20 research outputs found

    Measurement and correction of aberrations in light and electron microscopy

    Get PDF
    Imperfections in image formation, called aberrations, often preclude microscopes from reaching diffraction-limited resolution. Aberrations can be caused either by the microscope itself or by the sample and can be compensated for by using an active element integrated into the beam path which is functioning as a corrector. The optimal settings for this corrector need to be determined without excessive damage to the sample. In particular, for sensitive biological samples, the potential gain for signal and/or resolution needs to be weighed against sample damage. Here I present the development of a special type of optical coherence microscopy (called deep-OCM), which allows the precise determination of the average rat brain refractive index in vivo. The conclusion is that two-photon microscopy is affected by optical aberrations in this sample starting at depths around 200 micrometers. Deep-OCM is well suited for imaging myelinated nerve fibers. Individual fibers can be visualized in the living brain in unprecedented depths beyond 300 micrometers. In the second part of this thesis I describe the development and testing of an auto-focuser and auto-stigmator (called MAPFoSt) for a scanning electron microscope to ensure optimal imaging quality after switching samples or during long acquisition series. MAPFoSt determines the three focus and stigmation parameters from only two test images

    Mesure et correction des aberrations en microscopie optique et microscopie électronique

    No full text
    Imperfections in image formation, called aberrations, often preclude microscopes from reaching diffraction-limited resolution. Aberrations can be caused either by the microscope itself or by the sample and can be compensated for by using an active element integrated into the beam path which is functioning as a corrector. The optimal settings for this corrector need to be determined without excessive damage to the sample. In particular, for sensitive biological samples, the potential gain for signal and/or resolution needs to be weighed against sample damage. Here I present the development of a special type of optical coherence microscopy (called deep-OCM), which allows the precise determination of the average rat brain refractive index in vivo. The conclusion is that two-photon microscopy is affected by optical aberrations in this sample starting at depths around 200 m. Deep-OCM is well suited for imaging myelinated nerve fibers. Individual fibers can be visualized in the living brain in unprecedented depths beyond 300 m. In the second part of this thesis I describe the development and testing of an auto-focuser and auto-stigmator (called MAPFoSt) for a scanning electron microscope to ensure optimal imaging quality after switching samples or during long acquisition series. MAPFoSt determines the three focus and stigmation parameters from only two test imagesLa diffraction constitue une limite fondamentale en microscopie, mais souvent cette limite n est même pas atteinte. Des imperfections dans la formation d image, appelées aberrations, peuvent être induites par le microscope ou l échantillon. Un élément actif, dit correcteur, est intégré au chemin optique pour leur compensation. Les paramètres de ce correcteur doivent être déterminés sans dommage excessif pour l échantillon. Il faut comparer le gain en signal et/ou en résolution avec cet endommagement, surtout pour des échantillons biologiques fragiles. En première partie de cette thèse je présente une modalité particulière de la microscopie par cohérence optique (nommé deep-OCM). Ce développement a permis la mesure exacte et in vivo de l indice de réfraction moyen du cerveau du rat. Cette valeur implique que la microscopie bi-photonique est limitée par des aberrations optiques à partir d une profondeur de 200 m dans ce type d échantillon. Le deep-OCM est bien adapté à l imagerie de fibres nerveuses myélinisées. Des fibres individuelles peuvent être visualisées in vivo dans le cerveau à des profondeurs auparavant inaccessibles, supérieures à 300 m. Dans la deuxième partie de cette thèse je présente le développement d un autofocus et auto-stigmateur (nommé MAPFoSt) pour le microscope électronique à balayage qui permet d assurer la qualité maximale des images lors d un changement d échantillon ou pendant des séries d acquisitions de longue durée. MAPFoSt permet de déterminer avec précision les trois paramètres du focus et du stigmatisme en utilisant seulement deux images de testPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

    Mesure et correction des aberrations en microscopie optique et microscopie électronique

    No full text
    Imperfections in image formation, called aberrations, often preclude microscopes from reaching diffraction-limited resolution. Aberrations can be caused either by the microscope itself or by the sample and can be compensated for by using an active element integrated into the beam path which is functioning as a corrector. The optimal settings for this corrector need to be determined without excessive damage to the sample. In particular, for sensitive biological samples, the potential gain for signal and/or resolution needs to be weighed against sample damage. Here I present the development of a special type of optical coherence microscopy (called deep-OCM), which allows the precise determination of the average rat brain refractive index in vivo. The conclusion is that two-photon microscopy is affected by optical aberrations in this sample starting at depths around 200 ¬Ķm. Deep-OCM is well suited for imaging myelinated nerve fibers. Individual fibers can be visualized in the living brain in unprecedented depths beyond 300 ¬Ķm. In the second part of this thesis I describe the development and testing of an auto-focuser and auto-stigmator (called MAPFoSt) for a scanning electron microscope to ensure optimal imaging quality after switching samples or during long acquisition series. MAPFoSt determines the three focus and stigmation parameters from only two test imagesLa diffraction constitue une limite fondamentale en microscopie, mais souvent cette limite n'est m√™me pas atteinte. Des imperfections dans la formation d'image, appel√©es aberrations, peuvent √™tre induites par le microscope ou l'√©chantillon. Un √©l√©ment actif, dit correcteur, est int√©gr√© au chemin optique pour leur compensation. Les param√®tres de ce correcteur doivent √™tre d√©termin√©s sans dommage excessif pour l'√©chantillon. Il faut comparer le gain en signal et/ou en r√©solution avec cet endommagement, surtout pour des √©chantillons biologiques fragiles. En premi√®re partie de cette th√®se je pr√©sente une modalit√© particuli√®re de la microscopie par coh√©rence optique (nomm√© deep-OCM). Ce d√©veloppement a permis la mesure exacte et in vivo de l'indice de r√©fraction moyen du cerveau du rat. Cette valeur implique que la microscopie bi-photonique est limit√©e par des aberrations optiques √† partir d'une profondeur de 200 ¬Ķm dans ce type d'√©chantillon. Le deep-OCM est bien adapt√© √† l'imagerie de fibres nerveuses my√©linis√©es. Des fibres individuelles peuvent √™tre visualis√©es in vivo dans le cerveau √† des profondeurs auparavant inaccessibles, sup√©rieures √† 300 ¬Ķm. Dans la deuxi√®me partie de cette th√®se je pr√©sente le d√©veloppement d'un autofocus et auto-stigmateur (nomm√© MAPFoSt) pour le microscope √©lectronique √† balayage qui permet d'assurer la qualit√© maximale des images lors d'un changement d'√©chantillon ou pendant des s√©ries d'acquisitions de longue dur√©e. MAPFoSt permet de d√©terminer avec pr√©cision les trois param√®tres du focus et du stigmatisme en utilisant seulement deux images de tes

    Measuring aberrations in the rat brain by coherence-gated wavefront sensing using a Linnik interferometer

    Get PDF
    International audienceAberrations limit the resolution, signal intensity and achievable imaging depth in microscopy. Coherence-gated wavefront sensing (CGWS) allows the fast measurement of aberrations in scattering samples and therefore the implementation of adaptive corrections. However, CGWS has been demonstrated so far only in weakly scattering samples. We designed a new CGWS scheme based on a Linnik interferometer and a SLED lightsource, which is able to compensate dispersion automatically and can be implemented on any microscope. In the highly scattering rat brain tissue,where multiply scattered photons falling within the temporal gate of the CGWS can no longer be neglected, we have measured known defocus andspherical aberrations up to a depth of 400őľm

    Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy.

    No full text
    International audienceMyelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-őľm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis

    A functional model, eigenvalues, and finite singular critical points for indefinite Sturm-Liouville operators

    Full text link
    Eigenvalues in the essential spectrum of a weighted Sturm-Liouville operator are studied under the assumption that the weight function has one turning point. An abstract approach to the problem is given via a functional model for indefinite Sturm-Liouville operators. Algebraic multiplicities of eigenvalues are obtained. Also, operators with finite singular critical points are considered.Comment: 38 pages, Proposition 2.2 and its proof corrected, Remarks 2.5, 3.4, and 3.12 extended, details added in subsections 2.3 and 4.2, section 6 rearranged, typos corrected, references adde
    corecore