Optical coherence tomography (OCT) has been shown to be a versatile three-dimensional imaging tool in diagnostic medicine, combining micrometre-scale resolutions with fast acquisition times. This imaging modality uses the interference between light backscattered from a sample and light that has traversed a known reference path delay to determine the scattering profile over penetration depths of up to several millimetres in tissue. A novel OCT system is presented that uses nonlinear optics to process the backscattered light in the optical domain prior to standard Fourier-domain OCT acquisition and processing. The nonlinear optical effects experienced between short light pulses are strongly intensity-dependent, occurring only significantly when the pulses are temporally and spatially overlapped. These conditions allow for the creation of a user-controlled time gate that restricts the light backscattered from the sample to a narrow (~100 micrometres) depth field of view prior to detection. When strong and weak scattering interfaces exist across the sample depth range, the signal-to-noise ratio of the weaker scattering sites can be limited by the finite detector dynamic range in Fourier-domain OCT systems. By aligning the time gate temporal delay to the backscatter from the weak interfaces of interest, a user can completely remove the strong backscattered light and enhance imaging contrast. The nonlinear effect used in the current time-gated OCT design is sum-frequency generation, which provides an additional advantage of imaging at near infrared (1280 nm) wavelengths, used for long penetration depths in tissue, while detection is performed in the visible (504 nm) with silicon-based camera technology. With the reduced depth field of view, the number of sampling points required per depth scan is also proportionately reduced, permitting faster acquisition rates for the time-gated region of interest. A complete description of the time-gated OCT system design is presented, along with proof-of-concept images demonstrating contrast enhancement and operation in a highly scattering biological medium. Based on its successful initial performance, future development of this system is expected for its eventual use in many OCT imaging applications. / Thesis (Master, Physics, Engineering Physics and Astronomy) -- Queen's University, 2008-01-15 20:05:41.665 / This work was funded by the Natural Sciences and Engineering Research Council and the Cancer Imaging Network of Ontario, supported by Cancer Care Ontario

Cette thèse étudie l'utilisation de la microscopie FRET en décalage temporelle pour la détection de co-localisation des deux protéines membranaires E- et N-cadhérine. Ces protéines sont importantes pour les contacts cellule-cellule et jouent un rôle important dans la transition épithélio-mésenchymateuse (EMT), un processus clé dans la métastase du cancer. Dans EMT cellules perdent leurs marqueurs épithéliaux (par exemple E-cadhérine) et acquièrent des marqueurs mésenchymateuses (par exemple N-cadhérine), ce qui augmente leur motilité et le caractère invasif pour échapper à la tumeur primaire dans la circulation sanguine en tant que ce qu'on appelle des cellules tumorales circulantes (CTC). Ce manuscrit porte sur la détection des CTC qui ont subi une EMT partielle, montrant un phénotype hybride (épithélio-mésenchymateuse) et co-expriment E et N-cadhérine, par des études de co-localisation en utilisant le FRET sur une lignée modèle de cellules. FRET (Transfer d’énergie par résonance de type Förster) est un transfert d'énergie non-radiatif entre deux molécules qui sont en résonance et à proximité (environ 1-20 nm). Une co-localisation d’E- et N-cadhérine en clusters serait donc détectable par FRET. La coloration des cadhérines qui a été fait en utilisant des anticorps spécifiques marqués avec une donneur qui a un longue durée de vie de fluorescence, le complexe de terbium Lumi4-Tb (TbL4) de Lumiphore, Inc., et diverses accepteurs. La longue durée de vie du donneur et la longue durée de vie d’accepteur sensibilisé (FRET) pourraient être imagés dans une configuration de microscopie en décalage temporelle. L’imagerie en décalage temporelle présente plusieurs avantages par rapport à l'imagerie stationnaire en termes de suppression efficace du bruit de fond dans des échantillons biologiques. L'installation décrite dans ce manuscrit est basée sur l'utilisation d'une caméra CCD intensifiée, une source d'excitation laser pulsé en UV et un décalage temporel défini de quelques microsecondes entre l'excitation et l'acquisition d'image. L’imagerie de FRET en décalage temporelle a été utilisée pour étudier des différents échantillons biologiques (intracellulaire et située à la membrane). Bien que les deux marqueurs protéiques pourraient imager correctement sur les mêmes cellules, FRET entre E- et N-cadhérine ou E- et E-cadhérine ne pouvaient pas être détectés. Des expériences de contrôle avec des anticorps contre le même anticorps primaire ont révélé des signaux de FRET forts à cause de la reconnaissance des anticorps donneurs et accepteurs aux mêmes anticorps primaires. Ces résultats de FRET entre deux anticorps différents séparés par quelques nanomètres démontrent la faisabilité de mesurer des interactions protéine-protéine et la co-localisation sur des membranes à l'aide de l’imagerie de FRET en décalage temporelle en utilisant TbL4. Imagerie en décalage temporelle de quelques microsecondes est particulièrement intéressante pour l'enquête des interactions protéine-protéine dans des échantillons biologiques hautement autofluorescentes, tels que les tissus cancéreux. / This thesis investigates the use of time-gated FRET microscopy for detection of colocalization of two membrane proteins, E- and N-cadherin. These proteins are important for cell-cell contacts and have an important role in the epithelial to mesenchymal transition (EMT), a key process in cancer metastasis. In EMT cells lose their epithelial markers (such as E-cadherin) and gain mesenchymal markers (such as N-cadherin), increasing their motility and invasiveness, enabling escape from the primary tumor into the bloodstream as so called circulating tumor cells (CTCs). This manuscript focuses on the detection of CTCs that have undergone partial EMT, displaying a hybrid phenotype (epithelial-mesenchymal) and co-express E- and N-cadherin, by FRET co-localization studies on a model cell line. FRET (Förster resonance energy transfer) is a non-radiative energy transfer between two molecules that are in resonance and in close proximity (ca. 1-20 nm). A co-localization of E- and N-cadherin in clusters would therefore be detectable by FRET. The staining of the cadherins was done by using specific antibodies labelled with a long lifetime donor, the terbium complex Lumi4-Tb (TbL4) from Lumiphore, Inc., and various acceptors. The long lifetime donor and long lifetime sensitized acceptor emission (FRET) could be imaged in a time-gated microscopy setup. Time -gated imaging has several advantages compared to steady state imaging in terms of efficient background suppression in biological samples. The setup described in this manuscript is based on the use of an intensified CCD camera, a pulsed UV-laser excitation source, and a defined (µs) delay between excitation and image acquisition. In addition to the E- and N-cadherin FRET experiments the time-gated FRET imaging microscopy was used to investigate different biological samples (intracellular and membrane located). Although both protein markers could be successfully imaged on the same cells, FRET between E- and N-cadherin or E- and E-cadherin could not be detected. Control experiments with antibodies against the same primary antibody revealed strong time-gated FRET signals due to binding of both donor and acceptor antibodies to the same primary antibodies. The successful time-gated imaging of two different antibodies separated by a few nanometers demonstrates the feasibility of probing protein-protein interaction and co-localization at membranes using TbL4 based time-gated FRET imaging. Microsecond time-gated imaging is especially interesting for the investigation of protein-protein interactions in highly autofluorescent biological samples such as cancer tissues.

Thesis (M.Sc.). / Written for the Medical Physics Unit. Title from title page of PDF (viewed ). Includes bibliographical references.

Le Sarcome d'Ewing est un cancer pédiatrique rare, principalement dû à l'expression de l'oncogène de jonction EWS-Fli1, et dont les traitements médicamenteux ont peu évolué au cours des dernières décennies. Nous nous intéressons à une nouvelle approche thérapeutique utilisant des siRNA, ciblant spécifiquement l'oncogène EWS-Fli1, et permettant l'inhibition de la croissance tumorale. Durant mon travail de thèse, j'ai utilisé des nanocristaux de diamant issus soit de détonation (DND), soit de synthèse haute pression-haute température (NDHPHT) pour vectoriser les siRNA, accrochés par interaction électrostatique. Pour ce faire, les NDs ont été rendus cationiques par différentes méthodes: (i) hydrogénation assistée par plasma, (ii) par recuit thermique, ou (iii) par traitement chimique pour les DNDs, ou (iv) greffage covalent d'un polymère cationique sur des NDHPHT (COP-NDHPHT).Mes travaux ont comporté deux axes: (i) étude in vitro des complexes ND:siRNA (caractérisations physico-chimiques des NDs et étude de l'efficacité d'inhibition de l'oncogène par les complexes); (ii) distribution tissulaire de COP-NDHPHT, injectés dans des souris, grâce à des NDHPHT fluorescents, contenant des défauts azote-lacune. Pour les détecter individuellement dans des coupes d'organes de souris portant une tumeur xénogreffée sous-cutanée, nous avons développé un système d'imagerie en épifluorescence à grande ouverture numérique, et résolu en temps afin de rejeter l'autofluorescence tissulaire (de durée de vie plus courte que celle des NDs). Nous avons quantifié le nombre, l'état d'agrégation et la localisation cellulaire de ces vecteurs (grâce à un marquage histopathologique imagé simultanément) 24h après injection. Les NDs ont été clairement détectés dans les différents organes, dont la tumeur, ouvrant la voie à un contrôle de la progression tumorale grâce au siRNA. / Ewing Sarcoma is a rare pediatric cancer, caused in the majority of the cases by the expression of the fusion oncogene EWS-Fli1. Current treatments have not much evolved over the past decades. We are investigating a new therapy based on siRNA specifically targeting the oncogene and inhibiting the tumor growth. During my PhD thesis, I have tested different types of synthetic nanodiamonds (ND) used to vectorize siRNA electrostatically bound at their surface: ND produced by detonation (DND) or by High Pressure-High Temperature synthesis (NDHPTH). Their surfaces have been cationized by various processes: (i) plasma or (ii) thermal hydrogenation, (ii) chemical treatment, or (iv) covalent grafting of a copolymer (COP-NDHPHT).My PhD work included two main axis: (i) in vitro study of ND:siRNA complexes (NDs physico-chemical characterization and oncogene inhibition efficacy by the complexes); (ii) tissue distribution of COP-NDHPHT, injected into mice, using fluorescent NDHPHT containing nitrogen-vacancy defects. To detect them individually in sections of mouse organs carrying a subcutaneous xenograft tumor, we developed an epifluorescence imaging system with large numerical aperture and resolved in time to reject tissue autofluorescence (of a shorter lifetime than NDs). We quantified the number, the aggregation state and the cell localization (thanks to simultaneous histopathological imaging) of these vectors 24 hours after injection. NDs have been clearly detected in different organs, including the tumor, paving the way for tumor progression control with siRNA.

No description available.

Na prática intervencionista, a ultrassonografia intravascular (USIV) é usada para se obter informações quantitativas e qualitativas do acometimento aterosclerótico, de forma complementar à angiografia. Esta tese teve como objetivos explorar a característica tomográfica do exame de USIV, bem como sua dinâmica dentro do ciclo cardíaco. Para isso, desenvolvemos técnicas de processamento de imagens médicas. Primeiramente, investigamos a reconstrução tridimensional da coronária baseando-nos apenas nas imagens de USIV, ou seja, sem a angiografia, como é feita a reconstrução atualmente. Na análise da dinâmica, fizemos um estudo para dispor volumes da coronária em diferentes fases do ciclo cardíaco de forma que estivessem alinhados espacialmente. Como consequência dos tratamentos propostos anteriormente, realizamos estudos sobre a quantificação de propriedades mecânicas dentro das condições oferecidas no intervalo de um ciclo cardíaco. As metodologias propostas foram aplicadas em simulações numéricas desenvolvidas neste trabalho e em exames reais. Obtivemos resultados compatíveis com os objetivos iniciais para reconstrução tridimensional da USIV em simulações numéricas. Na análise da dinâmica, a reconstrução de volumes em diferentes fases do ciclo e o alinhamento espacial possibilitaram a quantificação da variação setorial de volume da luz do vaso durante o ciclo cardíaco / In percutaneous coronary interventions, intravascular ultrasound (IVUS) examination is used to retrieve quantitative and qualitative information about the atherosclerotic plaque progression, complementary to angiography examination. This thesis has as objectives to explore the tomographic characteristic of the IVUS examination, as well as its dynamics within a cardiac cycle. For that purpose, medical image processing techniques were developed. Firstly, we have investigated how to reconstruct the tridimensional coronary based only on IVUS images, that is, without angiography, as it is done nowadays. Regarding dynamic analysis, we have studied models to build volumes of the coronary in distinct phases of the cardiac cycle in a spatial aligned way. Conversantly, as a consequence of the previous image processing methods, we have studied the quantification of mechanical properties of the vessel wall within a cardiac cycle. The methodologies proposed were applied in numeric phantoms developed in this work and also in real IVUS examinations. As result, tridimensional reconstruction was successful in the numeric phantom approach. In dynamics analysis, the reconstruction in distinct cardiac phases and volumes spatial alignment enabled the quantification of lumen volume variation during the cardiac cycle

Na prática intervencionista, a ultrassonografia intravascular (USIV) é usada para se obter informações quantitativas e qualitativas do acometimento aterosclerótico, de forma complementar à angiografia. Esta tese teve como objetivos explorar a característica tomográfica do exame de USIV, bem como sua dinâmica dentro do ciclo cardíaco. Para isso, desenvolvemos técnicas de processamento de imagens médicas. Primeiramente, investigamos a reconstrução tridimensional da coronária baseando-nos apenas nas imagens de USIV, ou seja, sem a angiografia, como é feita a reconstrução atualmente. Na análise da dinâmica, fizemos um estudo para dispor volumes da coronária em diferentes fases do ciclo cardíaco de forma que estivessem alinhados espacialmente. Como consequência dos tratamentos propostos anteriormente, realizamos estudos sobre a quantificação de propriedades mecânicas dentro das condições oferecidas no intervalo de um ciclo cardíaco. As metodologias propostas foram aplicadas em simulações numéricas desenvolvidas neste trabalho e em exames reais. Obtivemos resultados compatíveis com os objetivos iniciais para reconstrução tridimensional da USIV em simulações numéricas. Na análise da dinâmica, a reconstrução de volumes em diferentes fases do ciclo e o alinhamento espacial possibilitaram a quantificação da variação setorial de volume da luz do vaso durante o ciclo cardíaco / In percutaneous coronary interventions, intravascular ultrasound (IVUS) examination is used to retrieve quantitative and qualitative information about the atherosclerotic plaque progression, complementary to angiography examination. This thesis has as objectives to explore the tomographic characteristic of the IVUS examination, as well as its dynamics within a cardiac cycle. For that purpose, medical image processing techniques were developed. Firstly, we have investigated how to reconstruct the tridimensional coronary based only on IVUS images, that is, without angiography, as it is done nowadays. Regarding dynamic analysis, we have studied models to build volumes of the coronary in distinct phases of the cardiac cycle in a spatial aligned way. Conversantly, as a consequence of the previous image processing methods, we have studied the quantification of mechanical properties of the vessel wall within a cardiac cycle. The methodologies proposed were applied in numeric phantoms developed in this work and also in real IVUS examinations. As result, tridimensional reconstruction was successful in the numeric phantom approach. In dynamics analysis, the reconstruction in distinct cardiac phases and volumes spatial alignment enabled the quantification of lumen volume variation during the cardiac cycle