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Super-resolution microscopy development for the detection of nano-structures and confocal study of the structural damage in gut cell nuclei due to total body irradiationHasan, Mehedi 25 November 2020 (has links)
Optical microscopy is the oldest form of microscopy that has been visually aiding scientific research. In our research, I have reported here two such optical microscopy techniques for two different projects. In the first project, we re-developed an instrumentation of a cost-effective, high-performing, single-molecular localization super-resolution microscopy setup that breaks the diffraction limitation barrier. Then we use a stochastic image capturing technique to capture the best precision image positions of gold nanoparticles. In our second project, we apply confocal microscopy technique to image DNA molecular nanoscale structural alterations of chromatin in cell nuclei of gut tissues caused by total body irradiation (TBI). We then quantify these alterations using a light localization technique called inverse participation ratio (IPR) using the confocal micrographs of the sample. Our results show radiation causes reduction and saturation of DNA spatial mass density fluctuations that were observed for different durations of post-irradiation.
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Quantitative single molecule imaging deep in biological samples using adaptive optics / Imagerie quantitative des molécules uniques en profondeur dans les échantillons biologique à l'aide d'optiques adaptativesButler, Corey 04 July 2017 (has links)
La microscopie optique est un outil indispensable pour la recherche de la neurobiologie et médecine qui permet l’étude des cellules dans leur environnement natif. Les processus sous-cellulaires restent néanmoins cachés derrière les limites de la résolution optique, ce qui rend la résolution des structures plus petites que ~300nm impossible. Récemment, les techniques de la localisation des molécules individuelles (SML) ont permis le suivi des protéines de l’échelle nanométrique grâce à l’ajustement des molécules uniques à la réponse impulsionnelle du système optique. Ce processus dépend de la quantité de lumière recueilli et rend ces techniques très sensibles aux imperfections de la voie d’imagerie, nommé des aberrations, qui limitent l’application de SML aux cultures cellulaires sur les lamelles de verre. Un système commercial d’optiques adaptatives est implémenté pour compenser les aberrations du microscope, et un flux de travail est défini pour corriger les aberrations dépendant de la profondeur qui rend la 3D SML possible dans les milieux biologiques complexes. Une nouvelle méthode de SML est présentée qui utilise deux objectifs pour détecter le spectre d’émission des molécules individuelles pour des applications du suivi des particules uniques dans 5 dimensions (x,y,z,t,λ) sans compromis ni de la résolution spatiotemporelle ni du champ de vue. Pour faciliter les analyses de manière quantitative des Go de données générés, le développement des outils biochimiques, numériques et optiques est présenté. Ensemble, ces approches ont le but d’amener l’imagerie quantitative des molécules uniques dans les échantillons biologiques complexes / Optical microscopy is an indispensable tool for research in neurobiology and medicine, enabling studies of cells in their native environment. However, subcellular processes remain hidden behind the resolution limits of diffraction-limited optics which makes structures smaller than ~300nm impossible to resolve. Recently, single molecule localization (SML) and tracking has revolutionized the field, giving nanometer-scale insight into protein organization and dynamics by fitting individual fluorescent molecules to the known point spread function of the optical imaging system. This fitting process depends critically on the amount of collected light and renders SML techniques extremely sensitive to imperfections in the imaging path, called aberrations, that have limited SML to cell cultures on glass coverslips. A commercially available adaptive optics system is implemented to compensate for aberrations inherent to the microscope, and a workflow is defined for depth-dependent aberration correction that enables 3D SML in complex biological environments. A new SML technique is presented that employs a dual-objective approach to detect the emission spectrum of single molecules, enabling 5-dimensional single particle imaging and tracking (x,y,z,t,λ) without compromising spatiotemporal resolution or field of view. These acquisitions generate ~GBs of data, containing a wealth of information about the localization and environment of individual proteins. To facilitate quantitative acquisition and data analysis, the development of biochemical, software and hardware tools are presented. Together, these approaches aim to enable quantitative SML in complex biological samples.
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Výpočetní metody v jednomolekulové lokalizační mikroskopii / Computational methods in single molecule localization microscopyOvesný, Martin January 2016 (has links)
Computational methods in single molecule localization microscopy Abstract Fluorescence microscopy is one of the chief tools used in biomedical research as it is a non invasive, non destructive, and highly specific imaging method. Unfortunately, an optical microscope is a diffraction limited system. Maximum achievable spatial resolution is approximately 250 nm laterally and 500 nm axially. Since most of the structures in cells researchers are interested in are smaller than that, increasing resolution is of prime importance. In recent years, several methods for imaging beyond the diffraction barrier have been developed. One of them is single molecule localization microscopy, a powerful method reported to resolve details as small as 5 nm. This approach to fluorescence microscopy is very computationally intensive. Developing methods to analyze single molecule data and to obtain super-resolution images are the topics of this thesis. In localization microscopy, a super-resolution image is reconstructed from a long sequence of conventional images of sparsely distributed single photoswitchable molecules that need to be sys- tematically localized with sub-diffraction precision. We designed, implemented, and experimentally verified a set of methods for automated processing, analysis and visualization of data acquired...
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The Copper(I)-catalyzed Azide–Alkyne Cycloaddition: A Modular Approach to Synthesis and Single-Molecule Spectroscopy Investigation into Heterogeneous CatalysisDecan, Matthew January 2015 (has links)
Click chemistry is a molecular synthesis strategy based on reliable, highly selective reactions with thermodynamic driving forces typically in excess of 20 kcal mol-1. The 1,3-dipolar cycloaddition of azides and alkynes developed by Rolf Huisgen saw dramatic rate acceleration using Cu(I) as a catalyst in 2002 reports by Barry Sharpless and Morten Meldal enabling its click chemistry eligibility. Since these seminal reports, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has become the quintessential click reaction finding diverse utility. The popularity of the CuAAC has naturally led to interest in new catalyst systems with improved efficiency, robustness, and reusability with particular focus on nanomaterial catalysts, a common trend across the field of catalysis. The high surface area of nanomaterials lends to their efficacy as colloidal and heterogeneous nanocatalysts, but the latter boasts the added benefit of easy separation and recyclability. With any heterogeneous catalyst, a common question arises as to whether the active catalyst species is truly heterogeneous or rather homogeneous through metal ion leaching. Differentiating these processes is critical, as the latter would result in reduced efficiency, higher cost, and inevitable environmental and heath side effects.
This thesis explores the CuAAC from an interdisciplary approach. First as a synthetic tool, applying CuAAC-formed triazoles as functional, modular building blocks in the synthesis of optical cation sensors by combining azide and alkyne modified components to create a series of sensors selective for different metal cations. Next, single-molecule spectroscopy techniques are employed to observe the CuNP-catalyzed CuAAC in real time. Combining bench-top techniques with single-molecule microscopy to monitor single-catalytically generated products proves to be an effective method to establish catalysis occurs directly at the surface of copper nanoparticles, ruling out catalysis by ions leached into solution. This methodology is extended to mapping the catalytic activity of a commercial heterogeneous catalyst by applying super-localization analysis of single-catalytic events. The approach detailed herein is a general one that can be applied to any catalytic system through the development of appropriate probes. This thesis demonstrates single-molecule microscopy as an accessible, effective, and unparalleled tool for exploring the catalytic activity of nanomaterials by monitoring single-catalytic events as they occur.
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Výpočetní metody v jednomolekulové lokalizační mikroskopii / Computational methods in single molecule localization microscopyOvesný, Martin January 2016 (has links)
Computational methods in single molecule localization microscopy Abstract Fluorescence microscopy is one of the chief tools used in biomedical research as it is a non invasive, non destructive, and highly specific imaging method. Unfortunately, an optical microscope is a diffraction limited system. Maximum achievable spatial resolution is approximately 250 nm laterally and 500 nm axially. Since most of the structures in cells researchers are interested in are smaller than that, increasing resolution is of prime importance. In recent years, several methods for imaging beyond the diffraction barrier have been developed. One of them is single molecule localization microscopy, a powerful method reported to resolve details as small as 5 nm. This approach to fluorescence microscopy is very computationally intensive. Developing methods to analyze single molecule data and to obtain super-resolution images are the topics of this thesis. In localization microscopy, a super-resolution image is reconstructed from a long sequence of conventional images of sparsely distributed single photoswitchable molecules that need to be sys- tematically localized with sub-diffraction precision. We designed, implemented, and experimentally verified a set of methods for automated processing, analysis and visualization of data acquired...
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Single-Molecule Metal-Induced Energy Transfer: From Basics to ApplicationsKaredla, Narain 02 June 2016 (has links)
No description available.
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Echantillonnage compressif appliqué à la microscopie de fluorescence et à la microscopie de super résolution / Compressive fluorescence microscopy for biological imaging and super resolution microscopy.Chahid, Makhlad 19 December 2014 (has links)
Mes travaux de thèse portent sur l’application de la théorie de l’échantillonnagecompressif (Compressed Sensing ou Compressive Sampling, CS) à la microscopie defluorescence, domaine en constante évolution et outil privilégié de la recherche fondamentaleen biologie. La récente théorie du CS a démontré que pour des signauxparticuliers, dits parcimonieux, il est possible de réduire la fréquence d’échantillonnagede l’information à une valeur bien plus faible que ne le prédit la théorie classiquede l’échantillonnage. La théorie du CS stipule qu’il est possible de reconstruireun signal, sans perte d’information, à partir de mesures aléatoires fortement incomplèteset/ou corrompues de ce signal à la seule condition que celui-ci présente unestructure parcimonieuse.Nous avons développé une approche expérimentale inédite de la théorie du CSà la microscopie de fluorescence, domaine où les signaux sont naturellement parcimonieux.La méthode est basée sur l’association d’une illumination dynamiquestructurée à champs large et d’une détection rapide à point unique. Cette modalitépermet d’inclure l’étape de compression pendant l’acquisition. En outre, nous avonsmontré que l’introduction de dimensions supplémentaires (2D+couleur) augmentela redondance du signal, qui peut être pleinement exploitée par le CS afin d’atteindredes taux de compression très importants.Dans la continuité de ces travaux, nous nous sommes intéressés à une autre applicationdu CS à la microscopie de super résolution, par localisation de moléculesindividuelles (PALM/STORM). Ces nouvelles techniques de microscopie de fluorescenceont permis de s’affranchir de la limite de diffraction pour atteindre des résolutionsnanométriques. Nous avons exploré la possibilité d’exploiter le CS pour réduiredrastiquement les temps d’acquisition et de traitement.Mots clefs : échantillonnage compressif, microscopie de fluorescence, parcimonie,microscopie de super résolution, redondance, traitement du signal, localisation demolécules uniques, bio-imagerie / My PhD work deals with the application of Compressed Sensing (or CompressiveSampling, CS) in fluorescence microscopy as a powerful toolkit for fundamental biologicalresearch. The recent mathematical theory of CS has demonstrated that, for aparticular type of signal, called sparse, it is possible to reduce the sampling frequencyto rates well below that which the sampling theorem classically requires. Its centralresult states it is possible to losslessly reconstruct a signal from highly incompleteand/or inaccurate measurements if the original signal possesses a sparse representation.We developed a unique experimental approach of a CS implementation in fluorescencemicroscopy, where most signals are naturally sparse. Our CS microscopecombines dynamic structured wide-field illumination with fast and sensitive singlepointfluorescence detection. In this scheme, the compression is directly integratedin the measurement process. Additionally, we showed that introducing extra dimensions(2D+color) results in extreme redundancy that is fully exploited by CS to greatlyincrease compression ratios.The second purpose of this thesis is another appealing application of CS forsuper-resolution microscopy using single molecule localization techniques (e.g.PALM/STORM). This new powerful tool has allowed to break the diffraction barrierdown to nanometric resolutions. We explored the possibility of using CS to drasticallyreduce acquisition and processing times.
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Development of advanced methods for super-resolution microscopy data analysis and segmentation / Développement de méthodes avancées pour l'analyse et la segmentation de données de microscopie à super-résolutionAndronov, Leonid 09 January 2018 (has links)
Parmi les méthodes de super-résolution, la microscopie par localisation de molécules uniques se distingue principalement par sa meilleure résolution réalisable en pratique mais aussi pour l’accès direct aux propriétés des molécules individuelles. Les données principales de la microscopie par localisation sont les coordonnées des fluorochromes, un type de données peu répandu en microscopie conventionnelle. Le développement de méthodes spéciales pour le traitement de ces données est donc nécessaire. J’ai développé les logiciels SharpViSu et ClusterViSu qui permettent d’effectuer les étapes de traitements les plus importantes, notamment une correction des dérives et des aberrations chromatiques, une sélection des événements de localisations, une reconstruction des données dans des images 2D ou dans des volumes 3D par le moyen de différentes techniques de visualisation, une estimation de la résolution à l’aide de la corrélation des anneaux de Fourier, et une segmentation à l’aide de fonctions K et L de Ripley. En plus, j’ai développé une méthode de segmentation de données de localisation en 2D et en 3D basée sur les diagrammes de Voronoï qui permet un clustering de manière automatique grâce à modélisation de bruit par les simulations Monte-Carlo. En utilisant les méthodes avancées de traitement de données, j’ai mis en évidence un clustering de la protéine CENP-A dans les régions centromériques des noyaux cellulaires et des transitions structurales de ces clusters au moment de la déposition de la CENP-A au début de la phase G1 du cycle cellulaire. / Among the super-resolution methods single-molecule localization microscopy (SMLM) is remarkable not only for best practically achievable resolution but also for the direct access to properties of individual molecules. The primary data of SMLM are the coordinates of individual fluorophores, which is a relatively rare data type in fluorescence microscopy. Therefore, specially adapted methods for processing of these data have to be developed. I developed the software SharpViSu and ClusterViSu that allow for most important data processing steps, namely for correction of drift and chromatic aberrations, selection of localization events, reconstruction of data in 2D images or 3D volumes using different visualization techniques, estimation of resolution with Fourier ring correlation, and segmentation using K- and L-Ripley functions. Additionally, I developed a method for segmentation of 2D and 3D localization data based on Voronoi diagrams, which allows for automatic and unambiguous cluster analysis thanks to noise modeling with Monte-Carlo simulations. Using advanced data processing methods, I demonstrated clustering of CENP-A in the centromeric regions of the cell nucleus and structural transitions of these clusters upon the CENP-A deposition in early G1 phase of the cell cycle.
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Microscopie super-résolutive aux synapses inhibitrices mixtes : régulation différentielle des GlyRs et des GABAARs par l’activité excitatrice / Glycine/GABA mixed inhibitory synapses studied with super-resolution microscopy : differential regulation of GlyRs and GABAARs by excitatory activityYang, Xiaojuan 10 September 2019 (has links)
La microscopie optique stochastique de reconstruction (STORM) contourne la limite de diffraction en enregistrant des signaux monomoléculaires spatialement et temporellement séparés, atteignant une résolution de ~10-40 nm. Dans mon étude, j'ai développé une stratégie d'imagerie et d'analyse de données dSTORM bicolore afin d'étudier l'ultrastructure des synapses inhibitrices mixtes. Mes résultats ont montré que les GlyRs, les GABAARs, la géphyrine et RIM1/2 présentent une organisation intra-synaptique hétérogène et forment des domaines sous-synaptiques (SSDs). Les GlyR et les GABAAR ne sont pas complètement mélangés, mais peuvent occuper des espaces différents à la densité post-synaptique (PSD). De plus, les SSD de géphyrine postsynaptique sont alignées avec les SSD de RIM1/2 pré-synaptiques, formant des nanocolonnes trans-synaptiques. Au cours d'une activité neuronale élevée par traitement 4-AP, la corrélation spatiale entre les GlyRs, les GABAARs et la géphyrine a augmentée au PSD. De plus, la corrélation spatiale des GlyRs et RIM1/2 a également augmenté, tandis que celle des GABAARs et RIM1/2 n'a pas changé. Le nombre de SSD par synapse pour ces protéines synaptiques n'est pas modifié par 4-AP. Cette étude fourni un nouvel angle de compréhension des mécanismes sous-jacents à la co-transmission GABAergique/glycinergique. / Stochastic optical reconstruction microscopy (STORM) bypasses the diffraction limit by recording spatially and temporally separated single molecule signals, achieving a resolution of ~10-40 nm. In my study, I have developed a two-color dSTORM imaging and data analysis strategy, in order to investigate the ultrastructure of mixed inhibitory synapses. My results show that GlyRs, GABAARs, gephyrin and RIM1/2 exhibit a heterogeneous intra-synaptic organization and form sub-synaptic domains (SSDs). GlyRs and GABAARs were not fully intermingled, but sometimes occupied different spaces at the post-synaptic density (PSD). In addition, post-synaptic gephyrin SSDs were aligned with pre-synaptic RIM1/2 SSDs, forming trans-synaptic nanocolumns. During elevated neuronal activity by 4-AP treatment, the spatial correlation between GlyRs, GABAARs and gephyrin was increased at the PSD. Moreover, the spatial correlation of GlyRs and RIM1/2 was also increased, while that of GABAARs and RIM1/2 did not change. The number of SSDs per synapse for these synaptic proteins was not changed by 4-AP. My study thus provides a new angle for understanding the mechanisms underlying GABAergic/glycinergic co-transmission.
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