Academic literature on the topic 'Super-resolution ; dSTORM ; fluorescence microscopy'

Au cours de ce travail de thèse expérimental, nous nous sommes intéressés à l’étude de la localisation nucléaire et nucléolaire de la protéine de la nucléocapside (NC) du VIH-1. Des études antérieures menées au laboratoire avaient mis en évidence une très forte accumulation de la NC dans les nucléoles. Ce compartiment nucléaire est connu pour être ciblé par de nombreux virus afin de promouvoir leur réplication. Des expériences de microscopie électronique avaient révélé la structure complexe du nucléole et montré qu’il est composé de trois sous-compartiments : les centres fibrillaires, le compartiment fibrillaire dense et le compartiment granulaire dans lesquels se déroule la synthèse des ribosomes. Afin de caractériser la localisation de la NC dans ces trois sous-compartiments, nous avons développé une approche de microscopie optique à haute résolution permettant d’obtenir des images à deux couleurs avec une résolution spatiale améliorée. Pour cela, nous avons mis au point un protocole qui permet d’utiliser simultanément une protéine fluorescente photocommutable et un fluorophore organique introduit par immunomarquage. Après avoir minimisé les aberrations optiques et corrigé les dérives mécaniques inhérentes au montage, nous avons visualisé simultanément la localisation de la NC surexprimée dans des cellules HeLa avec des marqueurs spécifiques des trois sous-compartiments nucléolaires (immunomarquage). La microscopie de fluorescence à haute résolution a permis de résoudre pour la première fois les différents compartiments et de montrer que la NC se localise préférentiellement dans le compartiment granulaire. Finalement, des expériences préliminaires avec des cellules vivantes ont permis de mettre en évidence que la NC est transportée de manière active dans le noyau et qu’elle pourrait interagir directement avec des protéines nucléolaires<br>During this experimental thesis work, we investigated the nuclear and nucleolar localization of the nucleocapsid protein (NC) of HIV-1. Previous studies performed in our laboratory evidenced a strong accumulation of NC in a subnuclear structure called nucleolus. Playing role in multiple cellular processes, nucleolus is often targeted by viruses to promote their replication. Electron microscopy revealed three nucleolar components (fibrillar centers, dense fibrillar component and granular component) associated to specific steps of the ribosome biogenesis. To characterize the distribution of the NC in these three sub-compartments and therefore shed light on the nucleolar localization of NC during the replication cycle, we developed a high-resolution optical microscopy approach. After having minimized the optical aberrations and corrected the mechanical drifts inherent to the imaging setup, the NC-mEos2 fusion protein overexpressed in HeLa cells was visualized simultaneously with immunolabeled nucleolar markers. The use of high-resolution fluorescence microscopy enabled us to resolve for the first time the three nucleolar compartments and to demonstrate the preferential localization of NC in the granular compartment of nucleolus. Finally, preliminary experiments performed with living cells showed that NC is actively transported in the nucleus and therefore may interact directly with nucleolar proteins

While super-resolution microscopy has brought a significant improvement in nano-scale imaging of molecular assemblies in biological media, its extension to imaging molecular orientation using fluorescence anisotropy has not yet been fully explored. Providing orientational order information at the nano-scale would be of considerable interest for the understanding of biological functions since they are intrinsically related to structural fundamental processes such as in protein clustering in cell membranes, supra-molecular polymerization or aggregation. In this thesis, we propose a super-resolution polarization-resolved microscopy technique able to image molecular orientation behaviors in static and dynamic environments, in order to report structural information at the single molecule level and at nanometric spatial scale. Using direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with polarized detection, fluorescence anisotropy images are reconstructed at a spatial resolution of a few tens of nanometers. We analyze numerically the principle of the method in combination with models for orientational order mechanisms, and provide conditions for which this information can be retrieved with high precision in biological samples based on fibrillar structures. Finally, we propose an alternative technique based on stochastic fluctuations of single molecules: polarized super-resolution optical fluctuation imaging (polar-SOFI), and compare this approach with the previous one in terms of information gained and spatial resolution. We illustrate both techniques on molecular order imaging in actin stress fibers and tubulin fibers in fixed cells, DNA fibers and insulin amyloid fibrils.<br>La microscopía de súper resolución ha aportado una mejora significativa en la imagen, a escala nanométrica, de ensambles moleculares en medios biológicos. Sin embargo, su extensión, mediante la utilización de la anisotropía de fluorescencia para la obtención de imágenes de orientación molecular, aún no ha sido explorada a fondo. El proporcionar información sobre la orientación molecular a escala nanométrica es de gran interés para la comprensión de las funciones biológicas. Esta información está intrínsecamente relacionada con la estructura de los ensamblajes de proteínas en las membranas celulares, la polimerización y la agregación supra molecular, entre otros. En esta tesis, proponemos una técnica de microscopía de luz polarizada de súper resolución, la cual permite visualizar el comportamiento de la orientación molecular en ambientes dinámicos y estáticos. El objetivo final es el de poder reportar información estructural a nivel de molécula única y escala espacial nanométrica. Utilizando microscopía de reconstrucción óptica estocástica (dSTORM) en combinación con detección polarizada, las imágenes de anisotropía de fluorescencia son reconstruidas con una resolución espacial de varias decenas de nanómetros. Además, el principio del método ha sido validado numéricamente en combinación con modelos de mecanismos de orientación molecular y delimitando las condiciones en que esta información se puede obtener con una precisión alta en muestras biológicas, principalmente en estructuras fibrilares. Así también, se propone una técnica alternativa basada en la emisión de fluctuaciones estocásticas de moléculas individuales: imagen de polarización con súper resolución de fluctuaciones (polar-SOFI). Además comparamos esta técnica con la anterior, en términos de la información obtenida y la resolución espacial. Finalmente, ilustramos ambas técnicas para la obtención de imágenes del orden molecular de fibras de estrés de actina y tubulina en células fijas, fibras de ADN y fibrillas de insulina amiloide.<br>Alors que la microscopie super-résolue a apporté une amélioration considérable en imagerie des assemblages moléculaires dans les milieux biologiques à l'échelle nanométrique, son extension à l'imagerie de l'orientation moléculaire, utilisant l'anisotropie de fluorescence, n'a pas encore été complètement explorée. Apporter une information sur l'orientation moléculaire à l'échelle nanométrique aurait un intérêt considérable pour la compréhension des functions biologiques, puisque celles-ci sont fortement reliée à la structure des assemblages de prot éines dans les membranes cellulaires, la polymérisation ou l'aggrégation supramol éculaire par exemple. Dans cette thèse, nous proposons une technique de microscopie super-résolution résolue en polarisation, capable d'imager les comportements d'orientation moléculaire dans des environnements statiques et dynamiques, dans le but de rapporter une information structurale à l'échelle de la molécule unique et à des échelles spatiales nanométriques. En utilisant la microscopie par reconstruction stochastique (dSTORM) en combinaison avec une détection polarisée, des images d'anisotropie de fluorescence sont reconstruites avec une résolution spatiale de quelques dizaines de nanomètres. Nous analysons numériquement le principe de la méthode en combinaison avec des modèles des mécanismes d'orientation moléculaire, et donnons les conditions auxquelles cette information peut être obtenue avec une grande précision dans des échantillons biologiques basés sur des structures fibrillaires. Enfin, nous proposons une technique alternative basée sur l'émission de molécules uniques en fluctuations stochastiques: l'imagerie super-résolue polarisée par fluctuations (polar-SOFI), et comparons cette approche avec la précédente en terme d'information gagnée et de résolution spatiale. Nous illustrons les deux techniques pour l'imagerie de l'ordre moléculaire dans des fibres de stress d'actin et de tubuline dans des cellules fixées, des fibres d'ADN et des fibrilles d'amyloid à base d'insuline.

Alors que la microscopie super-résolue a apporté une amélioration considérable en imagerie des assemblages moléculaires dans les milieux biologiques à l'échelle nanométrique, son extension à l'imagerie de l'orientation moléculaire, utilisant l'anisotropie de fluorescence, n'a pas encore été complètement explorée. Apporter une information sur l'orientation moléculaire à l'échelle nanométrique aurait un intérêt considérable pour la compréhension des fonctions biologiques. Dans cette thèse, nous proposons une technique de microscopie super-résolution résolue en polarisation, capable d'imager les comportements d'orientation moléculaire dans des environnements statiques et dynamiques, dans le but de rapporter une information structurale à l'échelle de la molécule unique et à des échelles spatiales nanométriques. En utilisant la microscopie par reconstruction stochastique (dSTORM) en combinaison avec une détection polarisée, des images d'anisotropie de fluorescence sont reconstruites avec une résolution spatiale de quelques dizaines de nanomètres. Nous analysons numériquement le principe de la méthode en combinaison avec des modèles des mécanismes d'orientation moléculaire. Enfin, nous proposons une technique alternative basée sur l'émission de molécules uniques en fluctuations stochastiques: l'imagerie super-résolue polarisée par fluctuations (polar-SOFI), et comparons cette approche avec la précédente. Nous illustrons les deux techniques pour l'imagerie de l'ordre moléculaire dans des fibres de stress d'actine et de tubuline dans des cellules fixées, des fibres d'ADN et des fibrilles d'amyloïde à base d'insuline<br>While super-resolution microscopy has brought a significant improvement in nanoscale imaging of molecular assemblies in biological media, its extension to imaging molecular orientation using fluorescence anisotropy has not yet been fully explored. Providing orientational order information at the nanoscale would be of considerable interest for the understanding of biological functions since they are intrinsically related to structural fundamental processes such as in protein clustering in cell membranes, supra-molecular polymerization or aggregation. In this thesis, we propose a super-resolution polarization-resolved microscopy technique able to image molecular orientation behaviors in static and dynamic environments, in order to report structural information at the single molecule level and at nanometric spatial scale. Using direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with polarized detection, fluorescence anisotropy images are reconstructed at a spatial resolution of a few tens of nanometers. We analyze numerically the principle of the method in combination with models for orientational order mechanisms, and provide conditions for which this information can be retrieved with high precision in biological samples based on fibrillar structures. Finally, we propose an alternative technique based on stochastic fluctuations of single molecules: polarized super-resolution optical fluctuation imaging (polar-SOFI), and compare this approach with the previous one. We illustrate both techniques on molecular order imaging in actin stress fibers and tubulin fibers in fixed cells, DNA fibers and insulin amyloid fibrils

Fluorescence microscopy is an important tool for biological research. However, the resolution of a standard fluorescence microscope is limited by diffraction, which makes it difficult to observe small details of a specimen’s structure. We have developed two fluorescence microscopy methods that achieve resolution beyond the classical diffraction limit. The first method represents an extension of localisation microscopy. We used nonnegative matrix factorisation (NMF) to model a noisy dataset of highly overlapping fluorophores with intermittent intensities. We can recover images of individual sources from the optimised model, despite their high mutual overlap in the original dataset. This allows us to consider blinking quantum dots as bright and stable fluorophores for localisation microscopy. Moreover, NMF allows recovery of sources each having a unique shape. Such a situation can arise, for example, when the sources are located in different focal planes, and NMF can potentially be used for three dimensional superresolution imaging. We discuss the practical aspects of applying NMF to real datasets, and show super-resolution images of biological samples labelled with quantum dots. It should be noted that this technique can be performed on any wide-field epifluorescence microscope equipped with a camera, which makes this super-resolution method very accessible to a wide scientific community. The second optical microscopy method we discuss in this thesis is a member of the growing family of structured illumination techniques. Our main goal is to apply structured illumination to thick fluorescent samples generating a large out-of-focus background. The out-of-focus fluorescence background degrades the illumination pattern, and the reconstructed images suffer from the influence of noise. We present a combination of structured illumination microscopy and line scanning. This technique reduces the out-of-focus fluorescence background, which improves the quality of the illumination pattern and therefore facilitates reconstruction. We present super-resolution, optically sectioned images of a thick fluorescent sample, revealing details of the specimen’s inner structure. In addition, in this thesis we also discuss a theoretical resolution limit for noisy and pixelated data. We correct a previously published expression for the so-called fundamental resolution measure (FREM) and derive FREM for two fluorophores with intermittent intensity. We show that the intensity intermittency of the sources (observed for quantum dots, for example) can increase the “resolution” defined in terms of FREM.

This thesis describes the design, construction and application of a novel magneto-optical tweezers with super-resolution fluorescence microscopy for manipulation, force/torque measurement and imaging of single biomolecules. The optical tweezers component offers force or position clamping in three dimensions. The 3D-printed magnetic tweezers is designated for rotation in the vertical plane. The separation of rotation from force transduction results in the capability of precise torque measurement. The filamentous biomolecules to be used in the device will lie in a transverse direction in the imaging plane to allow fluorescence imaging with techniques including Blinking assisted Localisation Microscopy (BaLM) and total internal reflection fluorescence microscopy (TIRF). Also included are features such as acousto-optic deflection and multiplexing of laser traps, interferometry based tracking with quadrant photodiode and piezoelectric actuated nanostage for active feedback. These tweezers have been developed to enable direct observation of molecular topological transformation and protein binding event localisation with mechanical perturbation, which traditional tweezers could not achieve.

The development of single-molecule and super-resolution fluorescence techniques has revolutionised biological imaging. Nano-scale cellular structures and heterogeneous dynamic processes are now able to be visualised with unprecedented resolution in both time and space. The achievable localisation precision and therefore the resolution is fundamentally limited by the number of photons a single-fluorophore can emit. The ideal super-resolution dye would emit a large number of photons over a short period of time. On the contrary, an optimal single-molecule tracking probe would be highly photostable and undergo no transient dark-state transitions. Single-molecule instrument development is beginning to reach technological saturation and as the frontiers of bioimaging expand, exorbitant demands are placed on the gamut of available probes that often cannot be met. Thus, the next key challenge in the field is the development of the better fluorophores that underlie these techniques; this includes both the synthesis of new chemical derivatives and alternative novel strategies to augment existing technologies. The results of this thesis are divided into two distinct parts; Project One details the development of new synthetic fluorescent probes for the study of amyloid protein aggregates implicated in neurodegenerative diseases. This includes a study of the photophysical and binding properties of a novel fluorophore library based on the amyloid dye Thioflavin-T. Following on from this, is the presentation of novel bifunctional dyes capable of simultaneously identifying hydrogen peroxide and amyloid aggregates by combining existing tools for the independent detection of these species. The sensing capabilities of these dyes are explored at the bulk and single-molecule levels. Project Two describes a new photo-modulatable fluorescent-protein fusion construct that can undergo Förster resonance energy transfer (FRET) to an organic dye molecule. This FRET cassette is comprised of a photoconvertible fluorescent protein donor, mEos3.2 and acceptor fluorophore, JF646. This strategy imparts a strong photostabilising effect on the fluorescent protein and a resistance to photobleaching. The functionality of this approach is demonstrated with in vitro single-molecule fluorescence studies and its biological applicability shown by tracking single proteins in the nuclei of live embryonic stem cells. Furthermore, initial characterisations of the excited state dynamics in effect are presented through the systematic modification of parameters.

High-Throughput imaging can reconstruct complex signalling networks, reveal unknown interactions and capture rare cellular events. Simultaneously, the development of Single Molecule Localization Super Resolution Microscopy has enabled molecular-level structural information to be obtained in a single cell. But the increase in resolution comes at a trade-off for the amount of molecular species that can be imaged and the time it takes to acquire data, all of which limit the applicability of super-resolution to high-throughput work-flows. The present work details a framework to address this. It combines three independent approaches: a microscope hardware design approach to increase the amount of data that can be obtained in a Super-Resolution experiment; an optofluidics platform that can be wholly synchronized with most microscopes; and a sequential labelling framework to increase the number of species that can be imaged in Super-Resolution in a single cell. The hardware design is validated by performing Single Molecule Localization of cytoskeleton components and its throughput is shown to be up to an order of magnitude larger than a corresponding commercial system. We demonstrate a complete optofluidics platform to integrate microfluidics with a microscope, enabling live imaging, drug application, fixation, and staining in single cells synchronized with imaging protocols. Finally, we show an efficient sequential labelling protocol that is compatible with the optofluidics platform, enabling several molecular species to be imaged in the same cells. Overall, our approach increases the speed and amount of data that can be acquired in a single of Super-Resolution experiment, as well as, by performing on-line fixation, considerably improves our capacity for High-Throughput experiments in Super-Resolution imaging.