abstract: Measuring molecular interaction with membrane proteins is critical for understanding cellular functions, validating biomarkers and screening drugs. Despite the importance, developing such a capability has been a difficult challenge, especially for small molecules binding to membrane proteins in their native cellular environment. The current mainstream practice is to isolate membrane proteins from the cell membranes, which is difficult and often lead to the loss of their native structures and functions. In this thesis, novel detection methods for in situ quantification of molecular interactions with membrane proteins are described. First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined. Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model. Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2018

Optical imaging techniques have improved much over the last fifty years since the invention of the laser. With a high brightness source many imaging applications which were once inaccessible to researchers have now become a reality. Among these techniques, the most beneficial one is the use of lasers for both wide-field and confocal imaging systems. The aim of this study was to design a laser imaging system based on the concept of laser scanning confocal microscopy. Specifically the optical system was based on optical fibers allowing the user to image remote areas such as the inner surface of rifled gun barrels and/or pipes with a high degree of precision (+/- 0.01 mm). In order to build such a system, initially the theoretical foundation for a confocal as well as a wide-field imaging system was analyzed. Using this basis a free-space optical confocal system was built and analyzed. The measurements support the fact that both the objective numerical aperture and pinhole size play an important role in the radial and axial resolution of the system as well as the quality of the images obtained. To begin construction of a confocal, optical-fiber based imaging system first an all fiber wide-field imaging system was designed and tested at a working wavelength of 1550 nm. Then an all fiber confocal system was designed at a working wavelength of 808 nm. In both cases results showed that while lateral resolution was adequate, axial resolution suffered since it was found that the design of the optical system needs to take into account under-filling of the objective lens, a result common with the use of laser beams whose divergence is not at all like that of a point source. The work done here will aid technology that will be used in the elimination process of faulty rifling fabrication in defense industry. The reason why the confocal technique is preferred to the conventional wide-field one is the need for better resolution in all directions. Theoretical concepts and mathematical background are discussed as well as the experimental results and the practical advantages of such a system.

This dissertation presents the development and use of a novel optical imaging system capable of monitoring changes in blood flow, oxygenated hemoglobin, deoxygenated hemoglobin, and absolute pO₂ in the brain. There are several imaging modalities capable of monitoring these parameters separately. Laser speckle contrast imaging (LSCI) and multi-spectral reflectance imaging (MSRI) have been used to monitor relative blood flow and hemoglobin changes respectively. Phosphorescence quenching, while not typically used for imaging, is capable of noninvasive measurements of pO₂. Combining these three techniques has led to the development of an imaging system that could ultimately lead to a better understanding of brain physiology. By combining techniques such as LSCI and MSRI, it becomes possible to estimate the cerebral metabolic rate of oxygen (CMRO₂), an important indicator of neuronal function. It is equally important to understand absolute pO₂ levels so that oxygen metabolism can be examined in context. Integrating phosphorescence quenching and a spatial light modulator into the imaging system allowed absolute pO₂ to be simultaneously measured in distinct regions. This new combined system was used to investigate pathophysiological conditions such as cortical spreading depression (CSD) and ischemia. The observed hemodynamic changes associated with these events were largely dictated by baseline oxygen levels and varied significantly in different regions. This finding highlighted the importance of having a system capable of monitoring hemodynamic changes and absolute pO₂ simultaneously while maintaining enough spatial resolution to distinguish the changes in different regions. It was found that animals with low baseline pO₂ were unable to deliver enough oxygen to the brain during events like CSD because of the high metabolic demand. In order for this technique to become more prevalent among researchers, it is essential to make it cost effective and simple to use. This was accomplished by replacing the expensive excitation sources with cheaper light emitting diodes (LEDs) and redesigning the software interface so that it was easier to control the entire device. The final system shows the potential to become a key tool for researchers studying the role of absolute pO₂ and other hemodynamic parameters during pathophysiological conditions such as CSD and ischemia. / text

No description available.

L'IRM (Imagerie par Résonance Magnétique) est une technique de choix pour l'imagerie moléculaire, soit la visualisation d'événements biologiques à l'échelle moléculaire. En effet, elle est non invasive et possède une haute résolution. Cependant, elle manque de sensibilité. L'utilisation d'agents de contraste commerciaux, qui sont pour la plupart des chélates de Gd(III), peut pallier à ce problème lors d'examens cliniques mais ils ne sont pas assez efficaces en vue de l'imagerie moléculaire. Leur efficacité, définie par leur relaxivité, peut être améliorée en optimisant les paramètres qui la gouvernent, comme le nombre de molécules d'eau coordinées, la vitesse d'échange de l'eau ou la dynamique de rotation du complexe. Deux stratégies sont envisagées pour améliorer la sensibilité : la première est de rassembler un grand nombre de complexes de Gd(III) possédant une haute relaxivité dans une seule entité ; la seconde est la conception de sondes bimodales IRM/imagerie optique, permettant de tirer profit à la fois de la haute résolution de l'IRM et de la très bonne sensibilité de l'imagerie optique.Ce travail de thèse a consisté à incorporer des agents de contraste à base de Gd(III) possédant des propriétés de relaxivité améliorées dans des systèmes macromoléculaires ou des nanobjets. Pour cela, des complexes ayant des paramètres de relaxivité optimisés connus au laboratoire ont été fonctionnalisés pour être greffés sur des quantum dots, pour le caractère bimodal et sur des oligonucléotides afin de réaliser des sondes ADN. L'utilisation de billes de silice a permis d'incorporer des complexes de Gd(III) de manière non covalente et d'obtenir à la fois une relaxivité par Gd(III) et par particule très élevée. L'incorporation simultanée de complexes d'Yb(III) permet également d'obtenir une sonde bimodale avec une émission dans le proche infrarouge. Tous ces systèmes ouvrent la possibilité au développement de sondes IRM avec des propriétés optimisées, prometteuses pour l'imagerie moléculaire. / Magnetic Resonance Imaging (MRI) is a widely used diagnostic method in medicinal practice. It is the ideal technique for molecular imaging, which allows biological events to be observed at the molecular scale thanks to its high resolution. However it has a very low sensitivity. Commercial contrast agents can palliate this lack of sensitivity, but their efficiency, defined as relaxivity, is too low for molecular imaging. The relaxivity of Gd(III) chelates depends on parameters such as the hydration number, the exchange rate and the rotational dynamics of the complex, which can be optimized to improve the relaxivity. Two strategies can be considered to improve the sensitivity: the first one is to gather a high number of complexes demonstrating an optimized relaxivity on a nanoobject ; the second one is the design of a bimodal probe MRI/Optical imaging to combine the high resolution of MRI and the high sensitivity of optical imaging.In this work we present the incorporation of gadolinium chelates into macromolecular systems and two different types of nanoobjects. Gd(III) chelates with improved relaxometric properties have been functionalized and grafted onto quantum dots for the design of a bimodal probe and into oligonucleotides for the conception of a DNA probe. The non-covalent incorporation of Gd(III) in silica spheres allows both a high relaxivity per Gd(III) and per particle. The simultaneous incorporation of Yb(III) complexes emitting in the near infrared permits the design of a bimodal probe. All of these systems lead to the development of a new class of promising probes for molecular imaging.

Cette thèse porte sur l'élaboration de nanoparticules (NPs) à luminescence persistante pour l'imagerie optique dans le domaine du rouge profond et du proche infrarouge. Leur utilisation présente un avantage indéniable, l'étape d'irradiation pour obtenir l'état excité pouvant être réalisée avant injection, il est possible d'éviter tout stress des tissus et autres phénomènes d'autofluorescence. Dans les compositions chimiques étudiées, elles sont par ailleurs ré-excitables in vivo. Nous présentons une synthèse originale pour l'élaboration de " petites " NPs de ZnGa2O4 :Cr3+ par voie hydrothermale assistée par chauffage micro-onde qui peut être suivi d'un traitement thermique. Les caractérisations avancées des propriétés de luminescence persistante et la structure fine du matériau a montré une corrélation directe entre la luminescence et l'ordre local des cations dans la structure (répartition et distorsion des sites cationiques). Ainsi, nous avons montré l'effet bénéfique d'une calcination sans frittage sur l'amélioration des propriétés de luminescence. Nous avons également mis en évidence le gain apporté par l'ajout d'un codopant comme l'ion Bi3+ ou la modification de la matrice avec la substitution d'une partie des ions Ga3+ par des ions Ge4+. Les limites d'optimisation de ce matériau étant atteinte, une toute nouvelle voie a été ouverte dans cette thématique en dopant la matrice avec des ions Ni2+ pour déplacer la luminescence persistante dans le domaine du proche infrarouge. L'exploitation de cette nouvelle fenêtre pour l'imagerie optique devrait permettre d'imager plus profond dans les tissus avec un meilleur rapport signal/bruit. / This thesis aims to develop persistent luminescent nanoparticles (NPs) for optical imaging in the deep red and near infrared range. Their use has a clear advantage as the irradiation step can be performed before injection, avoiding tissue stress and other autofluorescence phenomena. The chemical compositions studied are also re-excitable in vivo. We propose an original way of synthesis for the elaboration of “small” ZnGa2O4:Cr3+ NPs. Hydrothermal synthesis assisted by microwave heating followed by a heat treatment is used to obtain the NPs. The extensive characterization of the persistent luminescence properties and of the material’s fine structure showed a direct correlation between luminescence and local cation order in the structure (distribution and distortion of cationic sites). Thus, we have shown the benefits of a heat treatment without sintering on the improvement of luminescence properties. We have also highlighted the enhancement provided by the addition of a codopant such as Bi3+ ions or the modification of the matrix by substituting a part of Ga3+ ions by Ge4+ ions. The optimization limits of this material being reached, doping the matrix with Ni2+ ions to move the persistent luminescence in the near infrared range, opens a new pathway in this research field. Exploiting this new window for optical imaging should allow imaging deeper into tissue with a better signal-to-noise ratio.

Les ions lanthanides possèdent des propriétés photophysiques particulières, qui ont suscité une forte attention dans le développement de sondes biologiques luminescentes. En effet, les avantages que présentent leurs émissions en raies fines et caractéristiques, ainsi que leurs temps de vie de luminescence longs, permettent diverses applications en imagerie. Cependant, le faible coefficient d’absorption molaire de ces éléments conduit à préférer une excitation par le biais d’une antenne capable de transférer l’énergie au métal.Par ailleurs, l’utilisation de l’absorption biphotonique a montré de nombreux avantages, puisqu’elle permet une excitation de la molécule à une longueur d’onde deux fois plus grande que l’excitation à un photon : cette longueur d’onde peut alors se situer dans la fenêtre de transparence biologique (650 - 1100 nm).Les travaux de cette thèse visent à combiner les avantages des lanthanides avec ceux de l’excitation biphotonique. Les complexes synthétisés sont basés sur une plateforme triazacyclononane substituée par des antennes à deux-photons. Leurs propriétés spectroscopiques à un photon ont été étudiées, et ont alors permis de réaliser des images cellulaires en multiplexing à deux photons utilisant Eu3+ et Tb3+. Des sondes biphotoniques de brillance optimisée ont également été obtenues pour Sm3+, Tb3+ et Dy3+, et appliquées à l'imagerie. L’étude approfondie d’un des complexes de terbium(III) a de plus mis en évidence sa sensibilité à la viscosité. L’analyse des propriétés spectroscopiques de cette sonde a permis d'en rationaliser le comportement photophysique, ouvrant ainsi la voie à des applications originales en imagerie fonctionnelle. Enfin, nous avons montré une internalisation rapide de complexes de lanthanide cationiques de type cyclen ou cyclam par des cellules vivantes, et l'étude de ces nouvelles structures offre de nouvelles perspectives dans le développement de bio-sondes de lanthanides. / Lanthanide ions demonstrate peculiar photophysical properties that has attracted a substantial attention in the development of luminescent bioprobes. Indeed, the advantages stemming from their sharp and characteristic emissions, as well as their long luminescence lifetimes, enable various imaging applications. However, the weak molar extinction coefficient of these elements results in a preferential excitation through an antenna, which transfers its energy to the metal.On the other hand, biphotonic absorption showed numerous advantages, since excitation of a molecule can be achieved at a wavelength twice as high as using one-photon excitation. This wavelength may therefore reach the optical transparency window (650 - 1100 nm).The work carried out during this PhD thesis aims at combining the advantages of lanthanides with those of biphotonic excitation. The synthesized complexes are based on a triazacyclononane platform substituted with two-photon antennae. Their one-photon spectroscopic properties have been studied, and enabled to perform two-photon multiplexed cellular imaging, using Eu3+ and Tb3+. Biphotonic probes with an optimized brightness have been obtained for Sm3+, Tb3+ and Dy3+, and applied to cell imaging. The thorough study of one of the terbium(III) complexes has moreover displayed evidence of a sensitivity towards viscosity. Analyzing the spectroscopic properties of this probe has therefore allowed to rationalize its photophysical behavior, paving the way for original functional imaging applications. Lastly, a rapid internalization of cationic lanthanide complexes with a cyclen or cyclam framework by living cells has been proved. Thus, the study of these new structures presents new perspectives for the development of lanthanide bioprobes.

Ce travail de thèse porte sur la caractérisation et l'optimisation du spot de focalisation d'un objectif de microscope de grande ouverture numérique pour des applications d'imagerie super-résolue et de nanofabrication.Dans la première partie, nous avons systématiquement étudié les distributions de polarisation et d'intensité du faisceau laser dans la région du point focal en fonction de différents paramètres du faisceau incident tels que la phase, la polarisation, l’amplitude ainsi que l'influence du milieu de propagation, tel que l’indice de réfraction. Nous avons mis en oeuvre différentes méthodes théoriques pour contrôler et manipuler les distributions de polarisation et d'intensité du spot de focalisation. Ces prédictions théoriques sont vérifiées expérimentalement via un système optique confocal en mesurant l’image de fluorescence d’une nanoparticule d’or pour différentes caractéristiques.Dans la seconde partie de ce travail, une nouvelle microscopie basée sur le mécanisme d'absorption ultra-faible à un photon a été démontré théoriquement et expérimentalement. Le calcul théorique basé sur l'approche vectorielle de Debye, qui prend en compte l'effet d'absorption du matériau, montre qu'il est possible de focaliser le faisceau lumineux en profondeur à l'intérieur d'un matériau si celui-ci présente une absorption linéaire ultra-faible à la longueur d'onde d'excitation. Cette méthode, dite (LOPA), a ensuite permis de fabriquer des structures 2D et 3D submicrométriques, similaires à celles obtenues par la méthode utilisant l’absorption à deux photons. / Nowadays, far field optical microscopy is widely used in many fields, for fundamental research and applications. The low cost, simple operation, high flexibility are its main advantages. The key parameter of an optical microscope is the objective lens.This thesis's work focuses mainly on the characterization and optimization of the point spread function (PSF) of a high numerical aperture (NA) objective lens (OL) for applications of high resolution imaging and nano-fabrication.In the first part of the thesis, we have systematically investigated the dependency of polarization and intensity distributions of the focusing spot on numerous parameters, such as the phase, the polarization, and the beam mode of incident beam, as well as the refractive index mismatch. Then, we demonstrated theoretically different methods for manipulation of the polarization and intensity distributions of the focusing spot, which can have desired shapes and are useful for different applications. By using a home-made confocal microscope, we have experimentally verified some of the theoretical predictions, for example, vector properties of light beam under a tight focusing condition. In the second part of dissertation work, a new, simple and inexpensive method based on the one-photon absorption mechanism has been demonstrated theoretically and experimentally for 3D sub-micrometer imaging and fabrication applications. The theoretical calculation based on vectorial Debye approximation and taken into account the absorption effect of material shows that it is possible to focus the light tightly and deeply inside the material if the material presents a very low one-photon absorption (LOPA) at the excitation wavelength. We have then demonstrated experimentally that the LOPA microscopy allows to achieve 3D imaging and 3D fabrication with submicrometer resolution, similar to those obtained by two-photon absorption microscopy.

Background: Functional electrical stimulation (FES) has been found to be effective in restoring voluntary functions after spinal cord injury (SCI) and stroke. However, the central nervous system (CNS) changes that occur in as a result of this therapy are largely unknown. Objective: To examine the effects of FES on the restoration of voluntary locomotor function of the CNS in a SCI rat model. Methods: SCI rats were instrumented with chronic FES electrodes in the hindlimb muscles and were divided into two groups: (a) FES therapy and (b) sedentary. At day 7 post-SCI, the animals were assessed for locomotion performance by using a Basso, Beattie and Bresnahan (BBB) scale. They were then anesthetized for a terminal in vivo experiment. The lumbar spinal cord and somatosensory cortex were exposed and the instrumented muscles were stimulated electrically. Associated neurovascular responses in the CNS were recorded with an intrinsic optical imaging system. Results: FES greatly improved locomotion recovery by day 7 post-SCI, as measured by BBB scores (P < 0.05): (a) FES 10 ± 2 and (b) controls 3 ± 1. Furthermore, the FES group showed a significant increase (P < 0.05) of neurovascular activation in the spinal cord and somatosensory cortex when the muscles were stimulated between 1 and 3 motor threshold (MT). Conclusion: Hind limb rehabilitation with FES is an effective strategy to improve locomotion during the acute phase post-SCI. The results of this study indicate that after FES, the CNS preserves/acquires the capacity to respond to peripheral electrical stimulation.

No description available.