66 research outputs found

    Histologie massive basée sur la microscopie à tomographie par cohérence optique

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    L’histologie consiste en l’étude des tissus vivants à l’échelle microscopique. Cette discipline a permis de grandes avancées, tant en biologie qu’en médecine, allant de la découverte de la cellule, jusqu’à devenir l’outil de prédilection pour le diagnostic de nombreuses maladies. L’avènement de l’histologie s’est fait en parallèle avec le développement du microscope, nécessaire pour visualiser les tissus sous grossissement. La microscopie est par nature limitée à imager une très petite région d’intérêt et ne donne donc qu’un petit aperçu de l’échantillon en entier. Récemment, des microscopes tomographiques sériels combinant la microscopie au sectionnement du tissu ont permis d’imager automatiquement de larges échantillons de tissu à l’échelle du micron. Des algorithmes spécifiquement développés pour ces appareils permettent de reconstruire dans une grande matrice tridimensionnelle l’échantillon imagé. Dans le cadre de cette thèse, un tel système a été construit avec la tomographie par cohérence optique comme modalité d’imagerie. Cette technique d’imagerie, qui est basée sur la réflectance intrinsèque des tissus, permet une acquisition de données volumétriques à haut débit en plus d’offrir une haute résolution spatiale et une bonne pénétration dans les tissus. Le système développé a permis d’imager de façon fiable et répétable un organe de souris en l’espace de quelques heures avec un voxel d’une taille de (4,88 x 4,88 x 6,5) μm. L’objectif général de ce projet était d’utiliser le système développé pour faire des études de groupe sur la souris, pour répondre à des questions spécifiques. D’abord, une validation du système d’imagerie était de mise pour vérifier que la plateforme d’imagerie donne une représentation fidèle du tissu in vivo. En imageant une population de cerveaux de souris in vivo en imagerie par résonance magnétique, puis avec la plateforme d’imagerie, les déformations entre les modalités d’imageries furent quantifiées. L’utilisation d’outils d’analyses morphométriques développés pour l’imagerie par résonance magnétique a démontré que le sectionnement et la fixation ne déforment pas de façon significative le tissu et que les algorithmes permettant la reconstruction en un volume tridimensionnel donnent une représentation fidèle du cerveau in vivo. Cette démonstration ouvre la voie à des études de groupes s’intéressant à des altérations microscopiques dans un organe entier. Considérant la répétabilité et la fiabilité prouvée de la plateforme d’imagerie, l’effet du vieillissement normal sur le coeur de la souris a été examiné. En comparant les coeurs de souris jeunes à ceux de souris âgées, il a été mis en évidence que des changements dans l’architecture du myocarde s’opèrent en vieillissant. Chez les souris âgées la paroi du ventricule gauche s’épaissit résultant en une diminution du taux de changement d’orientation des fibres musculaires dans cette même paroi. En imageant préalablement la fonction cardiaque de ces mêmes coeurs in vivo, il fut démontré que ces changements microscopiques s’accompagnent de changements fonctionnels.----------ABSTRACT Histology consists in the study of biological tissue at the microscopic scale. This discipline has led to great advances in biology, such as permitting the discovery of the cell and in medicine, where it is to this day the gold standard to detect many diseases. The advent of histology has been brought to in parallel with the development of microscopy, necessary to visualize tissues under magnification. Microscopy is limited to imaging small field of views, thus giving only a small representation of the entire sample. Recently, serial scanning microscopes, combining light microscopy and tissue sectioning have allowed to automatically image large tissue samples, expanding the imaged region to the order of the centimeter while keeping micrometer scale resolution. Post processing algorithms, specifically developed for serial scanning microscopes, are used to reconstruct in large 3D datasets the imaged sample. In this thesis, a serial scanning optical coherence tomography microscope was developed. This imaging modality, based on the intrinsic reflectance of tissue, allows high-speed acquisition of volumetric datasets at micrometer scale spatial resolution and penetrates deep in biological tissue. The developed platform allowed reliable and repeatable imaging of whole mouse organs within a time lapse of a few hours with a voxel size of (4,88 x 4,88 x 6,5) μm. The general objective of this project was to use this developed imaging platform to perform group studies on mice, to answer specific questions on tissue morphology. First, a system validation was required to verify that the imaging platform gives reliable representation of in vivo tissue. By imaging a group of in vivo mice brains with magnetic resonance imaging before serial sectioning, inter-modality deformations were quantified. The use of morphometric analysis tools developed for magnetic resonance imaging demonstrated that tissue sectioning and fixation does not significantly deform tissue and that reconstruction algorithms to obtain a large 3D dataset give a reliable representation of the in vivo brain. This proof of concept investigation opens the way to further group studies looking at microscopic alterations in an entire small animal organ. Considering the previously demonstrated repeatability and reliability of the imaging platform, the effect of normal aging on the mouse heart was examined. By comparing hearts of young and old mice, it was shown that architectural changes in the myocardium occur with aging. In old mice, there was a thickening of the left ventricle wall, which showed a decrease of muscle fiber orientation change. Prior imaging of the cardiac function in vivo revealed that these microscopic changes in morphology were accompanied by changes in the heart function

    Multimodal Optical Medical Imaging Concepts Based on Optical Coherence Tomography

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    Optical medical imaging techniques in general exhibit outstanding resolution and molecule-specific contrast. They come however with a limited penetration in depth and small field of view. Multimodal concepts help to combine complementary strengths of different imaging technologies. The present article reviews the advantages of optical multimodal imaging concepts using optical coherence tomography (OCT) as core technology. In particular we first discuss polarization sensitive OCT, Doppler OCT and OCT angiography, OCT elastography, and spectroscopic OCT as intramodal concepts. To highlight intermodal imaging concepts, we then chose the combination of OCT with photoacoustics, and with non-linear optical microscopy. The selected multimodal concepts and their particular complementary strengths and applications are discussed in detail. The article concludes with notes on standardization of OCT imaging and multimodal extensions

    Multi-contrast Photoacoustic Microscopy

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    Photoacoustic microscopy is a hybrid imaging modality with high spatial resolution, moderate imaging depth, excellent imaging contrast and functional imaging capability. Taking full advantage of this powerful weapon, we have investigated different anatomical, functional, flow dynamic and metabolic parameter measurements using photoacoustic microscopy. Specifically, Evans-blue dye was used to enhance photoacoustic microscopy of capillaries; label-free transverse and axial blood flow was measured based on bandwidth broadening and time shift of the photoacoustic signals; metabolic rate of oxygen was quantified in vivo from all the five parameters measured by photoacoustic microcopy; whole cross-sectional imaging of small intestine was achieved on a double-illumination photoacoustic microscopy with extended depth of focus and imaging depth; hemodynamic imaging was performed on a MEMS-mirror enhanced photoacoustic microscopy with a cross-sectional imaging rate of 400 Hz. As a maturing imaging technique, PAM is expected to find new applications in both fundamental life science and clinical practice

    THE MOUSE BRAIN: A 3D ATLAS REGISTERING MRI, CT, AND HISTOLOGICAL SECTIONS IN THE THREE CARDINAL PLANES

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    Mouse brain atlases based on histology can be improved through the reconstruction of the 2D histological sections into a continuous 3D volume. Impediments to a continuous reconstruction include distortion caused by excision, fixation, and sectioning of the brain. In prior works, MR images have been used as a reference for global alignment of the sections and various methods have been implemented for local alignment. In this thesis, we offered an alternative method for local alignment and developed a method for registering orthogonal histological data sets into one coordinate system. As an end result we established a comprehensive mouse brain atlas with Nissl-stained histology images with 362 coronal, 162 horizontal, and 112 sagittal histological sections at 40 µm interval. For the global alignment, our MRI/CT population atlas was used to guide the alignment accuracy. The local alignment was performed using Large Deformation Diffeomorphic Metric Mapping (LDDMM) with a hierarchical approach to minimize structural discontinuity. Then the coordinate consistency was optimized by iteratively registering the three 3D volume data from the coronal, horizontal, and sagittal sections. The landmark-based analysis revealed the MRI-histology accuracy level was 0.1632 ± 0.1131 mm. This work established the coordinate link between the MRI/CT atlas and around 300 GB of histology data in the cellular-level anatomical information

    Development of High-speed Optical Coherence Tomography for Time-lapse Non-destructive Characterization of Samples

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    Optical coherence tomography (OCT) is an established optical imaging modality which can obtain label-free, non-destructive 3D images of samples with micron-scale resolution and millimeter penetration. OCT has been widely adopted for biomedical researches

    Magnetic Resonance Imaging of the Rat Retina: a Dissertation

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    The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed a superfluous choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layerspecific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway
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