The majority of fast excitatory neurotransmission in the brain occurs at glutamatergic synapses. The extensive dendritic arborisations of pyramidal neurons in the neocortex and hippocampus harbor thousands of synaptic connections, each formed on tiny protrusions called dendritic spines. Spine synapses are rapidly established during early postnatal development – a key period in neural circuit assembly – and are subject to dynamic activity-dependent plasticity mechanisms that are believed to underlie neural information storage and processing for learning and memory. Recent decades have seen remarkable progress in identifying diverse plasticity mechanisms responsible for regulating synapse structure and function, and in understanding the processes underlying computation of synaptic inputs in the dendrites of individual neurons. These advances have strengthened our understanding of the biological mechanisms underlying brain function but, not surprisingly, they have also raised many new questions. Using a combination of whole-cell electrophysiology, 2-photon imaging and glutamate uncaging in rodent brain slice preparations, I have helped to document the subtype-specific regulation of glutamate receptors during a homeostatic form of synaptic plasticity at CA1 pyramidal neurons of the hippocampus, and have discovered novel synaptic calcium dynamics during a critical period of neural circuit formation. First, we found that during a homeostatic response to prolonged inactivity, both AMPA and NMDA subtypes of glutamate receptors undergo a switch in subunit composition at synapses, but exhibit a divergence in their subcellular localization at extrasynaptic regions of the plasma membrane (this work was published in the Journal of Neuroscience in 2013). In separate series of experiments using 2-photon calcium imaging, I discovered a functional coupling between NMDA receptor activation and intracellular calcium release at dendritic spines and dendrites that is selectively expressed during a critical period of synapse formation. This synaptic calcium signaling mechanism enabled the transformation of distinct spatiotemporal patterns of synaptic input into salient biochemical signals, and is thus apt to locally regulate synapse development along individual dendritic branches. Consistent with this hypothesis, I found evidence for non-random clustering of synapse development between neighboring dendritic spines. Together, these experimental results expand the current understanding of the dynamics of synapse function during homeostatic plasticity and early postnatal development. --- Les synapses glutamatergiques soutiennent la majorité de la neurotransmission excitatrice rapide du cerveau. Des milliers de ces synapses, localisées sur de minuscules saillies appelées épines dendritiques, décorent les vastes arborisations dendritiques des neurones pyramidaux du néocortex et de l'hippocampe. Ces synapses sont formées tôt lors du développement postnatal et sont soumises à des mécanismes dynamiques de plasticité qui sous-tendent, croit-on, les capacités d'apprentissage et de mémoire du cerveau. Les dernières décennies ont vu des progrès remarquables dans l'identification de divers mécanismes de régulation de la structure et de la fonction des synapses sur différentes échelles de temps, et dans la compréhension des processus qui régissent l’intégration des inputs synaptiques au niveau des dendrites individuelles. Ces progrès ont renforcé notre compréhension des éléments fondamentaux régissant la fonction cérébrale et ont ouvert de nouvelles voies d’investigations neurophysiologiques. En utilisant une combinaison d’électrophysiologie cellulaire, d'imagerie à deux-photons et de photolibération de glutamate sur des neurones pyramidaux de la région CA1 de l'hippocampe de rats, j’ai contribué à la découverte et à la caractérisation de nouvelles régulations des récepteurs du glutamate durant la plasticité synaptique homéostatique. J’ai également découvert un nouveau type de dynamique de calcium synaptique relié à une organisation spatiale du développement des synapses pendant une période critique de l’ontogénie des circuits neuronaux. Dans la première étude, nous avons constaté que lors d'une plasticité de type homéostatique induite par une inactivité prolongée, les récepteurs de glutamate de types AMPA et NMDA sont soumis à un changement important dans la composition de leurs sous-unités. De plus, nous avons observé un ciblage différentiel de ces récepteurs vers des compartiments subcellulaires spécifiques des neurones. Dans une série d'expériences séparée utilisant l’imagerie calcique à deux-photons, j’ai découvert un couplage fonctionnel durant le développent entre l'activation des récepteurs NMDA et une libération de calcium intracellulaire qui envahit tant les épines dendritiques que les dendrites. J’ai également trouvé que ce mécanisme de signalisation de calcium synaptique transforme des motifs spatiotemporels d’activités synaptiques spécifiques en signaux biochimiques post-synaptiques de manière à potentiellement réguler l’organisation spatiale des synapses durant le développement. Conformément à cette hypothèse, j’ai observé des manifestations fonctionnelles claires de regroupement dans l’espace de synapses de forces similaires le long de branches dendritiques individuelles. Ensemble, ces résultats expérimentaux élargissent notre compréhension actuelle de de la fonction des synapses durant la plasticité homéostatique ainsi que durant le développement postnatal du cerveau. En étudiant les mécanismes neurophysiologiques de base, il sera possible d'avoir un aperçu plus profond du fonctionnement du cerveau et de ses pathologies.

Polarization-dependence provides a unique handle for extending the dimensionality of optical microscopy, with particular benefits in nonlinear optical imaging. Polarization-dependent second order nonlinear optical processes such as second harmonic generation (SHG) provide rich qualitative and quantitative information on local molecular orientation distribution. By bridging Mueller and Jones tensor, a theoretical framework was introduced to experimentally extend the application of polarization-dependent SHG microscopy measurements toward in vivo imaging, in which partial polarization or depolarization of the beam can complicate polarization analysis. In addition, polarization wavefront shaping was demonstrated to enable a new quantitative phase contrast imaging strategy for thin transparent samples. The axially-offset differential interference contrast microscopy (ADIC) was achieved as a combination of classic Zernike phase contrast and Nomarski differential interference contrast (DIC) methods. The fundamentally unique manner of this strategy also inspired rapid volumetric analysis in time dimension that is accessible for most existing microscopy systems. Finally, the dimensionality of high speed twophoton fluorescence imaging was extended to the spectral domain by spatial/spectral multiplexing, enabling beam scanning two photon fluorescence microscopy with 17 frames per second rate and over 2000 effective spectral data points.<br>

L’étude des phénomènes biologiques et notamment du rôle des protéines au sein d’un mécanisme cellulaire est un défi pour les biologistes. L’avènement de la microscopie de fluorescence à excitation biphotonique et des techniques « super-résolutives » a permis l’amélioration des performances des techniques de microscopie classiques et l’application l’imagerie in vivo pour l’analyse des tissus biologiques. Ces techniques requièrent cependant l’emploi de sondes aux propriétés photophysiques optimisées en complément de la spécificité vis-à-vis de la ou des cible(s) biologique(s). L’epicocconone, une molécule naturelle profluorescente de la famille des azaphilones, est employée en protéomique pour la détection des protéines sur gel d’électrophorèse. Ce composé a la faculté de réagir avec les amines des résidus lysine des protéines pour former un adduit covalent énaminone hautement fluorescent dans le proche infrarouge (610 nm) sous irradiation UV (395 nm) ou visible (520 nm). Différents analogues synthétisés au sein du laboratoire ont permis d’étudier la relation structure-fluorescence de ces composés capables de détecter non spécifiquement les protéines du milieu étudié. Afin d’améliorer la spécificité de ces molécules en vue d’applications en imagerie, la synthèse de sondes polyfonctionnelles, via une réaction de cycloaddition 1,3 dipolaire azoture-alcyne catalysée au cuivre, associant un analogue de l’epicocconone à un agent de reconnaissance possédant une affinité particulière pour une cible biologique a été étudiée. La synthèse d’analogues de l’epicocconone optimisés pour l’absorption biphotonique a également été réalisée lors de cette thèse. Les propriétés optiques linéaires et non linéaires de ces composés ont été étudiées afin de sélectionner le meilleur composé pour des applications en imagerie par microscopie de fluorescence à excitation biphotonique. Enfin, la réactivité particulière des azaphilones a servi de point de départ au développement d’une stratégie d’identification et d’isolement de nouvelles molécules naturelles azaphiles grâce à l’utilisation de sondes à produits naturels capables de cibler spécifiquement ce type de composés. / Understanding biological processes that involve proteins is a challenge for biologists. Two-photon excitation and super-resolution microscopy have improved drastically bioimaging techniques allowing in vivo deep tissue analysis. However those techniques require the use of optimized and selective fluorescent probes. Epicocconone is a natural profluorescent azaphilone widely used in proteomics for detecting proteins on electrophoresis gels. This compound can react reversibly with primary amines from lysines forming a covalent enaminone adduct that emits near infrared fluorescence light (610 nm) upon UV (395 nm) or visible (520 nm) excitation. Different analogues, that non-selectively bind to proteins, were previously synthesized in order to understand the structure-fluorescence relationship. The synthesis of polyfunctionnal probes was studied using copper-catalyzed azide-alkyne cycloaddition to connect the epicocconone scaffold to a recognition moiety, which can specifically recognise one biological target. New analogues optimized for two-photon absorption were synthesized. Their linear and non-linear optical properties were determined to select the most suitable molecule for two-photon excitation microscopy.In a last part, the particular reactivity of azaphilones was also regarded as a useful strategy for designing probes which could react specifically with azaphilic natural products in order to identify and isolate new ones

Fluorescent proteins (FPs) are the workhorses of biological molecular imaging. Important imaging modalities (such as polarization microscopy or FRET imaging) exploit anisotropic optical properties of fluorescent proteins. In this thesis, we present the results of our polarization microscopy and X-ray diffraction experiments on FP crystals, as well as mathematical interpretation of these results, yielding information on the directionality of one- and two-photon absorption within the investigated fluorescent protein molecules. For the anisotropy of one-photon absorption, we determine the transition dipole moment (TDM) orientations in three representative fluorescent proteins. Validation with available quantum mechanical predictions values and an experimentally determined TDM orientation of the GFP gives confidence to the results obtained. For the two-photon absorption, we first test our hypothesis that two-photon absorptivity tensors of representative FPs exhibit vector-like behaviour and then examine the applicability of this simplification as a basis for the interpretation of our two-photon polarization microscopy data.

Printing of 3-dimensional nanostructures with high-resolution by two-photon polymerization has gained significant attention recently. Isopropyl thioxanthone (ITX) has been studied and used as a photoinitiator because of its unique property in initiating and depleting polymerization, but to further improve the resolution of 3D structures, new photoinitiating materials are necessary to decrease the power requirements especially in industrial world. In this dissertation, different new types of thioxanthone-based photoinitiators were synthesized and our new initiators possessed a clear enhancement in terms of excitation over ITX. To clearly reveal the writing mechanism behind it, the behavior of the initiators was evaluated by several methods such as low temperature phosphorescence spectroscopy and density functional theory (DFT) calculations. The first type of new molecules with alkyne bridge will be discussed in chapter 2 and the further developed initiators with electron donating and withdrawing groups will be discussed in chapter 3. By modifying the structure of ITX, we have revealed and proposed an important pathway to guide future development of photoinitiators in direct laser writing.

The effects of surface plasmon for InGaN/GaN multi-quantum wells and ZnO nanoparticles optical linear and nonlinear emission efficiency had been experimentally studied. Due to the critical design for InGaN MQWs with inverted hexagonal pits based on GaN, both contribution of surface plasmon effect and image charge effect at resonant and off resonant frequencies were experimentally and theoretically investigated. With off- resonant condition, the InGaN MQWs emission significantly enhanced by metal nanoparticles. This enhancement was caused by the image charge effect, due to the accumulation of carriers to NPs region. When InGaN emission resonated with metal particles SP modes, surface Plasmon effect dominated the emission process. We also studied the surface plasmon effect for ZnO nanoparticles nonlinear optical processes, SHG and TPE. Defect level emission had more contribution at high incident intensity. Emissions are different for pumping deep into the bulk and near surface. A new assumption to increase the TPE efficiency was studied. We thought by using Au nanorods localized surface plasmon mode to couple the ZnO virtual state, the virtual state’s life time would be longer and experimentally lead the emission enhancement. We studied the TPE phenomena at high and near band gap energy. Both emission intensity and decay time results support our assumption. Theoretically, the carriers dynamic mechanism need further studies.

Rewritable photochrome-based 3D optical data storage requires photochromic molecules with high two-photon absorption (2PA) cross sections. Currently, the low value of two-photon absorption cross sections of existing photochromes makes them unsuitable for practical application in 3D data storage. Worldwide attempts to increase the cross section of photochromic molecules by altering the chemical structure have yielded poor results. In this work, two ways to increase the two-photon absorption cross sections of photochromes were investigated. In the first method, partial success demonstrated by extending the conjugation of a photochromic molecule, a high two-photon absorption cross section of the closed form isomer and high photoconversion to the closed form were realized. At the same time, a decrease in photoswitching quantum yield and low photoconversion to open form was observed. A discussion is provided to explain the results, suggesting that the proposed method of extending the conjugation may not solve the problem. For this reason a new method for effective two-photon absorption cross section enhancement of photochromes was proposed. As a proof of principle, a new two-photon absorbing dye with a hydrogen bonding moiety was synthesized and used for the formation of supramolecular structures with a photochromic compound. Theoretical reasoning and experimental demonstration of energy transfer from the dye to the photochrome under one and two-photon excitation confirmed the practical value of the method. The effects of a 2PA dye on the photochromic properties of a diarylethene were investigated using a model compound to simplify data analysis. Formation of supramolecular structures was revealed using ¹H NMR spectroscopic methods. The model compound, having the same hydrogen bonding moiety as 2PA dye, has been demonstrated to bind with photochrome molecules at very low concentrations. Photochromic properties of 2,3-bis(2,4,5-trimethyl-3-thienyl)maleimide, including conversions at the photostationary state, extinction coefficients, photoisomerization reaction rates and quantum yields, were shown to be affected by hydrogen bonding with the model compound - 2,6-bis-(acetamido)pyridine. The extent of this change was determined and discussed, demonstrating a balanced supramolecular strategy to modulate photochemical and photophysical properties of this important class of photochromic material.

Metal Organic Materials (MOM) are composed of transition metal ions as connectors and organic ligands as linkers. MOMs have been found to have high porosity, catalytic, and optical properties. Here we study the gas adsorption, color change, and non-linear optical properties of MOMs. These properties can be predicted using theoretical methods, and the results may provide experimentalists with guidance for rational design and engineering of novel MOMs. The theory levels used include semi-empirical quantum mechanical calculations with the PM7 Hamiltonian and, Density Functional Theory (DFT) to predict the geometry and electronic structure of the ground state, and Time Dependent DFT (TD-DFT) to predict the excited states and the optical properties. The molecular absorption capacity of aldoxime coordinated Zn(II) based MOMs (previously measured experimentally) is predicted by using PM7 Theory level. The 3D structures were optimized with and without host molecules inside the pores. The absorption capacity of these crystals was predicted to be 8H2 or 3N2 per unit cell. When going beyond this limit, the structural integrity of the bulk material becomes fractured and microcrystals are observed both experimentally and theoretically. The linear absorption properties of Co(II) based complexes are known to change color when the coordination number is altered. In order to understand the mechanism of this color change TD-DFT methods are employed. The chromic behavior of the Co(II) based complexes studied was confirmed to be due to a chain in coordination number that resulted in lower metal to ligand distances. These distances destabilize the occupied metal d orbitals, and as a consequence of this, the metal to ligand transition energy is lowered enough to allow the crystals to absorb light at longer wavelengths. Covalent organic frameworks (COFs) present an extension of MOM principles to the main group elements. The synthesis of ordered COFs is possible by using predesigned structures andcarefully selecting the building blocks and their conditions for assembly. The crystals formed by these systems often possess non-linear optical (NLO) properties. Second Harmonic Generation (SHG) is one of the most used optical processes. Currently, there is a great demand for materials with NLO optical properties to be used for optoelectronic, imaging, sensing, among other applications. DFT calculations can predict the second order hyperpolarizability ?2 and tensor components necessary to estimate NLO. These calculations for the ?2 were done with the use of the Berry's finite field approach. An efficient material with high ?2 was designed and the resulting material was predicted to be nearly fivefold higher than the urea standard. Two-photon absorption (2PA) is another NLO effect. Unlike SHG, it is not limited to acentric material and can be used development of in vivo bio-imaging agents for the brain. Pt(II) complexes with porphyrin derivatives are theoretically studied for that purpose. The mechanism of 2PA enhancement was identified. For the most efficient porphyrin, the large 2PA cross-section was found to be caused by a HOMO-LUMO+2 transition. This transition is strongly coupled to 1PA allowed Q-band HOMO-LUMO states by large transition dipoles. Alkyl carboxyl substituents delocalize the LUMO+2 orbital due to their strong ?-acceptor effect, enhancing transition dipoles and lowering the 2PA transition to the desirable wavelengths range. The mechanism 2PA cross-section enhancement of aminoxime and aldoxime ligands upon metal addition of is studied with TD-DFT methods. This mechanism of enhancement is found to be caused by the polarization of the ligand orbitals by the metal cation. After polarization an increase in ligand to ligand transition dipole moment. This enhancement of dipole moment is related to the increase in 2PA cross-sections.

The pursuit of more suitable drugs intended for possible biological applications are a continuously growing topic of research within the scientific community. One of these suitable qualities includes the need for hydrophilicity and or some appropriate delivery system for the drug to enter into biological systems. A system of analyzing and following these compounds would then, however, be necessary to conduct any kind of mechanistic or interaction studies for he said drug within the biological system. Just to name a few, fluorescence and X-ray computed tomography (CT) methods allow for imaging of biological systems but require the need of compounds with specific qualities. Finally, even with a means of entering and following a oaded drug, it would not be complete without a way of targeting its intended location. Herein, the first chapter reports the synthesis and characterization of a fluorene-based pyridil bis-?-diketone compound with suitable one- and two-photon fluorescent properties and its encapsulation into Pluronic F127 micelles for the possible application of tracking lysosomes. Next the synthesis and characterization of a BODIPY-based fluorophore with excellent fluorescence ability is reported. This compound was conjugated to two triphenylphosphine (TPP) groups and is shown as a potential mitochondria probe within HCT-116 cells. Finally, the synthesis and characterization of diatrizoic acid (DA) based derivatives conjugated to silica nanoparticles, as well as unconjugated, are reported as potential CT contrast agents. The derivatives were also functionalized with maleimide moieties facilitating subsequent potential bioconjugation of a targeting protein via a thiol group.

Understanding cell migration is a key step in unraveling many physiological phenomena and predicting several pathologies, such as cancer metastasis. In particular, confinement has been proven to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists’ analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical and spatial stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely the soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in vitro migration studies under spatial confinement in microfluidic device