Дисертації з теми "Microscope multiphotonique"
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Guillemant, Marie. "Development of a three-photon microscope for awake and behaving non-human primates." Electronic Thesis or Diss., université Paris-Saclay, 2023. http://www.theses.fr/2023UPASL025.
Повний текст джерелаMulti-photon microscopy has become a standard technique to study the structural and functional activity in mice but it faces obstacles to be applied in larger animals. It would be particularly advantageous to be able to apply it to macaque monkeys, as they are the animal model of choice to understand the neural mechanisms of high-level cognitive functions such as selective attention, working memory and consciousness. One of the main limiting factors for imaging in larger animals is the dura mater. This tough and opaque layer of tissue protects the brain but is so thick in larger animals that it obstructs imaging. It is therefore commonly removed but this leads to a highly invasive and unstable preparation. The main aim of the current work is to investigate the possibility to record functional activity from the cortex of the rhesus macaque monkey through the natural dura.A multi-photon microscopy setup has been designed with a two-photon and a three-photon microscopy optical paths to record from awake macaque monkeys. The repetition rate of the laser is 2MHz which allows a maximum imaging depth inside the cortex of 520µm at 960nm and 715µm at 1300nm with an additional 120µm-thick layer of dura mater at the surface. Resonance-galvo scanning is used to allow a maximal frame rate of 15.6Hz at a field of view of 620x630µm². In addition to the setup, surgical implants have been developed for long-term and awake imaging.Using an ex vivo study of dura mater from a macaque monkey, the induced optical aberrations are studied by measuring the decrease in spatial resolution of the setup for a varying thickness of dura mater. This reveals that it has no significant impact on the spatial resolution for a thickness up to 150µm at 1300nm. The effective attenuation length of the dura mater is estimated to be 56.5±10.1µm at 960nm and 80.7±5.3µm at 1300nm. These measurements are used to model the maximum imaging depth that can be reached according to the repetition rate of the laser and the thickness of the dura.This model is adjusted and validated using in vivo data from two non-human primates. The effective attenuation length of the natural dura mater and of a regrowth of tissue following a durectomy (called a 'neomembrane') are investigated. Functional recordings have been performed in mice and preprocessed using Suite2P. Viral injection parameters have been tested in three macaque monkeys and we have so far recorded the in vivo structural and functional activity of neurons in one. Finally, the comparison between the use of two- and three-photon microscopy to study non-human primates is discussed. In conclusion, we have set up and optimized a multi-photon microscope for long-term awake imaging of the cortex of non-human primates and shown that it was possible to record down to over 700µm into the cortex (which corresponds to the layers L2/L3) while imaging through the natural dura mater or a neomembrane
Thériault, Gabrielle. "Développement d'un microscope à grande profondeur de champ pour l'imagerie fonctionnelle de neurones dans des échantillons épais." Doctoral thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/25740.
Повний текст джерелаOne of the greatest challenges of modern neuroscience that will lead to a better understanding and earlier diagnostics of brain sickness is to decipher the details of neuronal interactions in the living brain. To achieve this goal, we must be capable of observing populations of living cells in their original matrix with a good resolution, both spatial and temporal. Two-photon microscopy offers the right tools for this since it presents with a spatial resolution in the order of the micron. Unfortunately, this very good three-dimensional resolution lowers the temporal resolution because the optical sectioning caused by the microscope's small depth of field forces us to scan thick samples repeatedly when acquiring data from a large volume. In this doctoral project, we have designed, built and characterized a two-photon microscope with an extended depth of field with the goal of simplifying the functional imaging of neurons in thick samples. To increase the laser scanning microscope's depth of field, we shaped the laser beam entering the optical system in such a way that a needle of light is generated inside the sample instead of a spot. We modify the laser beam with an axicon, a cone-shaped lens that transforms a gaussian beam into a quasi non-diffractive beam called Bessel-Gauss beam. The excitation beam therefore maintains the same transverse resolution at different depths inside the sample, eliminating the need for many scans in order to probe the entire volume of interest. In this thesis, we demonstrate that the extended depth of field microscope effectively works as we designed it, and we use it to image calcium dynamics in a three-dimensional network of live neurons. We also present the different advantages of our system in comparison with standard two-photon microscopy.
Canonge, Rafael. "Imagerie moléculaire 3D quantitative des tissus en utilisant la microscopie Raman cohérente sans marquage." Thesis, Ecole centrale de Marseille, 2017. http://www.theses.fr/2017ECDM0010/document.
Повний текст джерелаThis thesis focuses on multiphotonic microscopy techniques development and use in order to image human biological samples. A multiphotonic imaging setup using label-free nonlinear contrasts mechanisms such as two-photons fluorescence, second harmonic generation, or stimulated Raman effect (CARS or SRS) has been designed and developped during this PhD, and I present the experimental work in two main research topics.In a first part, we compare label-free 3D imaging with classic histological imaging using colorimetric labels in human digestive system. We show that multiphotonic technics allow to reconstruct the organization and discern the molecular compounds inside the tissues, in order to get a caratérization of the cancerous tumors developpement.The second part is related to the application of our multimodal setup to the quantitative study of real active molecular compounds real time penetration into in vivo human skin. We show that multiphotonic microscopy make possible to mesure active molecules in depth 3D concentration in the skin in order to understand transcutaneous diffusion mechanisms in cosmetic and pharmacological applications
Strupler, Mathias. "Imagerie du collagène par microscopie multiphotonique." Phd thesis, Ecole Polytechnique X, 2008. http://pastel.archives-ouvertes.fr/pastel-00004540.
Повний текст джерелаLeray, Aymeric. "Microscopie multiphotonique appliquée à la biologie." Rennes 1, 2005. http://www.theses.fr/2005REN1S156.
Повний текст джерелаSlimani, Amel. "Photonic approach for the study of dental hard tissues and carious lesion detection." Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT125.
Повний текст джерелаPhotonic properties of dental hard tissues allowed us to proceed to in vitro analysis of enamel and dentin on a molecular level. Confocal Raman microscopy has been used to produce a mapping of collagen cross-link and crystallinity of human dentin–enamel junction (DEJ) with a spatial resolution not achieved up to now. The method is a non-invasive, label-free and a high spatial resolution imaging technique. This chemical analysis of DEJ led us to redefine a wider width of this transition zone and advance our understanding of dental histology. A study on the intrinsic fluorescence changes of sound and carious tissues using conventional fluorescence microscopy suggests the involvement of protoporphyrin IX and pentosidine in the fluorescence red-shift observed in carious tissues. Multiphoton microscopy allowed to detect nonlinear optical signal changes during caries process using second harmonic generation (SHG) and two-photon excitation fluorescence (2PEF). Our studies led us to propose the ratio SHG/2PEF as valuable parameter to monitor caries lesion. Collectively, advances described in this thesis show the potential of photonic properties of enamel and dentin using Raman and multiphoton microcopies for molecular investigations on sound as much as on carious tissues. It opens new perspective in dental research and clinical applications
Guilbert, Thomas. "Microscopie multiphotonique de protéines fibrillaires : application à l’étude de la fibrose hépatique." Rennes 1, 2010. http://www.theses.fr/2010REN1S191.
Повний текст джерелаOptical imaging techniques based on nonlinear effects of photon-matter interactions, are increasingly applied in biology. Multiphoton microscopy provides an in-depth information at a micrometric scale in tissues, with low photo-toxicity. Moreover, the various sources of endogenous contrast in biological tissues allow to visualize, without any staining, the two photon excited fluorescence emission (TPEF), and second harmonic generation (SHG) of non-centrosymmetric molecules in non-centrosymmetric media, such as fibrillar proteins like collagen and myosin. These two endogenous contrasts enable a wide range of applications along with embryology, neuroscience or oncology. In this context, multiphoton microscopy appears as a promising tool for the study of fibrillar proteins present in biological tissues. In this document we first present the basis of multiphoton microscopy, the two contrasts involved in this imaging technique, and a tool based on the modulation of polarization of the exciting laser for the characterization of two fibrillar proteins, collagen and myosin. We also present a preliminary study of cattle skeletal muscle by multiphoton microscopy. In a second step, we use the specificity of SHG for imaging collagen to develop a method of scoring of fibrillar collagen deposits in human liver fibrosis. After an in-depth study of our collagen SHG scoring method, it is evaluated and compared to usual technics used to characterise fibrosis. This method which uses low NA lenses, could be extended to SHG endoscopes, when virtual biopsies will be possible
Thibon, Louis. "Méthodes d'augmentation de résolution en microscopie optique exploitant le modelage de faisceau laser et la déconvolution." Doctoral thesis, Université Laval, 2019. http://hdl.handle.net/20.500.11794/34695.
Повний текст джерелаLaser scanning microscopy is limited in lateral resolution by the diffraction of light. Superresolution methods have been developed since the 90s to overcome this limitation. However, superresolution is generally achieved at the cost of a greater complexity (high power lasers, very long acquisition times, specic uorophores) and limitations on the observable samples. In some cases, such as Structured Illumination Microscopy (SIM) and Switching Laser Modes (SLAM), a more modest improvement in resolution is obtained with a reduced complexity and fewer limitations. We propose here methods which improve the resolution while minimizing the experimental constraints and keeping most of the advantages of classical microscopy. First, we show that we can improve by twenty percent the resolution of confocal microscopy by using Bessel-Gauss beams, and by having the right pinhole size (1 Airy Unit), compared to conventional Gaussian beam based confocal microscopy. The advantages of this strategy include simplicity of installation and use, linear polarization compatibility, possibility to combine it with other resolution enhancement and superresolution strategies. We demonstrate the resolution enhancement capabilities of Bessel-Gauss beams both theoretically and experimentally on nano-spheres and biological tissue samples with a resolution of 0.39. We achieved these resolutions without any residual artifacts coming from the Bessel-Gauss beam side lobes. We also show that the resolution enhancement of Bessel-Gauss beams leads to a better statistical colocalization analysis with fewer false positive results than when using Gaussian beams. We have also used Bessel-Gauss beams of different orders to further improve the resolution by combining them in SLAM microscopy achieving a resolution of 0.17 (90 nm with a wavelength of 532 nm). In a second step, we propose a method to improve the resolution of confocal microscopy by combining different laser modes and deconvolution. Two images of the same eld are acquired with the confocal microscope using different laser modes and are used as inputs to a deconvolution algorithm. The two laser modes have different Point Spread Functions and thus provide complementary information leading to an image with enhanced resolution compared to using a single confocal image as input to the same deconvolution algorithm. By changing the laser modes to Bessel-Gauss beams, we were able to improve the effciency of the deconvolution algorithm and to obtain images with a residual Point Spread Function having a width smaller than 100 nm. The proposed method requires only a few add-ons to the classic confocal or two photon microscopes. Finally, we propose a three dimensional tomography reconstruction method using Bessel-Gauss beams as projection tools in two-photon microscopy. While focussing Bessel-Gauss beams at an angle in two photon microscopy, we can obtain a series of projections that can be used for tomography reconstruction. The aim is to test the practicality of the methods allowing to reconstruct a volume while using fewer images than plane by plane acquisitions as in classic two-photon microscopy.
Nguyen, Anh Dung. "Amélioration de la résolution spatiale en microscopie multiphotonique par saturation de la fluorescence." Doctoral thesis, Universite Libre de Bruxelles, 2015. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/221923.
Повний текст джерела-----------------------Résumé-----------------------Depuis la prédiction de Maria Göppert-Mayer dans les années 30 de la possibilité pour une molécule fluorescente d'être excitée simultanément par plusieurs photons et, plus récemment, depuis le développement des lasers pulsés, la microscopie multiphotonique s'est peu à peu développée pour finalement s'imposer aujourd'hui comme un des outils d'observation par fluorescence les plus performants pour les études de tissus épais diffusants, ou encore pour l'observation in vivo d'animaux. Que ce soit pour des études neurologiques, physiologiques ou morphologiques, l'aspect non invasif et la limitation du volume excité au volume focal ont rendu cet outil de microscopie indispensable aux biologistes.Cependant, dans un monde où les études biologiques nécessitent toujours de meilleurs microscopes et où la résolution spatiale en particulier doit toujours être améliorée, il convient de proposer des techniques permettant d'obtenir une meilleure résolution dans les trois dimensions et d'aller au-delà de la limite de diffraction définie par Ernst Abbe il y a plus d'un siècle.Dans cette thèse, la technique de saturation de l'excitation de la fluorescence est adaptée à la microscopie multiphotonique. Cette méthode permet d'obtenir des images de superrésolution en modulant temporellement l'intensité laser d'excitation et en démodulant les harmoniques supérieures présentes dans le signal saturé de fluorescence. La démonstration de principe sur des microsphères fluorescentes a été réalisée montrant une amélioration de la résolution latérale et axiale. Alors que l'utilisation de la troisième harmonique produit déjà une meilleure résolution, ce travail de thèse montre qu'une amélioration supplémentaire peut être obtenue en utilisant une combinaison linéaire particulière des harmoniques démodulées.Au final, un quasi doublement de la résolution a pu être observé tant dans les directions latérales que dans la direction axiale. Cette amélioration correspond à l'amélioration prédite dans l'analyse théorique et mathématique réalisée également dans ce travail.De plus, le passage aux études in vitro a été réalisé avec succès en observant des microsphères fluorescentes incorporées dans des cellules HeLa. Des améliorations de la résolution latérale et axiale ont également été observées montrant que cette technique de superrésolution peut être appliquée à l'étude d'échantillons biologiques. Les forces et les faiblesses de cette méthode sont également analysées et détaillées afin de voir dans quel créneau d'études biologiques la technique de saturation de l'excitation de fluorescence pourrait se faire une place. A cette fin, ses caractéristiques sont comparées aux autres méthodes de superrésolution et de superlocalisation détaillées dans la première partie de ce travail.Il en resort que l'importante profondeur d'imagerie, l'aspect non invasif et la limitation du volume excité de la microscopie multiphotonique couplés à la simplicité d'implémentation et les relativement faibles puissances utilisées pour saturer l'excitation font de cette technique un excellent candidat pour des études in vivo dans des zones en profondeur dans des milieux diffusants comme la peau.
Doctorat en Sciences de l'ingénieur et technologie
info:eu-repo/semantics/nonPublished
Quémener, Mireille. "Conception, fabrication et caractérisation d'un GRIN-axicon pour une application en microscopie multiphotonique." Master's thesis, Université Laval, 2021. http://hdl.handle.net/20.500.11794/68755.
Повний текст джерелаTechnological advances in microscopy have led to the creation of a wide variety of optical systems dedicated to the investigation of the dynamic behavior of cells in vivo. In neuroscience, the challenge lies in the observation of interactions between labeled neurons located at different depths in the tissue. In order to achieve this, it is necessary to scan the sample on several transverse planes to fully cover its volume. Since this procedure decreases the temporal resolution, it has been proposed to use an axicon to increase the depth of field of the microscope and reduce the number of scans to be performed. However, the axicon is di cult to manufacture and usually has defects on the tip of the cone, thus degrading the quality of the component. In order to replace the axicon by another optical component easier to manufacture, the use of a graded index lens coupled to a single lens (GRIN-axicon) was considered. Simulations have shown that the GRIN-axicon has the potential to produce a good quality Bessel beam. However, experimental tests have been very limited and it is necessary to further investigate the behaviour of this new component in the laboratory. The objective of this master's project is therefore to design, manufacture and characterize a GRIN-axicon for application in multiphoton microscopy. As a secondary objective, we wish to deepen the theory related to this new component.
Hayek, Ali. "Ingénierie, synthèse et caractérisation de nouveaux chromophores absorbants multiphotoniques : Applications en imagerie biologique." Université Louis Pasteur (Strasbourg) (1971-2008), 2006. https://publication-theses.unistra.fr/public/theses_doctorat/2006/HAYEK_Ali_2006.pdf.
Повний текст джерелаCellular imaging is more and more used for the investigation of cells structures, functions and communications. For that purpose, fluorescence confocal microscopy has become a routine technique since it permits to investigate the details of a variety of physiological behaviors in the cell. More recently two-photon excited microscopy (TPEM) has emerged in biomedical research, for example in cellular and small animal imaging. This method takes advantages of the nonlinear optical (NLO) properties of chromophores: the emission occurs only at the focal point of the laser (quadratic dependence upon intensity) and the laser used induces a low bleaching out of focus (use of IR light instead of the more energetic UV light). Wide efforts are therefore being undertaken in order to develop chromophores with high two-photon absorption cross-section (σ2), good fluorescence efficiency, and low bleaching. The aim of our research is the elaboration and characterization of new water-soluble chromophores, optimized for two-photon induced fluorescence in biological systems for bio-imaging. This technique (TPEM) can be combined to other type of imaging like the Magnetic Resonance Imaging (MRI) or some kind of therapy like Boron Neutron Capture Therapy (BNCT). For these aims, chromophores containing paramagnetic atom (gadolinium in our case) and boron atoms were designed, synthesized an characterized
Labroille, Guillaume. "Imagerie tridimensionnelle multiphotonique des tissus biologiques à l'aide d'impulsions façonnées." Phd thesis, Ecole Polytechnique X, 2011. http://pastel.archives-ouvertes.fr/pastel-00695044.
Повний текст джерелаPena, Ana-Maria. "Génération de seconde harmonique par le collagène et application à l'étude de fibroses par microscopique multiphoton." Palaiseau, Ecole polytechnique, 2006. http://www.theses.fr/2006EPXX0026.
Повний текст джерелаBoksebeld, Maxime. "Elaboration et caractérisation de nanoparticules hybrides pour la microscopie multiphotonique et la thérapie ciblée du cancer." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSEC029/document.
Повний текст джерелаThis thesis describes the synthesis of new nanoprobes with properties allowing their use for cancer-targeted multiphotonic microscopy and cancer phototherapy. On the one hand, this work was focused on the synthesis of nanoparticles with non-linear optical and phototherapeutic properties. Different nanoparticles were synthesized and used like gold nanorods, silicon carbide or potassium niobate nanoparticles, and nanohybrids coupling these previous nano-building blocks. These nanoparticles were functionalized with biomolecules like folic acid to provide specific cancer-targeting properties. The surface chemistry of these nanoparticles was carefully evaluated through advanced characterization techniques such as infrared spectroscopy, XPS and ToF-SIMS. On the other hand, optical and therapeutic properties of these nanoparticles were studied. These nanoprobes were successfully used to perform healthy cells labelling and cancer cells targeting for multiphotonic microscopy. Phototherapeutic properties of our nanoparticles were also used to induce light-triggered cancer therapy
Dufour, Pascal. "Utilisation d'axicons pour la microscopie à deux photons." Thesis, Université Laval, 2012. http://www.theses.ulaval.ca/2012/28480/28480.pdf.
Повний текст джерелаSadetsky, Gregory. "Perceptual hashing-based movement compensation applied to in vivo two-photon microscopy." Thesis, Université Laval, 2014. http://www.theses.ulaval.ca/2014/30446/30446.pdf.
Повний текст джерелаAnimal movement during in vivo two-photon microscopy experiments hinders efforts at observing biological phenomena and the subsequent analysis of the acquired video streams. One of the reasons for this is that, due to optical sectioning, any displacement in the z-axis (perpendicular to the plane of imaging) dramatically changes the collected image and thus provides the experimenter with an unstable view of the imaged sample. By applying a hashing function on the acquired video frames, we produce vectors embodying the images’ perceptual qualities; these vectors can then be used to compare the frames one to another, in real-time. These comparisons allow us to group similar images in clusters corresponding to distinct z-planes. In effect, the process of perceptually hashing, comparing and grouping video frames provides us with software-based, real-time movement compensation which can be used in a biological laboratory setting.
Pakula, Guillaume. "Stratégies avancées d'imagerie multiphoton pour l'étude de processus périodiques rapides in vivo : des cils motiles au cœur battant." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLX018.
Повний текст джерелаOscillations are abundant at every scale of the living world. In development biology, the beating heart and the motile cilium are two oscillators operating at temporally and spatially analogous scales. Their dynamic visualization is a major experimental challenge, which would shed new lights on the biological processes they are involved in. In this manuscript, we will extract their biophysical features or image their three-dimensional beating pattern in vivo, in a vertebrate model organism: the zebrafish. To circumvent current limitation in imaging speed, we will use the common periodic property of their motion to develop innovative strategies for fast multiphoton imaging.First, the cellular cilium is a cytoplasmic extension found in almost all eukaryotic cells whose role is to generate a biological flow when motile. In recent years, their dysfunctions have been identified to be involved in a growing number of pathologies, known as ciliopathies. In particular, they are present in large numbers in the zebrafish left-right organizer, the Kupffer vesicle: the directional flow they generate is responsible, at early stages, for the breaking of the left-right symmetry, the last biological axis to specify in vertebrates. In this manuscript, we will describe a systematic approach to extract cilia biophysical features, using the scanning artifacts observed in point scanning multiphoton microscopy when imaging their periodic beating. The systematic worfkflow we developed has enabled us to unveil the establishment of a novel form of asymmetry in the Kupffer's vesicle.Similarly, the study of the beating heart and the visualization of its rapid and three-dimensional periodic motion remains a challenge, even using state-of-the-art fluorescence microscopy techniques. In this work, we will propose a fast point-scanning imaging method that takes advantage of the periodic property of the beating heart. It relies on the retrospective synchronization of fast scanned line sequences. We will show how this approach can reach unprecedented temporal resolutions (in the order of 1000 volumes per second), with label-free contrast, and without compromising any other acquisition parameters, such as SNR or spatial resolution. In particular, we applied the method to the tracking of contracting cardiac sarcomeres in vivo, using second harmonic generation (SHG), and achieved reconstruction the 3D motion of the cardiac valves, the fastest-moving structure of the heart
Fabre, Aurélie. "Voies des peptides endogènes sérotonine et somatostatine et application de la microscopie multiphoton dans la fibrose pulmonaire." Paris 7, 2007. http://www.theses.fr/2007PA077237.
Повний текст джерелаIdiopathic pulmonary fibrosis is characterised by inflammation and fibrosis within the pulmonary interstitium. The bleomycin induced lung fibrosis animal model allows the study of potentially therapeutic molecules, and the application of new techniques of tissue microscopy. In this model, we have studied two endogenous peptides, serotonin (5-HT) and somatostatin which display pro fibrotic and ant anti-fibrotic properties respectively, using specific antagonists or analogues to cellular receptors. Blockage of 5-HT2A and 5-HT2B receptors - which are preferentially expressed in the lung, - with specific antagonists (ketanserin and SB215505 respectively), reduced lung fibrosis (total soluble collagen and procollagen I and III mRNA) and the expression of profibrotic mediators known to be involved in the pathophysiology of lung fibrosis (TGF-B1, CTGF, PAI-1). The somatostatin pathway appears also involved, and its receptors sstl, sst2, sst3, ssst4 and sst5 are expressed in the normal lung, with and an increase of sst-2 receptor following bleomycin instillation. Daily subcutaneous single dose injection of SOM230, a new somatostatin analogue, in bleomycin mice, improves mice survival, decreases the pulmonary concentration in collagen and the expression of procollagen I, as well as the expression of pro fibrotic factors TGF-IÎ1 and CTGF, and increases the expression of the protective factors HGF and KGF. Finally, the application of multiphoton microscopy in this model describes for the first time the tridimensional features on the normal and fibrotic lung, and allows volumetric quantitative and qualitative analysis of collagen deposition of unstained fresh lung, opening new perspectives in tissue assessment of this disease both in vive and ex vivo
DULIEU, FRANCOIS. "Photodetachement d'ions negatifs d'halogenes : distributions angulaires multiphotoniques en polarisation elliptique ; microscopie au seuil dans un champ electrique uniforme." Paris 11, 1996. http://www.theses.fr/1996PA112193.
Повний текст джерелаPuech, Magali. "Standardisation de la mesure du contenu nucléaire en ADN par microscopie quantitative." Université Joseph Fourier (Grenoble), 1996. http://www.theses.fr/1996GRE19007.
Повний текст джерелаLeclerc, Pierre. "Développement d’un endomicroscope multiphotonique à deux couleurs pour l’imagerie du métabolisme énergétique cellulaire." Thesis, Limoges, 2017. http://www.theses.fr/2017LIMO0053/document.
Повний текст джерелаNonlinear microscopy is a cutting edge imaging modality leading to remarkable step forward in biology but also in the clinical field. To use it at its full potential and at the very heart of clinical practice, there has been several development of fiber-based micro-endoscope. The application for those probes is now limited by few major restrictions, such as the impossibility to collect auto-fluorescence signal from tissues theses being inherently weak such as the fluorescence from NADH or FAD. This limitation reduces the usefulness of the micro-endoscope effectively restraining it to morphological imaging modality requiring staining of the tissue. Our aim is to go beyond this limitation, showing cellular metabolism monitoring, in real time, without any staining. The experimental setup is an upgrade of our precedent one where the reflection- based Grism stretcher is replace with a new generation transmission-based Grism stretcher. Another Laser was also added in order to tune the first laser at 860nm to allow FAD imaging and the second one to 760nm for NADH. The results prove that we assess and image the level of NADH and FAD at subcellular resolution through a five-meter-long fiber. Thus we demonstrate that we are capable of measuring the optical redox ratio in a micro-endoscopic configuration
Bordeau, Guillaume. "Nouvelles tryarylamines pour la microscopie biphotonique : application au marquage des acides nucléiques." Paris 6, 2009. http://www.theses.fr/2009PA066358.
Повний текст джерелаFluorescent labelling of biomolecules, in particular DNA, has become increasingly important for biological studies. Multiphoton microscopy is additionally a powerful technique for biological imaging. In this context, we have designed new fluorescent DNA probes for two-photon excited fluorescence microscopy. Prior to this work, a series of red emitting vinylpyridinium triphenylamines (TP-Py) had shown high two-photon absorption properties and nuclear DNA staining in confocal and two-photon excitation microscopy. In this regard, we developed several new series of analogues to optimize the properties of TP-Py and to study the relationships between structure and optical properties. In addition, a completely new family, trinaphthylamines, was developed. Furthermore, TP-Py conjugates were developed for covalent DNA labelling for hybridization studies and in vivo optical tracking of plasmids. Polyamine conjugates targeting cancerous cells were also prepared. A number of TP-Py derivatives for grafting to gold nanoparticles were synthesized in order to study the gold surface-fluorophore interactions and to enhance their fluorescence
Samson, Karen. "Imagerie multiphotonique de la sérotonine par contraste endogène : Vers un outil pour évaluer la concentration de la sérotonine in vivo." Thesis, Université Laval, 2012. http://www.theses.ulaval.ca/2012/28895/28895.pdf.
Повний текст джерелаSerotonin (5-HT) is a neurotransmitter regulating several basic functions of the body: thermoregulation, sexual and food behavior, the sleep-wake cycle, perception of pain, anxiety, motor control, but is best known for control of mood. The lack of serotonin in the central nervous system is associated with mental illness. These diseases are depression, bipolar disorder, anxiety, panic disorder, phobias, obsessive-compulsive disorder and schizophrenia. A detection tool of serotonin and its precursors would allow us to study the mechanism of various diseases involving an imbalance of this neurotransmitter. In the central nervous system, the biosynthesis of serotonin occurs in specific neurons of the brainstem. Serotonin, like its precursor tryptophan (Trp), is highly fluorescent in comparison to other endogenous molecules. Multiphoton fluorescence excitation has been used in this project to detect serotonin without exogenous labels. This fluorescence imaging technique provides a good specificity as well as optical sectioning for imaging in cells and tissues. Different excitation processes (two- and three-photon) under different conditions (fixed cells or not) were explored to optimize the detection of autofluorescence of serotonin. We therefore first developed an imaging system capable of detecting the fluorescence of molecules involved in the biosynthesis of serotonin, excluding tryptophan. We are able to detect these isolated species in solution at concentrations near a millimolar. The method was tested in two models containing serotonin (cells and slices). The measurements have shown a lack of specificity and sensitivity when used in systems more complex than simple solutions. This lack of specificity and sensitivity is discussed with possible improvements for future project including the use of other models with better control of serotonin concentrations, of other more advanced optical techniques such as fluorescence lifetime imaging.
Ait, el Madani Hassan. "Microscopie multiphotonique in vivo chez l'homme : application à l'analyse dynamique des modifications structurales et fonctionnelles cutanées et à l'évaluation de la réponse thérapeutique." Paris 7, 2011. http://www.theses.fr/2011PA077214.
Повний текст джерелаInvestigation of the 3D structure of human skin with a sub-cellular resolution. Multiphoton microscopy imaging has generated new qualitative information in dermatology. However, only very few published data deal with quantitative analysis from multiphoton images. The aim of this thesis was to contribute to the developpement of multiphoton images analysis, by a rationalization of the visual approach and the validation of new quantitative parameters. Two clinical approaches were performed. The first one addressed the assessment and the monitoring of cutaneous side effects induced by topical corticotherapy. A preliminary pilot study allowed demonstration that multiphoton microscopy was able to objectivate morphological and pigmentary modifications in epidermis in a non invasive way. A more comprehensive second study focusing on two age groups showed that multiphoton microscopy could objectivate age-related cutaneous modifications. Differences in the effect induced by the corticoids were characterized between young and old volunteers. Quantitative parameters were validated by comparison to already known histological features. The second clinical approach dealt with a physiological situation: analysis of different skin color typologies. This study comforted the pertinence of quantitative parameters related to pigmentation assessment. Discrimination of various skin color typologies was performed thanks to quantitative methods based on use of the specific fluorescence lifetime of the melanin or its high fluorescence signal
Debarre, Delphine. "Microscopie par génération de troisième harmonique appliquée à la biologie." Phd thesis, Ecole Polytechnique X, 2006. http://pastel.archives-ouvertes.fr/pastel-00001978.
Повний текст джерелаMahou, Pierre. "Microscopie non linéaire de tissus biologiques : excitation multicouleur, faisceaux de Bessel, et excitation en nappe de lumière." Phd thesis, Ecole Polytechnique X, 2012. http://pastel.archives-ouvertes.fr/pastel-00840186.
Повний текст джерелаTerras, Fériel. "Développement d’une méthode de mesure et d’analyse du transport intraneuronal dans le cerveau de larve de poissons-zèbre par suivi de nanocristaux non-linéaires en microscopie de second-harmonique : application à l’étude d’anomalies de transport chez des poissons portant une mutation retrouvée dans des neuropathies humaines." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASP050.
Повний текст джерелаMolecular transport in neurons plays an essential role in their development and the maintenance of their functions, due in particular to their long branches. The consequences of axonal transport abnormalities have long been associated with Alzheimer's disease, such as the swelling of axons caused by the accumulation of transported organelles. However, there are few tools allowing the detailed long-term study of this transport in culture and even less in vivo.The team in which this work was conducted has developed a method for measuring intraneuronal transport in mouse cultured neurons based on the tracking of endosomes having spontaneously internalized fluorescent nanodiamonds. This technique was able to detect genetic risk factors of neuropsychiatric diseases, proving its high sensitivity.The objective of my thesis was to extend this measurement method to the neuronal circuit of the zebrafish larvae’s brain. For this purpose, I have used nanocrystals (NCs) with non-linear optical response, which can be excited in a tissue transparency window at a wavelength ≈1 µm, and generate a second harmonic signal. We have used the multiphoton microscope of the Emerg'In facility (INRAE, Jouy-en-Josas), which is installed next to a fish farming and has resonant galvanometric scanners and hybrid detectors, which allowed us to maintain a frame rate of 20 fps despite the need to scan the laser beam, unlike in culture studies conducted in wide field.We have developed a new measurement protocol, from the injection of the NC to the extraction of transport parameters from the videos data. To do this, I have optimized and automated the data analysis by developing two programs written in Python: one that automatically reconstructs the trajectories of the NCs as faithfully as possible and the other that segments the trajectories into motion and pause phases in order to extract transport parameters.I have applied these new tools to study, in a transgenic zebrafish model, the functional impact of a mutation present in neuropathies including hereditary spastic paraplegia and peripheral Charcot-Marie-Tooth disease. In patients suffering from one of these diseases, several mutations have been identified in the KIF5A gene coding for the heavy chain of the kinesin 1 molecular motor which ensures anterograde movements (from the cell body to the periphery). We have measured endosome transport in axons of neurons in the brain of transgenic zebrafish larvae bearing a mutation on kif5a, and we observed subtle changes in this transport. More than 500 endosome trajectories (acquired at a depth of ≈100 µm below the surface of the head) were analyzed from 40 larvae.As a control, we also studied endosomal transport in mutants deprived of dynein motors, the only ones to ensure retrograde transport, and observed that bi-directional transport was totally stopped in the mutants.This methodology can be used to screen for functional abnormalities resulting from genetic factors of neurological or neurodegenerative disorders
Bélanger, Erik. "Développement et utilisation d'une plateforme d'imagerie optique quantitative, multimodale et non linéaire de la moelle épinière chez les animaux vivants." Doctoral thesis, Université Laval, 2013. http://hdl.handle.net/20.500.11794/24192.
Повний текст джерелаOptical microscopy in living animals is a promising research tool for the evolution of neurobiology. Intravital imaging offers a live preview of how individual cells respond to the nervous system damages. Applying in vivo microscopy to a panoply of transgenic mice used with different animal models of neurodegenerative diseases promotes the understanding of the progress of pathologies and the comprehension of how therapies work. It is thus essential to promote the emergence of optical microscopy technologies in living animals because it is a strategy with great potential. Therefore, the project described in this doctoral thesis focuses on the development and use of a microscopy platform for quantitative, multimodal and nonlinear imaging of the spinal cord in living animals. First, we alleviated the polarization dependence of the coherent anti-Stokes Raman scattering (CARS) signal intensity. This strategy makes images more amenable to histological interpretation. With this technique, we studied the histology of myelin in the rat spinal cord. Secondly, we proposed a new image analysis procedure compatible with live animals imaging in order to achieve the histology of myelinated axons. We quantified the demyelination proximal, and remyelination distal to the crush site ex vivo and in vivo respectively. Third, we showed that CARS imaging of the spinal cord in living mice can be achieved with a microendoscope, and this while maintaining compatibility with the two-photon excitation fluorescence signal. Finally, we discuss a digital image processing strategy that reduces imaging artifacts related to movement of the animal. This technique allows the histological study of myelin and the quantification of the motility of microglial cells in their native environment. Ultimately, this thesis demonstrates that in vivo CARS microscopy progresses gradually towards a robust tool for research in neurobiology.
Jaouën, Alexandre. "Etablissement d'un protocole haut débit d'acquisition et d'analyse d'images pour les études précliniques par microscopie bi-photonique intravitale multispectrale : application à l'étude de la neuroinflammation provoquée par un modèle murin de sclérose en plaque." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0619/document.
Повний текст джерелаMultiple sclerosis is a chronic auto-immune neurodegenerative disease characterized by the appearance of inflammatory plaques in the central nervous system. To characterize the innate immune response I have developed nonlinear optical tools for intravital multispectral imaging as well as semi-automated image processing solutions. I also contributed to the development of a flow cytometry protocol allowing the identification of immune cell phenotypes. Applied to study of Experimental Autoimmune Encephalomyelitis, a mouse model of MS, these tools have allowed to analyze the immune cascade thanks to immunolabelings and transgenic expression of fluorescence, describe the distributions of cell populations in the CNS with regard to neuron degeneration on time scales ranging from seconds to weeks. On a transgenic mouse line Thy1-CFP/Cd11c-EYFP/LysM-EGFP, I have followed by two-photon microscopy the evolution of fluorescent cells densities in the same area of the spinal cord for several weeks. I conclude that axonal degeneration and motor deficits are correlated with neutrophils and monocytes infiltration from the meninges. The monocytes differentiate in situ in monocyte-derived dendritic cells (moDCs) along with the recruitment of activated microglia. These cellular events correlated with a stabilization/remission phase of the disease. MoDCS maturation thus seems involved in the dampening of inflammation. Methodology and tools are now set for further investigations with other models. The microscope optimization for multicolor excitation allowed me to access simultaneously to the endogenous CARS contrast to visualize the myelin sheath
Mettra, Bastien. "Ingénierie, photophysique et fonctionnalisation de chromophores pour la bio-photonique non linéaire in-vivo." Thesis, Lyon, École normale supérieure, 2015. http://www.theses.fr/2015ENSL1038/document.
Повний текст джерелаThe 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