L’utilisation de chromophores absorbant à deux photons (ADP) pour des applications en photothérapie dynamique (PDT) et en imagerie de fluorescence présente de nombreux avantages. Les propriétés non-linéaires de ces chromophores permettent notamment d’améliorer la longueur de pénétration dans les organismes vivants ainsi que la résolution. Pour des applications in-vivo la biocompatibilité de ces chromophores lipophiles doit aussi se poser. Une étude d’ingénierie pour le développement de chromophores pour la PDT-ADP en utilisant des atomes de brome comme groupe générateur d’oxygène singulet est décrite. Différents paramètres dont le nombre et la position des atomes de brome sur la chaîne carbonée, la longueur de conjugaison, la géométrie des chromophores ont été étudiés. Cette étude permet de mettre en évidence l’importance de la position des substituants bromes et de la symétrie sur le rendement de croisement inter-système.Les observations spectroscopiques faites lors de l’étude d’ingénierie ont permis de développer des chromophores pour la microscopie de fluorescence à deux photons. La biocompatibilité est apportée grâce à un polymère d’(hydroxyethyl)acrylate. Ce polymère permet de créer une coquille hydrosolubilisante covalente. Ces chromophores ont été utilisés pour faire de l’imagerie de vascularisation cérébrale de haute résolution. Une observation particulière sur un chromophore, marquage des cellules endothéliales des parois des vaisseaux sanguins intravitaux ainsi que les applications en résultant sont présentées. Des stratégies visant l’amélioration de la sélectivité des systèmes polymères/chromophores pour des applications intravitales, comme le traitement des tumeurs cancéreuses sont décrites. Une stratégie de modification des fonctions hydroxy des chaînes polymères par des groupements imidazoliums est présentée. L’étude de complexation des polymères avec l’ADN et les études spectroscopiques in-cellulo ont été réalisées. / The use of two-photon absorbing (TPA) chromophore for applications in photodynamic therapy (PDT) and fluorescence imaging provides many advantages. The non-linear properties make it possible to increase both observation depth in animals and 3D resolution. Nevertheless, for in-vivo applications, improving bio-compatibility of these inherently lipophilic chromophore is a challenge. The development of new chromophores for PDT-TPA using a molecular engineering approach using bromide substituents as singlet oxygen generators is described. Parameters like position and number of bromide, the conjugated length and chromophore symmetry are studied. The study shows the importance of bromide atom position and of the symmetry on the inter system crossing efficiency. During the engineering study, spectroscopic observation and rationalization permit to envision the design of new chromophores for two photon laser scanning fluorescent microscopy. Bio-compatibility of these chromophores is provided by (hydroxyethyl)acrylate polymer, which provides a covalent water-soluble shell. These chromophores are used to make high resolution image of cerebral vascularization. One of these chromophores shows intravital specific interaction with endothelial cells in blood vessels. Some applications of the chromophore are described. Strategies to increase the intravital selectivity of polymer/chromophores units towards cancer cells and tumor are presented. A modification of hydroxyl function by imidazolium group is described. This new chromophore is evaluated towards its complexation properties with DNA and in cellulo spectroscopic studies.

This thesis deals with the preparation, characterization methods and in vitro imaging of nanoparticles with great potential for medical diagnostics. A key role in this work occupy nanoparticles comprising a core of calcium tungstate, CaWO4, covered by a natural polysaccharide together serve as a suitable model contrast agent for cell imaging techniques. CaWO4 natural properties such as the absorption of X-rays, natural fluorescence capabilities and capacities for doping fluorescently active lanthanides, makes from nanoparticles suitable candidates for the position of contrast agents for imaging in the diagnosis of oncological diseases. Moreover, by coverage of the inorganic core by natural polysaccharide nanoparticles reach the desired size and the possibility of active targeting to specific or nonspecific interactions with the cell membrane, which are key factors in in vitro cell studies. It was found the most suitable synthesis both doped and undoped core of this model reaching the formation of spherical particles of colloidal dimensions. Doping was realized by red fluorescent trivalent europium, Eu3+ , and green fluorescent trivalent terbium, Tb3+ , and neither of these dopants affected the original crystalline structure CaWO4. In the next stage, these fluorescent nuclei successfully dispersed...

This thesis explores and expands upon theoretical means of quantifying a number of nonlinear spectroscopies, including two-photon absorption, resonant-inelastic x-ray scattering, Jones birefringence, and magnetic circular dichroism. On top of that, detailed information is given for the derivation and program implementation of damped cubic response functions. Complex-valued cubic response functions have been implemented in the quantum chemistry package DALTON, based on working equations formulated for an approximate-state wave function. An assessment of the implementation, such that for small frequencies the second-order hyperpolarizability should behave according to an analytic function that depends quadratically on the optical frequencies. It is demonstrated how two-photon absorption (TPA) can be described either through second-order transition moments or via the damped cubic response function. A few calculated TPA profiles are produced for a set of smaller molecules, in order to display the capability of the cubic response function in the x-ray frequency region. Resonance-inelastic x-ray scattering (RIXS) is explored in a similar manner as two-photon absorption. It is shown how the second-order hyperpolarizability can represent RIXS in the limit of intermediate-state and final-state resonances. Complications emerging from the complex dispersion of the hyperpolarizability are discussed. Moreover, linear birefringences, with focus on the Jones birefringence, are investigated for noble gases, monosubstituted benzenes, furan homologues, and a pure acetonitrile liquid. A linear relation between the Jones birefringence and the empirical para-Hammett constant as well as the permanent electric dipole moment is presented. Estimations of three linear birefringences --- Kerr, Cotton--Mouton, and Jones ---are obtained by averaging over a set of liquid snapshots. The Jones effect for acetonitrile turns out to be unusually large inmparison to the other two investigated linear birefringences. The final chapter of the thesis investigates magnetically induced circular dichroism (MCD). A question regarding relative stability of the first set of excited states for DNA-related molecular systems is resolved through MCD by exploiting the signed nature of circular dichroisms. Furthermore, to what extent solvent contributions affect MCD spectra is explored. The effect on uracil MCD spectrum due to thionation is studied, for which the degree of redshifting for systems 2-thiouracil and 4-thiouracil can be seen to be addative as compared to the 2,4-dithiouracil system. / <p>QC 20171129</p>

La limitation optique (LO) est un processus de protection des détecteurs optiques (yeux, caméras…) vis-à-vis des agressions laser. Le développement rapide de ces derniers aux fréquences des télécommunications a encouragé l’ingénierie moléculaire pour la LO dans cette fenêtre spectrale, en particulier à 1500 nm. Un des principaux mécanismes de la LO est l’absorption multiphotonique et plus particulièrement l’absorption à deux photons (ADP). Il s’agit d’un phénomène d’optique non-linéaire du troisième ordre au cours duquel un électron est promu de l’état fondamental vers l’état excité par absorption simultanée de deux photons. De plus, des travaux récents ont mis en évidence que le phénomène de LO peut être exalté par des processus d’absorption à l’état excité (AEE) induits par ADP. Dans ce contexte, cette thèse décrit la synthèse d’une nouvelle famille de chromophores de type aza-dipyrrométhène de bore (Aza-Bodipy), fonctionnalisés par des groupements donneur ou accepteur d’électrons. Ces molécules ont un comportement de limiteur optique sur toute la gamme spectrale voulue, avec un maximum d’efficacité vers 1300 nm. Les résultats ont été interprétés sur la base de deux phénomènes : l’ADP, suivie de l’AEE pour un procédé d’absorption globale à trois photons. Ces deux processus ont été mesurés séparément et des relations structure-activité ont été établies, afin de rationnaliser les données de LO. L’importance du transfert de charge et la superposition spectrale des deux phénomènes sont alors apparues comme les paramètres clefs à optimiser. En vue d’une application dans un dispositif optique réel, il est nécessaire que les chromophores soient incorporés dans un matériau solide, qui peut être poli et transformé. Ainsi, les composés synthétisés ont été insérés avec succès dans une matrice sol-gel. Pour la première fois, des matériaux de qualité optique ont été réalisés, possédant des propriétés de LO dans l’infra-rouge très performantes et meilleures qu’en solution. / The optical power limiting (OPL) is a protection process of detectors (eyes, cameras…) against laser aggression. The rapid development of frequency-tunable pulsed lasers up to telecommunication wavelengths led to the design of new materials for nonlinear absorption in this spectral range, in particular at 1500 nm. Mutiphotonic absorption, such as the two-photon absorption (TPA) is one of the mechanisms involved in the OPL. TPA is a third order nonlinear phenomenon that promotes a molecule to an excited state by the simultaneous absorption of two photons. Moreover, the overall OPL efficiency can be enhanced by excited state absorption (ESA). In this context, this thesis describes the synthesis of a new family of versatile near infra-red dyes, namely aza-borondipyrromethene (Aza-Bodipy) featuring several sites of functionalization by electro-donating or electro-withdrawing moieties. These molecules present the typical behavior of optical limiters at telecommunication wavelengths, with a maximal efficiency around 1300 nm. OPL curves were interpreted on the basis of two phenomena: TPA, followed by ESA for an overall 2+1 photons absorption. These two processes have been measured independently and structure-activity relationships have been established in order to rationalize OPL experiments. The charge transfer effect and the spectral overlap between TPA and ESA are therefore the key parameters. For practical use of OPL functions, it is required for the chromophores to be introduced into a solid material, which enables polishing and other post-processing. Therefore, Aza-Bodipy dyes have been successfully incorporated to a solid matrix using the sol-gel technique. For the first time, optical quality materials have been prepared featuring OPL properties in the infra-red, which are better than the ones in solution.

Les astrocytes protoplasmiques de la matière grise corticale sont des cellules gliales dont les prolongements très fins et ramifiés sont en contact avec les éléments neuronaux pré- et post-synaptiques d’une part, et les vaisseaux sanguins d’autre part. Ils expriment plusieurs récepteurs des neurotransmetteurs, entre autres des récepteurs purinergiques dont l'activation facilite l’activité calcique astrocytaire et la libération de gliotransmitters (par exemple, le glutamate, le GABA, l'ATP, et la D sérine) qui régulent l’activité des neurones et des cellules gliales situées au voisinage. L’objectif de ma thèse était d’étudier in situ l’activité calcique des astrocytes et de leurs prolongements en réponse à l’application des agonistes purinergiques. Lors de ma thèse, j’ai tout d'abord testé la possibilité d’induire l’expression spécifique de gènes d’intérêt par les astrocytes corticaux de souris adultes par la technique de recombinaison Cre-LoxP. J’ai comparé les performances d’un virus adeno-associé de type 5 (AAV5) flexé (AAV5.FLEX.EGFP) et d’une souris qui exprime un indicateur calcique (GCaMP3) sous contrôle de la recombinase (souris Rosa-CAG-LSL-GCaMP3). L’injection d’AAV5.FLEX.EGFP dans le cortex d’une souris hGFAPcre n’a pas permis l’expression spécifique d’EGFP. La combinaison des souris exprimant le cre recombinase sous contrôle d’un promoteur sélectif des astrocytes (GLAST-CreERT2 et Cx30-CreERT2) avec le AAV5.FLEX.EGFP ou avec une lignée des souris Rosa-CAG-LSL-GCaMP3 permet l’expression spécifique des gènes d’intérêt (EGFP et GCaMP3) par les astrocytes corticaux. J’ai ensuite analysé l’activité calcique des astrocytes qui expriment GCaMP3. J’ai utilisé la microscopie biphotonique et enregistré l’activité calcique spontanée et évoquée par application d’agonistes purinergiques sur des tranches de cortex somatosensoriel primaire de souris adultes GLAST-CreERT2. L’activité calcique spontanée est complexe, généralement locale et désynchronisée, répartie dans les prolongements et la région somatique. Les régions actives ont été identifiées à partir d’une carte de corrélation temporale calculée en MATLAB, et leurs caractéristiques (amplitude, durée, position, fréquence) mesurées grâce à des routines établies sous IGOR. La fréquence et l’amplitude de l’activité calcique paraissent augmenter lors de l’enregistrement, ce qui suggère une sensibilité significative et une photoactivation des astrocytes, en imagerie biphotonique. La durée des impulsions laser modulerait ce phénomène. En présence d'adénosine (1-100 µM) et d’ATP (100 µM), et de façon marginale en présence d’un agoniste P2X7 non sélectif (BzATP 50-100 µM), une activité calcique synchronisée accrue est visible dans le soma et les prolongements astrocytaires en présence de tétrodotoxine qui bloque les potentiels d'action et minimise l’activité synaptique. Le mécanisme de ces réponses synchronisées reste à étudier. Aucun effet significatif n’a été observé en présence d’un agoniste spécifique P2Y1 (MRS2365 50 uM). Mon travail a permis le développement : i) de modèles murins pour l’adressage sélectif de protéines d’intérêt au niveau des astrocytes protoplasmiques ; ii) d’outils d’analyse des signaux calciques astrocytaires au niveau sub-cellulaire. Il a mis en évidence des limites possibles des protocoles standards d'enregistrement de l’activité calcique des astrocytes en imagerie biphotonique. Il confirme l’importance de l’ATP et de l’adénosine pour la signalisation astrocytaire. / Grey matter protoplasmic astrocytes are compact glial cells with highly branched processes, enwrapping synapses, and one or two endfeet contacting the blood vessels. Several neurotransmitter receptors are expressed by astrocytes, among them purinergic receptors. Upon activation of these receptors, intracellular calcium (Ca2+) transients can be induced, that, in turn, trigger gliotransmitter release (e.g. glutamate, GABA, ATP, D-serine) and participate in astrocyte-to-astrocyte signaling as well as in the communication between astrocytes and neurons or other glia. During my PhD work, I first implemented and validated several approaches for targeting transgene expression specifically to cortical astrocytes and employed them to study purinergic signaling in astrocytes. To achieve astrocyte-specific transgene expression, I used either floxed adeno-associated viral (AAV) vectors or a Cre-dependent mouse line and several mouse lines expressing the Cre recombinase under astrocyte-specific promoters. Intracerebral injections of a Cre-dependent AAV serotype 5 containing the ubiquitous CAG promoter and an enhanced green fluorescent protein (AAV5.CAG.flex.EGFP) in adult mice expressing Cre recombinase under the human glial fibrillary protein (hGFAP) promoter resulted in a non-astrocyte specific expression in the cortex. Combining inducible mouse lines expressing Cre recombinase under the glutamate aspartate transporter (GLAST) promoter with the same AAV vector resulted in a virtually astrocyte-specific expression of the reporter gene. As an alternative approach for astrocyte-specific transgene expression, we used a Cre-dependent mouse line expressing the genetically encoded Ca2+ indicator GCaMP3. Crossing this mouse line with the above described GLAST-CreERT2 mouse line or a Connexin30 (Cx30)-CreERT2 line led to selective GCaMP3 expression in cortical astrocytes. Second, I investigated both spontaneous and agonist-evoked Ca2+ transients in astrocytic processes, the investigation of which has presented a major challenge in earlier studies, due to the unspecific and weak labeling by membrane-permeable chemical Ca2+ indicators. Using the strategy developed in the first part of my work allowing an astrocyte-specific expression of the genetically encoded Ca2+ indicator GCaMP3. Using two-photon excitation fluorescence (2PEF) imaging in acute slices of the primary somatosensory cortex, I recorded Ca2+ transients in the astrocytic soma and processes. By aid of a custom-made MATLAB routine based on a temporal Pearson correlation coefficient, active regions could be identified in an unbiased manner. Evoked Ca2+ transients were quantified using custom IGOR routines. Spontaneous desynchronized Ca2+ transients occurred in the processes and rarely in the soma. Ca2+ signals appeared localized in distinct microdomains. Their frequency appeared to increase during long recordings of several hundred images, suggesting that fine astrocytes are vulnerable to photodamage under imaging conditions routine in 2PEF microscopy. The possibility to minimize photodamage, by varying the length of the femtosecond laser pulses is under investigation. Bath application of adenosine (1-100 µM) and adenosine-triphosphate (ATP, 100 µM), as well as the application of the non-selective P2X7 receptor agonist (2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate, BzATP, 50-100 µM), in the presence of tetrodotoxin to block neuronal action potentials, evoked synchronized Ca2+ rises in the soma and the processes of astrocytes. The effect of adenosine was dose-dependent. No significant effect of the specific P2Y1 agonist (MRS2365, 50 µM) was seen. Altogether, my work sets up a powerful and versatile toolbox for studying astrocytic Ca2+ signaling at the sub-cellular level. It also pinpoints possible limits of standard two-photon recording protocols to investigate the local Ca2+ signals in fine astrocytic processes.

Le moment orbital angulaire (OAM) de la lumière est une grandeur quantifiée associée à la phase d’un vortex optique et est actuellement une des variables explorées pour les technologies quantiques.Dans ce contexte, cette thèse étudie expérimentalement la conversion de vortex optiques par une vapeur de rubidium, via la transition Raman stimulée à deux photons 5S₁/₂ − 5D₅/₂. Quand les atomes sont soumis à deux lasers respectivement à 780 nm et 776 nm, ils génèrent des rayonnements cohérents, infrarouge à 5,23 μm et bleu à 420 nm. On examine le rayonnement bleu lorsque l’un des lasers ou les deux sont des vortex, en particulier des modes de Laguerre-Gauss. Dans une première partie nous montrons que si l’OAM est porté par le laser à 776 nm, alors le rayonnement bleu émis porte un OAM qui respecte l’accord de phase azimutale et de phase de Gouy. Nous montrons aussi que la conversion est efficace sur une grande plage d’OAM allant de -50 à +50, que l’efficacité est gouvernée par le produit des intensités des lasers incidents et que le rayonnement bleu se comporte comme un mode de Laguerre-Gauss pur. Dans une deuxième partie nous montrons qu’il est possible de convertir une superposition de vortex ou une paire de vortex coaxiaux et que l’OAM du rayonnement bleu émis obéit à la règle de somme des OAM incidents. Pour les cas étudiés, nous proposons un modèle de mélange à quatre ondes qui établit les règles de sélection du processus de conversion d’OAM. Ce travail ouvre la voie vers la conversion d’OAM utilisant des transitions vers des niveaux atomiques plus élevés. / The orbital angular momentum of light (OAM) is a quantized quantity arising from the azimuthal phase carried by optical vortices and is well-known for quantum technology applications. Its set of values is theoretically infinite.In this context this thesis experimentally study the conversion of optical vortices in a rubidium vapor through the 5S₁/₂ − 5D₅/₂ stimulated Raman transition. When the atoms are illuminated with laser beams at 780 nm and 776 nm they generate two coherent light beams at 5,23 μm and 420 nm. We investigate the blue light when one laser or both are optical vortices, in particular Laguerre-Gaussian modes. In a first part we show that if the laser at 776 nm carries an OAM the blue light is an optical vortex with an OAM which respects azimutal and Gouy phase matchings. We further show that the conversion is efficient on a large set of OAM from -50 to +50, that the efficiency is governed by the product of the input laser intensities and that the blue light behaves like a pure Laguerre-Gaussian mode. In a second part we demonstrate the conversion of a vortex superposition or a pair of coaxial vortices and that the OAM of the emitted light obeys the conservation rule of total OAM. For each studied case we propose a four wave mixing model establishing selection rules for the conversion process. This work opens possibilities towards OAM conversion using higher atomic levels.

V oblasti nových nízkomolekulárních organických materiálů patří deriváty difenyldiketopyrrolopyrrolu (DPP), používané dříve jako barviva a pigmenty, k objektům vysokého zájmu pro jejich potencionální aplikace v moderních technologiích. Studium jejich optických vlastností ve vztahu k jejich chemické struktuře umožní využití jejich vysokého potenciálu ve vývoji pokročilých inteligentních materiálů. Přehled chemických a fyzikálních vlastností DPP derivátů a zhodnocení současného stavu řešené problematiky jsou uvedeny v teoretické části této práce. Tři hlavní procesy studované v této práci jsou: klasická absorpce a emise, dvoufotonová absorpce (TPA) a zesílená spontánní emise (ASE). Výsledky budou diskutovány a shrnuty ve dvou částech: první zahrnuje první dvě výše zmíněné oblasti a druhá problematiku zesílené spontánní emise.

<div>Vibrational spectroscopic imaging has become an emerging platform for chemical visualization of biomolecules and materials in complex systems. For over a century, both Raman and infrared spectroscopy have demonstrated the capability to recognize molecules of interest by harnessing the characteristic features from molecular fingerprints. With the recent development of hyperspectral vibrational spectroscopy imaging, which records the chemical information without sacrificing the spatial-temporal resolution, numerous discoveries has been achieved in the field of molecular and cellular biology. Despite the ability to provide complimentary chemical information to Raman-based approaches, infrared spectroscopy has not been extensively applied in routine studies due to several fundamental limitations: 1). the poor spatial resolution; 2). inevitable strong water absorption; 3). lack of depth resolution.</div><div>Mid-infrared photothermal (MIP) microscopy overcame all the above mentioned problems and for the first time, enabled depth-resolved in vivo infrared imaging of live cells, microorganisms with submicrometer spatial resolution. The development of epi-detected MIP microscopy further extends its application in pharmaceutical and materials sciences. With the deployment of difference frequency generation and other nonlinear optical techniques, the spectral coverage of the MIP microscopy was significantly enhanced to enable chemical differentiation in complex systems across the broad mid-infrared region. In addition to the efforts to directly improve the performance of MIP microscopy, a novel quantitative phase imaging approach based on polarization wavefront shaping via custom-designed micro-retarder arrays was developed to take advantage of the highly sensitive phase measurement in combination with the photothermal effect. Besides, the extended depth-of-field and multifocus imaging enabled by polarization wavefront shaping could both improve the performance of MIP microscopy for volumetric imaging.</div>

The development of therapies promoting recovery after spinal cord injury is a challenge. Alginate hydrogels offer the possibility to develop biocompatible implants with mechanical properties tailored to the nervous tissue, which could provide a permissive environment for tissue repair. Here, the effects of non-functionalized soft calcium alginate hydrogel were investigated in a rat model of thoracic spinal cord hemisection and compared to lesioned untreated controls. Open field locomotion tests were employed to evaluate functional recovery. Tissue analysis was performed with label-free multiphoton microscopy using a multimodal approach that combines coherent anti-Stokes Raman scattering to visualize axonal structures, two-photon fluorescence to visualize inflammation, second harmonic generation to visualize collagenous scarring. Treated animals recovered hindlimb function significantly better than controls. Multiphoton microscopy revealed that the implant influenced the injury-induced tissue response, leading to decreased inflammation, reduced scarring with different morphology and increased presence of axons. Demyelination of contralateral white matter near the lesion was prevented. Reduced chronic inflammation and increased amount of axons in the lesion correlated with improved hindlimb functions, being thus relevant for locomotion recovery. In conclusion, non-functionalized hydrogel improved functional outcome after spinal cord injury in rats. Furthermore, label-free multiphoton microscopy qualified as suitable technique for regeneration studies.

In the last decades, additive manufacturing (AM), also called three-dimensional (3D) printing, has advanced micro/nano-fabrication technologies, especially in applications like lightweight engineering, optics, energy, and biomedicine. Among these 3D printing technologies, two-photon polymerization (TPP) offers the highest resolution (even at the nanometric scale), reproducibility and the possibility to create monolithically 3D complex structures with a variety of materials (e.g. organic and inorganic, passive and active). Such active materials change their shape upon an applied stimulus or degrade over time at certain conditions making them dynamic and reconfigurable (also called 4D printing). This is particularly interesting in the field of medical microrobotics as complex functions such as gentle interactions with biological samples, adaptability when moving in small capillaries, controlled cargo-release profiles, and protection of the encapsulated cargoes, are required. Here we review the physics, chemistry and engineering principles of TPP, with some innovations that include the use of micromolding and microfluidics, and explain how this fabrication schemes provide the microrobots with additional features and application opportunities. The possibility to create microrobots using smart materials, nano- and biomaterials, for in situ chemical reactions, biofunctionalization, or imaging is also put into perspective. We categorize the microrobots based on their motility mechanisms, function, and architecture, and finally discuss the future directions of this field of research.