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Advanced optical microscopy for three dimensional deformation, profile and tomography measurementPan, Zhipeng 27 May 2016 (has links)
Deformation, profile and tomography measurement is critical for engineering materials characterization and engineering structure component design, analysis and biomedical application. The current existing 3D measurement method, such as stylus based profilometry, 3D optical stereo imaging and focus stacking, either suffers from low sampling speed from spatial scanning or maximum thickness of the specimen that could be imaged due to physical constraints. This thesis is dedicated to develop a hybrid 3D measurement method that can be easily implemented with fast imaging speed for dynamic process at the microscale. Also, at the microscale, the reduced depth of focus of existing microscope system greatly limits the maximum depth of the specimen that could be imaged, especially at high magnification. In this study, a 3D tomography system will be developed with extended depth of focus and improved axial resolution.
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Energetic Considerations and Structural Characterization of Twinning in NanowiresWu, Chun-Hsien 08 May 2013 (has links)
Twins are a pair of adjoining crystal grains related to each other by a special symmetry. They are frequently observed in bulk materials and nanomaterials. The formation of twins is an important topic in materials science and engineering because it affects material behaviors such as plastic deformation of metals, yield strength, and band gap energy in nanoscale semiconductors. Because of these unique phenomena and properties that the twinning can bring to the materials, it is of interest to investigate the formation of twins. Our primary objective in this dissertation is to study twinning in nanowires.
Both gold and platinum <111> oriented nanowires were fabricated by similar solution-phase chemical synthesis methods. High-resolution transmission electron microscopy and electron diffraction patterns were carried out to analyze the structures of the nanowires. Nanodiffraction was used to demonstrate twinning is a general structural feature of the growth of gold nanowires growing in a <111> direction. A model was proposed to explain the conditions under which twinning is energetically favored during nanowire growth. The model, which is based on a maximum rate hypothesis, considers the nanowire geometry and the relative surface and stacking fault energies and predicts twins should appear in gold nanowires but not in platinum nanowires, in agreement with experimental observations.
During the structural characterization of gold nanowires, our interest is to resolve 3D structure of twinning. However, the structure of twinning in gold nanowires is very fine and the average spacing between twin boundaries is only 0.57nm (+/- 0.38 nm); therefore, regular 3D electron microscopy technique is unable to reconstruct these defected structures. Here we present a stereo vision technique to reconstruct 3D atomic non-periodic structures containing defects. The technique employs intrinsic atomic planes as epipolar planes to achieve the alignment accuracy needed to reconstruct a crystal with atomic resolution. We apply it to determine the 3D geometry and atomic arrangements of twinning in gold nanowire.
In addition, an iterated cross-correlation algorithm was developed to analyze electron diffraction fully automatically to facilitate structural analysis of nanowires. A time-temperature-transformation diagram of platinum nanowires in chemical synthesis was determined to help optimize the fabrication process of the nanowires. / Ph. D.
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Sidestepping mechanism of yeast kinesin-8, Kip3Mitra, Aniruddha 07 March 2018 (has links) (PDF)
Kinesin-8 motors regulate the lengths of microtubules in cells. In previous studies, these motors have been shown to utilize their highly processive plus-end directed motility to reach microtubule plus-ends where they act as a microtubule depolymerase. The superprocessive motility importantly allows Kip3 motors to depolymerize microtubules in a length-dependent manner, the underlying mechanism of which has been described by an antenna model. During such long runs, motors in vivo are expected to frequently encounter roadblocks, such as microtubule associated proteins. The adaptions in the stepping mechanism that allow kinesin-8 motors to navigate around roadblocks to reach microtubule ends is not well understood. In this work, in vitro techniques were utilized to understand the navigation strategy of yeast kinesin-8, Kip3.
Three-dimensional stepping motility of Kip3 on the surface of microtubules can be inferred (i) indirectly from rotational motion of microtubules gliding along a surface coated with Kip3 and (ii) directly by three-dimensional tracking of Kip3 on freely suspended microtubules. Firstly, an impact-free method to detect rotations of gliding microtubules was established based on fluorescent speckles within the microtubule structure in combination with fluorescent interference contrast microscopy. Secondly, a suspended microtubule assay was established to obtain three- dimensional trajectories of single Kip3 motors, using Parallax, a dual-focus imaging technique.
The motility assays performed in this work revealed that Kip3 motors undergo left-handed helical motion around the microtubule lattice. This indicates that Kip3 employs a directed sidestepping strategy which is attributed to the motor having a flexible neck and/or a long neck linker. Interestingly, further analysis of the rotational motion revealed that the sidestepping of Kip3 is not directly coupled to the forward stepping. Based on these observations, it is hypothesized that the motor can transition from a two-head-bound conformation to a one-head-bound conformation while waiting for ATP. Whereas the motor can step forward from both states, sidestepping is strongly favored from the one-head-bound conformation. This hypothesis was confirmed through experiments as well as numerical simulations where the transition from the two-head-bound conformation to the one-head-bound conformation was enhanced by either prolonging the ATP waiting time or increasing the transition rate (by reducing the motor-microtubule interaction).
Finally, Kip3 based motility assays were performed using microtubules decorated with rigor binding kinesin-1 motors acting as roadblocks. While gliding assays using roadblock-decorated microtubules indicated a left-biased sidestepping strategy for Kip3, stepping assays revealed an additional diffusive component in the stepping motility of Kip3, along with the leftward bias. Taken together, it is hypothesized that Kip3 has a dual-mode roadblock circumnavigation strategy. Upon encountering a roadblock, the motor circumnavigates it (i) by shifting to the adjacent left microtubule protofilament using the biased sidestepping mechanism or (ii) by shifting microtubule protofilaments in an unbiased diffusive manner upon switching out of the step cycle. Therefore, the biophysical properties of Kip3 are fine-tuned to ensure that the motor reaches the microtubule plus-end to perform its depolymerase activity.
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Phenotype characterization of lung structure in inbred mouse strains using multi modal imaging techniquesNamati, Jacqueline Thiesse 01 May 2009 (has links)
Research involved in modeling human lung disease conditions has provided insight into disease development, progression, and treatment. In particular, mouse models of human pulmonary disease are increasingly utilized to characterize lung disease conditions. With advancements in small animal imaging it is now possible to investigate the phenotypic differences expressed in inbred mouse strains in vivo to investigate specific disease conditions that affect the lung.
In this thesis our aim was to generate a comprehensive characterization of the normative mouse lung phenotypes in three of the most utilized strains of mice, C57BL/6, A/J, and BALB/c, through imaging techniques. The imaging techniques that we utilized in this research included micro-CT, a custom Large Image Microscope Array (LIMA) system for 3D microscopy, and classical histology. Micro-CT provided a non-destructive technique for acquiring in vivo and fixed lung images. The LIMA 3D microscopy system was utilized for direct correspondence of the gold standard histology images as well as to validate the anatomical structures and measurements that were extracted from the micro-CT images. Finally, complete lung histology slices were utilized for assessment of the peripheral airspace structures that were not resolvable using the micro-CT imaging system.
Through our developed imaging acquisition and processing strategies we have been able to successful characterize important phenotypes in the mouse lung that have not previously been known as well as identify strain variations. These findings will provide the scientific community with valuable information to be better equipped and capable of pursuing new avenues of research in investigating pulmonary disease conditions that can be modeled in the mouse.
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Etude de mécanismes de type prion impliqués dans la maladie d’Alzheimer sur un modèle de mini-cerveaux humains avec exploration par microscopie 3D / Study of Prion-Like Mechanisms Involved in Alzheimer's Disease on Human Mini-Brains with 3D Microscopy ExplorationNassor, Férid 13 May 2019 (has links)
Les principales maladies neurodégénératives humaines, qui reposent toutes sur des mécanismes de type prion (auto-propagation d’agrégats protéiques pathogéniques), représentent un risque sociétal majeur avec une augmentation de leur prévalence directement corrélée avec l’augmentation de la longévité de la population mondiale. Il n’existe à ce jour aucun traitement curatif ni aucun modèle expérimental suffisamment pertinent pour ces maladies.L'objectif de ce travail a été d'utiliser le potentiel des mini-cerveaux humains comme modèle in vitro auto-assemblé en trois dimensions (3D) capable de restituer la complexité du cortex cérébral et d’étudier les mécanismes de type prion en développant une méthodologie de validation basée sur la microscopie 3D. Ces nouvelles structures 3D qui peuvent être obtenues à partir de cellules adultes reprogrammées de patients en cellules souches pluripotentes induites (iPSC), offrent des possibilités uniques pour accéder, observer et perturber les processus biologiques dans le cerveau humain sans les biais ni les complications des modèles animaux ou des échantillons de cerveau humain ex vivo. Ce modèle permet notamment d’observer l’apparition d’agrégats d’Aß et de Tau phosphorylée, deux protéines qui s’accumulent et se propagent de cellule en cellule dans la maladie d’Alzheimer.Nous avons été en mesure de rendre le modèle des mini-cerveaux compatible avec une future approche de criblage de molécules thérapeutiques en modifiant la méthodologie de différenciation pour augmenter leur rendement de production. D’autre part, nous avons pu tester différentes modalités de modélisation pour la Maladie d’Alzheimer, la maladie de Parkinson, la Démence Fronto-Temporale et la maladie de Creutzfeldt-Jakob : à l’aide de molécules d’induction chimique, à l’aide de cellules issues de patients, par modification génétique et par mise en contact de matériel infectieux. Ces différentes approches nous ont permis d’établir que le modèle d’organoïde cérébral permet de reproduire des aspects-clés retrouvés dans l’apparition de la pathologie chez les patients. Pour compenser l’hétérogénéité de notre modèle, nous avons réalisé une analyse in toto par imagerie, c’est-à-dire dans sa totalité sans coupes préalables. La modalité retenue pour cette acquisition est la microscopie à feuillet de lumière utilisée après marquage et clarification des mini-cerveaux. Pour ce faire, nous avons évalué et développé différentes stratégies en vue d’obtenir une plateforme d’analyse à haut contenu pour nos organoïdes cérébraux.Cette plateforme centrée autour de l’organoïde cérébral, sous-tendue par l’analyse en microscopie 3D, a été développée dans le cadre du projet « Investissements d’Avenir » 3DNeuroSecure. Ce projet a pour ambition d’apporter des solutions de calcul haute performance au domaine de la biologie, notamment avec la possibilité de traiter des informations de très grand volume, dit « exascale », telles que celles que nous obtenons avec la microscopie 3D. Le développement de cet aspect nous permettrait à terme de pouvoir établir les bases d’un outil de criblage thérapeutique par les organoïdes cérébraux pour les maladies neurodégénératives. Nous avons démontré que les mécanismes de type prion pouvaient être étudiés dans ce modèle de mini-cerveaux humains et de multiples voies de recherche fondamentale et appliquée sont désormais possibles. A plus long terme, une telle plateforme pourrait accueillir tout type d’organoïdes pour modéliser l’ensemble du corps humain et s’inscrire comme un compagnon biologique dans le cadre des futurs développements de la médecine personnalisée. / Human neurodegenerative diseases, which are all based on prion-like mechanisms (self-propagation of pathogenic protein aggregates), represent a major societal risk with the increase of their prevalence directly correlated to the increasing longevity of the world population. There is to date neither any cure nor any pertinent experimental model for their study.The objective of this work was to use the potential of human mini-brains, a self-assembled three-dimensional in vitro model able to restitute the complexity of the cerebral cortex. This model will allow us to study prion-like mechanism by developing a validation methodology based on 3D microscopy. These novel 3D structures can be obtained from reprogrammed adult cells into induced pluripotent stem cells (iPSCs) and offer unique possibilities to access, observe and disrupt biological processes in the human brain without bias nor complications as in animal models and ex vivo human brain samples. This model makes it possible to observe the development of aggregates of Aβ and hyper-phosphorylated Tau, two proteins that accumulate and propagate from cell to cell during Alzheimer’s disease.We have been to able to adapt the cerebral organoid model for a future screening approach by modifying the differentiation methodology to enhance its production ratio. We also have been able to test different modalities for disease modeling for Alzheimer’s disease, Parkinson’s disease, Fronto-Temporal Dementia and Creutzfeldt-Jakob disease: with chemical induction, with patient specific cells, through genetic modification and through contact with infectious material. These different approaches allowed us to validate that the cerebral organoid can indeed reproduce key aspects found during pathological development within patients. To compensate for the heterogeneity of the cerebral organoid, we performed an in toto analysis through microscopy, meaning in its totality without prior slicing. The chosen method of acquisition is fluorescence light-sheet microscopy used after staining and optical clearing of cerebral organoids. To do so, we have evaluated and established different strategies in order to obtain a high content screening platform for our cerebral organoid model.This platform centered around the cerebral organoid model, underpinned by 3D microscopy analysis, was developed during the “Investissements d’Avenir” project 3DNeuroSecure. This project has for ambition to bring high performance computing to the biological sciences, notably with the possibility to deal with large scale data, also called “exascale”, like the ones obtained with 3D microscopy. The development of this aspect would allow us to establish the basis for a therapeutic screening tool based on cerebral organoids for neurodegenerative diseases. We have demonstrated that prion-like mechanisms can be studied in a human mini-brain model and multiple research avenues are now opened for both fundamental and applied research. This platform could in turn become the basis for any kind of organoids derived from patients to model the whole human body and become a biological companion for personalized medicine.
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Sidestepping mechanism of yeast kinesin-8, Kip3Mitra, Aniruddha 18 December 2017 (has links)
Kinesin-8 motors regulate the lengths of microtubules in cells. In previous studies, these motors have been shown to utilize their highly processive plus-end directed motility to reach microtubule plus-ends where they act as a microtubule depolymerase. The superprocessive motility importantly allows Kip3 motors to depolymerize microtubules in a length-dependent manner, the underlying mechanism of which has been described by an antenna model. During such long runs, motors in vivo are expected to frequently encounter roadblocks, such as microtubule associated proteins. The adaptions in the stepping mechanism that allow kinesin-8 motors to navigate around roadblocks to reach microtubule ends is not well understood. In this work, in vitro techniques were utilized to understand the navigation strategy of yeast kinesin-8, Kip3.
Three-dimensional stepping motility of Kip3 on the surface of microtubules can be inferred (i) indirectly from rotational motion of microtubules gliding along a surface coated with Kip3 and (ii) directly by three-dimensional tracking of Kip3 on freely suspended microtubules. Firstly, an impact-free method to detect rotations of gliding microtubules was established based on fluorescent speckles within the microtubule structure in combination with fluorescent interference contrast microscopy. Secondly, a suspended microtubule assay was established to obtain three- dimensional trajectories of single Kip3 motors, using Parallax, a dual-focus imaging technique.
The motility assays performed in this work revealed that Kip3 motors undergo left-handed helical motion around the microtubule lattice. This indicates that Kip3 employs a directed sidestepping strategy which is attributed to the motor having a flexible neck and/or a long neck linker. Interestingly, further analysis of the rotational motion revealed that the sidestepping of Kip3 is not directly coupled to the forward stepping. Based on these observations, it is hypothesized that the motor can transition from a two-head-bound conformation to a one-head-bound conformation while waiting for ATP. Whereas the motor can step forward from both states, sidestepping is strongly favored from the one-head-bound conformation. This hypothesis was confirmed through experiments as well as numerical simulations where the transition from the two-head-bound conformation to the one-head-bound conformation was enhanced by either prolonging the ATP waiting time or increasing the transition rate (by reducing the motor-microtubule interaction).
Finally, Kip3 based motility assays were performed using microtubules decorated with rigor binding kinesin-1 motors acting as roadblocks. While gliding assays using roadblock-decorated microtubules indicated a left-biased sidestepping strategy for Kip3, stepping assays revealed an additional diffusive component in the stepping motility of Kip3, along with the leftward bias. Taken together, it is hypothesized that Kip3 has a dual-mode roadblock circumnavigation strategy. Upon encountering a roadblock, the motor circumnavigates it (i) by shifting to the adjacent left microtubule protofilament using the biased sidestepping mechanism or (ii) by shifting microtubule protofilaments in an unbiased diffusive manner upon switching out of the step cycle. Therefore, the biophysical properties of Kip3 are fine-tuned to ensure that the motor reaches the microtubule plus-end to perform its depolymerase activity.
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Restauration d'images de noyaux cellulaires en microscopie 3D par l'introduction de connaissance a priori / Denoising 3D microscopy images of cell nuclei using shape priorsBouyrie, Mathieu 29 November 2016 (has links)
Cette thèse aborde la problématique de la restauration d’images 3D de noyaux cellulaires fluorescents issues de la microscopie 2-photons à balayage laser d’animaux observés in vivo et in toto au cours de leur développement embryonnaire. La dégradation originale de ces images provient des limitations des systèmes optiques, du bruit intrinsèque des systèmes de détection ansi que de l’absorption et la diffusion de la lumière dans la profondeur des tissus. A la différence des propositions de “débruitage” de l’état de l’art, nous proposons ici une méthode qui prend en compte les particularités des données biologiques. Cette méthode, adaptation à la troisième dimension d’un algorithme utilisé dans l’analyse d’image astronomique, tire parti de connaissances a priori sur les images étudiées. Les hypothèses émises portent à la fois sur la détérioration du signal par un bruit supposé Mixe Poisson Gaussien (MPG) et sur la nature des objets observés. Nous traitons ici le cas de noyaux de cellules embryonnaires que nous supposons quasi sphériques.L’implémentation en 3D doit prendre en compte les dimensions de la grille d’échantillonnage de l’image. En effet ces dimensions ne sont pas identiques dans les trois directions de l’espace et un objet sphérique échantillonné sur cette grille perd cette caractéristique. Pour adapter notre méthode à une telle grille, nous avons ré-interprété le processus de filtrage, au coeur de la théorie originale, comme un processus physique de diffusion. / In this this document, we present a method to denoise 3D images acquired by 2-photon microscopy and displaying cell nuclei of animal embryos. The specimens are observed in toto and in vivo during their early development. Image deterioration can be explained by the microscope optical flaws, the acquisition system limitations, and light absorption and diffusion through the tissue depth.The proposed method is a 3D adaptation of a 2D method so far applied to astronomical images and it also differs from state-of the of-the-art methods by the introduction of priors on the biological data. Our hypotheses include assuming that noise statistics are Mixed Poisson Gaussian (MPG) and that cell nuclei are quasi spherical.To implement our method in 3D, we had to take into account the sampling grid dimensions which are different in the x, y or z directions. A spherical object imaged on this grid loses this property. To deal with such a grid, we had to interpret the filtering process, which is a core element of the original theory, as a diffusion process.
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Exprese CD47 a jeho topologie na povrchu primárních buněk karcinomu močového měchýře při interakci s makrofágy / Exprese CD47 a jeho topologie na povrchu primárních buněk karcinomu močového měchýře při interakci s makrofágyRajtmajerová, Marie January 2018 (has links)
CD47 is a so-called "don't eat me" signal, which protects cells from phagocytosis. Its high expresion on tumor cells brings new perspective to the tumor therapy. Monoclonal antibodies, which are these days undergoing clinical trials, prevent CD47 binding to the SIRPA inhibitory receptor on macrophages, and so they enhance their phagocytic functional capacity. In this way they enable phagocytic removal of tumor cells. Overall expression, structural conformation and stoichiometry of CD47 on a particular cell predestine whether it will be phagocytised. The aim of the thesis is to develop and test methods to characterise expression parameters of CD47 via flow cytometry (FCM), quantitative PCR (qPCR) and microscopy. To achieve this goal I performed competition tests of commercially available antibodies in order to characterise their binding epitopes on cell lines. After performing tSNE analysis of primary BCa patient samples I correlated CD47 expression with other cell surface markers. I focused on CD47 expression in various differentiation stages of the tumor. To better understand the relationship between CD47 expression and differentiation status of cells I performed qPCR analysis of particular transcription factors. Using cell lines I examined method for phagocytosis quantification, which will be...
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