction greater than 50%, was 33% for the ALA group and 83% for the MEALA group. The optical attenuation coefficients for groups with cancer were higher than those in the control group. The groups treated with PDT presented attenuation coefficient values that are close to the values obtained for healthy skin, showing the positive response to the treatment. This study showed that PDT was effective for both photosensitizers when initiated at the times determined in this study, and OCT technique proved to be a potential tool in the assessment of this treatment.