Neuro-imagerie multimodale et multirésolution de cerveaux de souris combinant l’histologie sérielle par tomographie en cohérence optique et l’IRM de diffusion

L’histologie sérielle est une technique d’imagerie permettant d’observer des échantillons entiers à haute résolution. Cette technique consiste à trancher de fines couches de tissu, puis à déplacer l’échantillon sous un objectif de microscope afin d’acquérir autant d’images que nécessaire pour couvrir toute la surface révélée par la coupe. Ce processus est automatisé et est répété jusqu’à ce que tout l’échantillon soit imagé, c’est-à-dire un cerveau de souris dans cette thèse. Couplée à un microscope par tomographie en cohérence optique (OCT), cette modalité est capable de cartographier la distribution spatiale de la matière blanche dans des cerveaux entiers de souris. L’objectif principal de cette thèse était de développer les méthodes de reconstruction nécessaires à l’assemblage en un seul volume des milliers d’images acquises par un système d’histologie massive. De plus, dans cette première phase du projet, des méthodes permettant d’aligner les données sur des images IRM acquises pour les mêmes animaux ont été développées. Cela a permis de mieux comprendre l’origine du contraste optique dans le cerveau et cela offre maintenant la possibilité d’intégrer l’histologie massive dans les études de neuro-imagerie employant des groupes d’animaux. Dans une seconde phase du projet, un microscope à cohérence optique haute résolution a été ajouté au système d’histologie par OCT existant. Cette nouvelle plateforme d’imagerie utilise les images à basse résolution comme repère pour localiser au sein du cerveau les images à haute résolution du second microscope. L’utilité d’une telle plateforme réside dans le fait qu’il est maintenant possible de cibler des régions spécifiques à observer en détail sans avoir à imager un cerveau entier à cette grande résolution, ce qui représenterait plusieurs semaines de mesurage et des quantités immenses de données à assembler. Les données mesurées avec la nouvelle plateforme ont été intégrées à la procédure de reconstruction et d’alignement développé pour la première phase du projet. Ainsi, il a été possible de comparer les images à grande résolution avec les données d’IRM de diffusion acquises pour les mêmes cerveaux de souris. Ceci a permis de confirmer des hypothèses posées lors de l’analyse des données IRM de diffusion à partir de la microscopie. Les méthodes de reconstruction, d’alignement et d’analyse développées, ainsi que la nouvelle plateforme d’histologie sérielle bi-résolution par OCT, offrent enfin la possibilité d’utiliser cette modalité optique pour réaliser des études de groupes animales ou bien pour valider des mesures faites dans le cerveau avec d’autres modalités d’imagerie telle que l’IRM de diffusion.----------ABSTRACT Serial histology is an imaging technique able to observe whole samples at high resolution. This technique involves cutting thin tissue layers, followed by the positioning of the sample under a microscope objective and the acquisition of as many images as necessary to cover the entire area revealed by the cut. This process is automated and is repeated until the entire brain has been imaged. Coupled with an optical coherence tomography (OCT) microscope, this modality is able to map the spatial distribution of white matter in whole mouse brains. The main objective of this thesis was to develop the reconstruction methods necessary for the assembly into a single volume of the thousands of images acquired with a massive histology system. In addition, in this first project phase, methods for aligning data on MRI images acquired for the same animals have been developed. This has led to a better understanding of the optical contrast origin in the brain and it now offers the possibility of integrating massive histology into neuroimaging studies using animal groups. In a second phase of the project, a high resolution optical coherence microscope was added to the existing OCT histology system. This new imaging platform uses low-resolution images as a reference to locate the high-resolution images of the second microscope within the brain. The usefulness of such a platform lies in the fact that it is now possible to target specific regions to observe in detail without having to image an entire brain at this high resolution, which would represent several weeks for measurements and immense quantities of data to assemble. The data measured with the new platform have been incorporated into the reconstruction and alignment procedure developed for the first phase of the project. Thus, it was possible to compare the high resolution images with the diffusion MRI data acquired for the same mouse brains. This made it possible to confirm hypotheses posed during the analysis of diffusion MRI data. The methods of reconstruction, alignment and analysis developed during this thesis, as well as the new dual resolution serial OCT histology platform, finally offer the possibility of using this optical modality to carry out studies of animal groups or to validate measurements made in a brain with other imaging modalities such as diffusion MRI

Développement d’outils de vectorisation d’angiographies obtenues par microscopie 2-photons dans le contexte du vieillissement du cerveau

RÉSUMÉ Les pathologies affectant les petits vaisseaux de la neurovasculature sont-elles à l’origine des effets cognitifs qui apparaissent au cours du vieillissement? Pour répondre à cette question, il faut d’abord posséder des outils permettant d’extraire la microvasculature du néocortex à partir d’angiographies acquises par microscopie à fluorescence deux-photons. Une meilleure compréhension de l’évolution de la microvasculature du cerveau avec l’âge et de l’effet de ces modifications sur les fonctions des aires corticales constitue en effet une étape essentielle vers la mise en place de nouveaux biomarqueurs du vieillissement du cerveau. Des modèles réalistes de la vasculature du cerveau peuvent servir de base à des modélisations du débit sanguin et à la simulation de signal IRM. En utilisant des vasculatures vectorisées, de nouvelles voies de recherche pourront donc être explorées, dont l’effet de différents types de pathologies des petits vaisseaux sur le signal IRM dépendant du niveau d’oxygène sanguin (BOLD). L’objectif de ce projet de recherche est donc le développement d’une méthode de segmentation des vaisseaux sanguins et d’un outil d’interaction permettant de corriger et de modifier le réseau vasculaire extrait. Ces outils sont utilisés pour comparer la microvasculature du néocortex de rats provenant de deux cohortes d’âges différents formés de 12 jeunes rats (âge = 11-15 semaines) et 12 rats âgés (âge = 23-25 mois) de type Long-Evans. Ces méthodes ont été développées en utilisant principalement la plateforme de programmation MATLAB, le module de gestion de pipeline de traitement PSOM et l’outil de traitement d’image FIJI. La méthode est semi-automatique, nécessitant une correction manuelle des graphes extraits des angiographies après la segmentation. L’approche modulable adoptée permet l’ajout de nouvelles fonctions et de nouveaux outils, ce qui pourra améliorer sa robustesse et l’automatisation de l’extraction des vaisseaux sanguins. En analysant les masques des vasculatures issus du prétraitement des données, il a été montré que la densité des capillaires dans le néocortex sensorimoteur de rats Long-Evans diminue avec l’âge, en passant de pour les jeunes rats à pour les rats âgés, ce qui représente une baisse statistiquement significative de 20 %. Une analyse utilisant les graphes nettoyés semble également aller dans ce sens en montrant que la densité linéaire des vaisseaux décroît au cours du vieillissement. Cette mesure est liée aux capacités de perfusion de la vasculature, et pourrait indiquer que l’efficacité d’apport en nutriment et en oxygène décroît dans le néocortex sensorimoteur de rats au cours du vieillissement.----------ABSTRACT Are the conditions affecting the small vessels of the neurovasculature the cause of cognitive impairments that appear with aging? To answer this question, we must have tools to extract the neocortex microvasculature from angiograms acquired by two-photon fluorescence microscopy. A better understanding of the brain microvasculature evolution with age and the effect of those changes on the cortical areas functions is indeed an essential step towards the development of new biomarkers of brain aging. Realistic models of the brain vasculature can be used as a basis of blood flow modeling and to simulate MRI signal originating from these vessels. Using vectorized vasculatures, new research avenues can be explored, including the effect of different types of small vessels diseases on blood oxygen level-dependent (BOLD) MRI signal. The main objective of this research project is the development of a blood vessel segmentation method and of an interface to correct and modify the extracted vascular networks. These tools are used to compare the neocortex microvasculature of rats from two different age cohorts. These cohorts consist of 12 young (age = 11-15 weeks) and 12 old Long-Evans rats (age = 23-25 months). The tools have been developed using the MATLAB programming platform, the pipeline processing module PSOM and the image processing tool FIJI. The method is semi-automatic, requiring manual correction of the extracted angiogram graphs after segmentation. The modular approach allows the addition of new features and tools, which can improve the robustness and automation of the blood vessels extraction technique. By analyzing the vasculature masks obtained by the initial data preprocessing, it is found that the density of capillaries in the sensorimotor neocortex of Long-Evans rats decreases with age, from ρ = 6.8 ± 0.3 [%] in young rats to ρ = 5.4 ± 0.3 [%] in aged rats, which represents a statistically significant decrease of 20%. An analysis using the cleaned graphs also seems to go in this direction by showing that the linear density of vessels decreases with aging. This density is linked to the perfusion capacity of the vasculature, and may indicate that the efficiency of nutrient and oxygen distribution decreases with aging in rat’s sensorimotor neocortex

A review of intrinsic optical imaging serial blockface histology (ICI-SBH) for whole rodent brain imaging

In recent years, multiple serial histology techniques were developed to enable whole rodent brain imaging in 3-D. The main driving forces behind the emergence of these imaging techniques were the genome-wide atlas of gene expression in the mouse brain, the pursuit of the mouse brain connectome, and the BigBrain project. These projects rely on the use of optical imaging to target neuronal structures with histological stains or fluorescent dyes that are either expressed by transgenic mice or injected at specific locations in the brain. Efforts to adapt the serial histology acquisition scheme to use intrinsic contrast imaging (ICI) were also put forward, thus leveraging the natural contrast of neuronal tissue. This review focuses on these efforts. First, the origin of optical contrast in brain tissue is discussed with emphasis on the various imaging modalities exploiting these contrast mechanisms. Serial blockface histology (SBH) systems using ICI modalities are then reported, followed by a review of some of their applications. These include validation studies and the creation of multimodal brain atlases at a micrometer resolution. The paper concludes with a perspective of future developments, calling for a consolidation of the SBH research and development efforts around the world. The goal would be to offer the neuroscience community a single standardized open-source SBH solution, including optical design, acquisition automation, reconstruction algorithms, and analysis pipelines

Identification of the metallurgical parameters explaining the corrosion susceptibility in a 2050 aluminium alloy

The corrosion behaviour of a 2050 aluminium alloy was studied in a NaCl solution. The structure ofprecipitation did not fully explain the susceptibility to intergranular (in the -T34 state) and intragran-ular corrosion for the aged state (the -T8 state). A relationship between the nature of interfaces, thegrains characteristics (size, internal misorientation and orientation according to the plane exposed tothe electrolyte) on one hand and the corrosion susceptibility of the alloy on the other hand was clearlyestablished. Galvanic coupling between grains with different internal misorientations helped to explainthe intergranular corrosion susceptibility of the -T34 state

TRAIT2D: a Software for Quantitative Analysis of Single Particle Diffusion Data

Single particle tracking (SPT) is one of the most widely used tools in optical microscopy to evaluate particle mobility in a variety of situations, including cellular and model membrane dynamics. Recent technological developments, such as Interferometric Scattering microscopy, have allowed recording of long, uninterrupted single particle trajectories at kilohertz framerates. The resulting data, where particles are continuously detected and do not displace much between observations, thereby do not require complex linking algorithms. Moreover, while these measurements offer more details into the short-term diffusion behaviour of the tracked particles, they are also subject to the influence of localisation uncertainties, which are often underestimated by conventional analysis pipelines. we thus developed a Python library, under the name of TRAIT2D (Tracking Analysis Toolbox – 2D version), in order to track particle diffusion at high sampling rates, and analyse the resulting trajectories with an innovative approach. The data analysis pipeline introduced is more localisation-uncertainty aware, and also selects the most appropriate diffusion model for the data provided on a statistical basis. A trajectory simulation platform also allows the user to handily generate trajectories and even synthetic time-lapses to test alternative tracking algorithms and data analysis approaches. A high degree of customisation for the analysis pipeline, for example with the introduction of different diffusion modes, is possible from the source code. Finally, the presence of graphical user interfaces lowers the access barrier for users with little to no programming experience

Fully automated dual-resolution serial optical coherence tomography aimed at diffusion MRI validation in whole mouse brains

An automated dual-resolution serial optical coherence tomography (2R-SOCT) scanner is developed. The serial histology system combines a low-resolution ( 25 mu m / voxel ) 3 x OCT with a high-resolution ( 1.5 mu m / voxel ) 40 x OCT to acquire whole mouse brains at low resolution and to target specific regions of interest (ROIs) at high resolution. The 40 x ROIs positions are selected either manually by the microscope operator or using an automated ROI positioning selection algorithm. Additionally, a multimodal and multiresolution registration pipeline is developed in order to align the 2R-SOCT data onto diffusion MRI (dMRI) data acquired in the same ex vivo mouse brains prior to automated histology. Using this imaging system, 3 whole mouse brains are imaged, and 250 high-resolution 40 x three-dimensional ROIs are acquired. The capability of this system to perform multimodal imaging studies is demonstrated by labeling the ROIs using a mouse brain atlas and by categorizing the ROIs based on their associated dMRI measures. This reveals a good correspondence of the tissue microstructure imaged by the high-resolution OCT with various dMRI measures such as fractional anisotropy, number of fiber orientations, apparent fiber density, orientation dispersion, and intracellular volume fraction

Interaction of HmC1q with leech microglial cells: involvement of C1qBP-related molecule in the induction of cell chemotaxis

<p>Abstract</p> <p>Background</p> <p>In invertebrates, the medicinal leech is considered to be an interesting and appropriate model to study neuroimmune mechanisms. Indeed, this non-vertebrate animal can restore normal function of its central nervous system (CNS) after injury. Microglia accumulation at the damage site has been shown to be required for axon sprouting and for efficient regeneration. We characterized <it>Hm</it>C1q as a novel chemotactic factor for leech microglial cell recruitment. In mammals, a C1q-binding protein (C1qBP alias gC1qR), which interacts with the globular head of C1q, has been reported to participate in C1q-mediated chemotaxis of blood immune cells. In this study, we evaluated the chemotactic activities of a recombinant form of <it>Hm</it>C1q and its interaction with a newly characterized leech C1qBP that acts as its potential ligand.</p> <p>Methods</p> <p>Recombinant <it>Hm</it>C1q (r<it>Hm</it>C1q) was produced in the yeast <it>Pichia pastoris</it>. Chemotaxis assays were performed to investigate r<it>Hm</it>C1q-dependent microglia migration. The involvement of a C1qBP-related molecule in this chemotaxis mechanism was assessed by flow cytometry and with affinity purification experiments. The cellular localization of C1qBP mRNA and protein in leech was investigated using immunohistochemistry and <it>in situ </it>hybridization techniques.</p> <p>Results</p> <p>r<it>Hm</it>C1q-stimulated microglia migrate in a dose-dependent manner. This r<it>Hm</it>C1q-induced chemotaxis was reduced when cells were preincubated with either anti-<it>Hm</it>C1q or anti-human C1qBP antibodies. A C1qBP-related molecule was characterized in leech microglia.</p> <p>Conclusions</p> <p>A previous study showed that recruitment of microglia is observed after <it>Hm</it>C1q release at the cut end of axons. Here, we demonstrate that r<it>Hm</it>C1q-dependent chemotaxis might be driven via a <it>Hm</it>C1q-binding protein located on the microglial cell surface. Taken together, these results highlight the importance of the interaction between C1q and C1qBP in microglial activation leading to nerve repair in the medicinal leech.</p

Towards the Industrialization of New MDO Methodologies and Tools for Aircraft Design

An overall summary of the Institute of Technology IRT Saint Exupery MDA-MDO project (Multi-Disciplinary Analysis - Multidisciplinary Design Optimization) is presented. The aim of the project is to develop efficient capabilities (methods, tools and a software platform) to enable industrial deployment of MDO methods in industry. At IRT Saint Exupery, industrial and academic partners collaborate in a single place to the development of MDO methodologies; the advantage provided by this mixed organization is to directly benefit from both advanced methods at the cutting edge of research and deep knowledge of industrial needs and constraints. This paper presents the three main goals of the project: the elaboration of innovative MDO methodologies and formulations (also referred to as architectures in the literature 1) adapted to the resolution of industrial aircraft optimization design problems, the development of a MDO platform featuring scalable MDO capabilities for transfer to industry and the achievement of a simulation-based optimization of an aircraft engine pylon with industrial Computational Fluid Dynamics (CFD) and Computational Structural Mechanics (CSM) tools

Validating intravascular imaging with serial optical coherence tomography and confocal fluorescence microscopy

Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology