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Photophysics of fluorescent silver nanoclustersPatel, Sandeep A. 03 April 2009 (has links)
Fluorescence imaging has been increasingly relied upon as the method of choice for many biological and medical applications. As demands for more sensitive and higher resolution imaging are ever-increasing, it is critical that photostable, and robust fluorophores capable of delivering high emission rates are available. Fluorescent silver nanoclusters offer an attractive compromise between the photostability and brightness of quantum dots and the compact versatility of organic chromophores. They have been shown to be superior in many roles, including as single molecule fluorophores and bulk multiphoton biological staining agents. The two-photon absorption cross sections are several orders of magnitude larger than commercially-available dyes, and they have demonstrated superior photostability under high intensity irradiation. In addition to the endogenous effects of the cluster, its small size of only a few atoms renders it highly susceptible to surface and environmental effects, which manifests, for example, in the observed photoinduced charge transfer between the silver cluster and oligonucleotide. This state has been shown to be highly advantageous in imaging applications, as control of this state enables better control over the time-averaged emission rate of the molecule. The mechanism of charge transfer, and the possible means by which this state can be controlled will be also be investigated in this work.
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Genotyping and Mutation Detection In Situ : Development and application of single-molecule techniquesGrundberg, Ida January 2011 (has links)
The human body is composed of trillions of cells closely working together to maintain a functional organism. Every cell is unique in molecular composition and can acquire genetic variations that might cause it to turn pathological. It is essential to develop improved tools to better understand the development of normal and disease tissue, ideally enabling single-cell expression studies in preserved context of complex tissue with single-nucleotide resolution. This thesis presents the development and application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The described technique utilizes padlock probes and target-primed rolling circle amplification and is highly suitable for sensitive in situ analysis. First, a new strategy for directed cleavage of single stranded DNA was investigated, e.g. nucleic acid targets with extended 3´ ends, for successful initiation of rolling circle amplification. The presented cleavage strategy is simple and applicable for subsequent enzymatic reactions, e.g. ligation and polymerization. Specific cleavage of long target overhangs was demonstrated in synthetic oligonucleotides and in genomic DNA and the detection efficiency was substantially increased. For multiplex detection and genotyping of individual transcripts in single cells, a new in situ method was developed. The technique showed a satisfactorily detection efficiency and was later applied as a general mutation analysis tool for detection of KRAS point mutations in complex tumor tissue sections, e.g. formalin-fixed, paraffin-embedded tumor tissues and cytologic tumor imprints. Mutation status was assessed in patient samples by in situ padlock probe detection and results were confirmed by DNA-sequencing. Finally, the method was adapted for simultaneous detection of individual mRNA molecules and endogenous protein modifications in single cells using padlock probes and in situ PLA. This assay will be useful for gene expression analysis and exploration of new drugs with vague effector sites. To our knowledge, no other technique exists today that offers in situ transcript detection with single-nucleotide resolution in heterogeneous tissues. The method will especially be suitable for discrimination of highly similar transcripts, e.g. splice variants, SNPs and point mutations, within gene expression studies and for cancer diagnostics.
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Visualizing Interacting Biomolecules In SituWeibrecht, Irene January 2011 (has links)
Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication. In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously. In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes. This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures.
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Detection and Sequencing of Amplified Single MoleculesKe, Rongqin January 2012 (has links)
Improved analytical methods provide new opportunities for both biological research and medical applications. This thesis describes several novel molecular techniques for nucleic acid and protein analysis based on detection or sequencing of amplified single molecules (ASMs). ASMs were generated from padlock probe assay and proximity ligation assay (PLA) through a series of molecular processes. In Paper I, a simple colorimetric readout strategy for detection of ASMs generated from padlock probe assay was used for highly sensitive detection of RNA virus, showing the potential of using padlock probes in the point-of-care diagnostics. In Paper II, digital quantification of ASMs, which were generated from padlock probe assay and PLA through circle-to-circle amplification (C2CA), was used for rapid and sensitive detection of nucleic acids and proteins, aiming for applications in biodefense. In Paper III, digital quantification of ASMs that were generated from PLA without C2CA was shown to be able to improve the precision and sensitivity of PLA when compared to the conventional real-time PCR readout. In Paper IV, a non-optical approach for detection of ASMs generated from PLA was used for sensitive detection of bacterial spores. ASMs were detected through sensing oligonucleotide-functionalized magnetic nanobeads that were trapped within them. Finally, based on in situ sequencing of ASMs generated via padlock probe assay, a novel method that enabled sequencing of individual mRNA molecules in their natural context was established and presented in Paper V. Highly multiplex detection of mRNA molecules was also achieved based on in situ sequencing. In situ sequencing allows studies of mRNA molecules from different aspects that cannot be accessed by current in situ hybridization techniques, providing possibilities for discovery of new information from the complexity of transcriptome. Therefore, it has a great potential to become a useful tool for gene expression research and disease diagnostics.
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Étude biochimique et biophysique de l’ARN hélicase UPF1 : un moteur moléculaire hautement régulé / Biochemical and biophysical study of the RNA helicase UPF1 : a highly regulated molecular motorKanaan, Joanne 09 July 2018 (has links)
UPF1 (Up-Frameshift 1) est une hélicase multifonctionnelle conservée chez tous les eucaryotes. Elle est essentielle à la voie de surveillance du NMD (Nonsense Mediated mRNA Decay), qui dégrade des ARNm portant un codon stop prématuré. UPF1 est l’archétype d’une famille d’hélicases qui partagent des corps similaires mais sont impliquées dans des voies cellulaires variées. Cependant, les relations structure-fonction et les caractéristiques biophysiques intrinsèques de ces moteurs moléculaires restent à ce jour peu connues. In vitro, le coeur hélicase d’UPF1 est hautement processif, il traverse des milliers de bases sur l’ARN ou l’ADN et déroule des doubles brins. Dans ce travail, nous avons cherché les facteurs clés régissant cette remarquable processivité en combinant des techniques de biochimie et de biophysique. En particulier, nous avons utilisé des pinces magnétiques pour étudier en temps réel des hélicases à l’échelle de la molécule unique. Contrairement à UPF1, l’hélicase IGHMBP2 de la famille UPF1-like n’est pas processive ; la processivité n’est donc pas un trait conservé au sein de la famille. Grâce à une étude fine de la structure 3D des deux hélicases, nous avons conçu divers mutants que nous avons utilisés pour identifier les éléments structuraux qui modulent la processivité. Notre approche révèle qu’UPF1 a une prise très ferme sur les acides nucléiques, garantissant de longs temps de résidence et d’action qui dictent sa haute processivité. Grâce à la variété de comportements des mutants, nous avons construit un modèle mécanistique expliquant le lien entre énergie d’interaction et processivité. Nous démontrons aussi que la processivité d’UPF1 est requise pour un processus de NMD efficace in vivo. Nous avons utilisé les mêmes outils biochimiques et biophysiques pour étudier une isoforme naturelle d’UPF1 humaine se déplaçant plus vite que l’isoforme majeure, et pour comparer la régulation d’UPF1 humaine et de levure par leurs domaines flanquants. Nous avons également caractérisé l'interaction d’UPF1 de levure avec de nouveaux partenaires. Nos travaux montrent comment la combinaison d'outils biochimiques, biophysiques, structuraux etin vivo offre des aperçus inattendus quant au mode de fonctionnement des moteurs moléculaires. / UPF1 (Up-Frameshift 1) is a multifunctional helicase that unwinds nucleic acids and is conserved throughout the eukaryote kingdom. UPF1 is required for the Nonsense Mediated mRNA Decay (NMD) surveillance pathway, which degrades mRNAs carrying premature termination codons, among other substrates. UPF1 is the archetype of a family of 11 helicases sharing similar cores but involved in various cellular pathways. However, the structure-function relationship and intrinsic biophysical properties of these molecular engines remain poorly described. In vitro, the UPF1 helicase core is highly processive, it travels along thousands of RNA or DNA bases and unwinds double-strands. In this work, we looked for key factors governing this remarkable processivity. We combined biochemical and biophysical techniques. In particular, we used magnetic tweezers to study helicases in real time at a single molecule scale. In contrast to UPF1, the related IGHMBP2 is not processive, thus processivity is not a shared family trait. Based on the 3D structures of both proteins, we designed various mutants and used them to identify structural elements that modulate processivity. Our approach reveals that UPF1 has a very firm grip on nucleic acids, guaranteeing long binding lifetimes and action times that dictate its high processivity. Thanks to the variety in mutant behaviors, we built a novel mechanistic model linking binding energy to processivity. Furthermore, we show that UPF1 processivity is required for an efficient NMD in vivo. In addition, we used the same biochemical and biophysical tools to investigate a natural human UPF1 isoform moving faster than the major isoform, and to compare the regulation of human andyeast UPF1 by their flanking domains. We also characterized the interaction of yeast UPF1 with new NMD partners. Our work shows how a combination of biochemical, biophysical, structural and in vivo tools can offer unexpected insights into the operating mode of molecular motors.
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2D and 3D optical nanoscopy of single molecules at cryogenic temperatures / Nanoscopie optique 2D et 3D de molécules uniques à températures cryogéniquesBaby, Reenu 17 July 2018 (has links)
Dans cette thèse, nous présentons le développement d'une méthode de nanoscopie optique superrésolue en trois dimensions pour résoudre des émetteurs quantiques uniques à température cryogénique. Cette méthode, appelée microscopie à saturation d'état excité (ESSat), est une technique d'imagerie confocale à balayage basée sur la saturation optique de la raie sans phonon de l'émetteur. Elle utilise un faisceau d’illumination structurée comprenant une zone d'intensité nulle au foyer de l'objectif de microscope, avec un gradient d'intensité important autour. En imageant des molécules fluorescentes aromatiques individuelles à 2 K, nous avons atteint une résolution de 28 nm dans la direction latérale et 22 nm dans la direction axiale, avec de faibles intensités laser d'environ dix kWcm-2, soit cinq ordres de grandeur inférieures à celles utilisées à température ambiante dans les méthodes de nanoscopie basées sur la déplétion par émission stimulée. Notre technique offre une opportunité unique de super-résoudre des molécules uniques séparées par des distances nanométriques et avec des résonances optiques qui se recouvrent. De plus, la méthode fournit une détermination directe de l'orientation des dipôles moléculaires à partir des images ESSat de fluorescence. La microscopie ESSat ouvre ainsi la voie à des études approfondies des interactions cohérentes dipôle-dipôle optiques entre émetteurs quantiques individuels, qui nécessitent des distances relatives nanométriques. En particulier, cette méthode permettra d'étudier les riches signatures spatiales et fréquentielles du système couplé et de manipuler leur degré d'intrication. / In this thesis, we present the development of a cryogenic super-resolution optical nanoscopy thatcan resolve molecules at nanometric distances, called the Excited State Saturation (ESSat)Microscopy.ESSat microscopy is a scanning confocal imaging technique based on the optical saturation of thezero phonon line of a single fluorescent molecule. It uses a patterned illumination beam thatcontains a ‘zero-intensity’ region at the focus of the microscope objective with a large intensitygradient around. We achieved a sub-10 nm resolution in the lateral direction and 22 nm resolutionin the axial direction with extremely low excitation intensities of few tens of kWcm-2. Comparedto other super-resolution imaging techniques, like STED, RESOLFT, etc., our technique offers aunique opportunity to super-resolve single molecules with overlapping optical resonances and thatare much closer than the diffraction limit. In addition, it is possible to determine the orientation ofmolecular dipoles from the fluorescent ESSat images. Since coherent dipole-dipole couplinginteractions between single quantum emitters have a very high coupling efficiency at short distancemuch smaller than the diffraction limit, it is important to resolve them well below it. ESSatmicroscopy thus paves a way to disclose the rich spatial and frequential signatures of the coupledsystem and to manipulate their degree of entanglement.
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Transfert d'énergie engendré par plasmon et imagerie de super-résolution en champ proche de milieux nano-structurés / Plasmon-mediated energy transfer and super-resolution imaging in the near field of nanostructured materialsBouchet, Dorian 27 November 2017 (has links)
Dans cette thèse, nous associons mesures expérimentales et modélisation des données pour étudier l'émission spontanée d'émetteurs fluorescents en environnement nano-structuré. Le mémoire est organisé en deux parties.Dans la première partie, nous étudions le transfert d'énergie entre émetteurs fluorescents en environnement plasmonique et sur des distances micrométriques. Pour commencer, nous caractérisons le transfert d'énergie entre deux ensembles d'émetteurs situés en champ proche d'une surface d'argent. Nous déterminons ainsi la dépendance en distance du taux de transfert d'énergie sur des distances micrométriques. Nous couplons ensuite une boite quantique et une bille fluorescente à un nano-fil d'argent et nous étudions le transfert d’énergie entre ces deux émetteurs, distants de plusieurs micromètres. Nous démontrons notamment le clignotement corrélé de ces deux émetteurs grâce à l'étude de la fonction de corrélation de leur intensité de fluorescence.Dans la seconde partie, nous sondons les variations spatiales de densité locale d'états électromagnétiques induites par des environnements nano-structurés grâce à différentes techniques de microscopie à super-résolution. A l'aide d'un microscope à balayage, nous réalisons tout d’abord une étude en trois dimensions de l’interaction de champ proche entre une bille fluorescente et différentes antennes en silicium. Nous introduisons ensuite une technique stochastique permettant de déterminer expérimentalement la position et le taux d'amortissement de molécules uniques photo-activées, avec une précision de localisation de l'ordre de 10 nm. Enfin, nous utilisons l'information de Fisher afin d'estimer les bornes inférieures de l'erreur type des estimations de positions et de taux d'amortissement réalisées dans le cadre de mesures sur molécules uniques. / In this thesis, we perform experimental measurements and data modelling to investigate spontaneous emission of fluorescent emitters in nanostructured environments. The manuscript is organised into two main parts.In the first part, we study micrometre-range energy transfer between fluorescent emitters in plasmonic environments. First of all, we characterise plasmon-mediated energy transfer between ensembles of fluorescent emitters located in the near field of a silver film. We thus determine the distance dependence of the energy transfer rate over micrometre distances. We then couple a single quantum dot and a fluorescent nanobead to a silver nanowire and we study evidences of the energy transfer between the two emitters, separated by several micrometres. We notably demonstrate a correlated blinking of the two emitters through the study of the correlation function of their fluorescence intensity.In the second part, we probe sub-wavelength spatial variations of the local density of electromagnetic states induced by nanostructured environments by means of different super-resolution microscopy techniques. To start with, we perform a three-dimensional study of the near-field interaction between a fluorescent nanobead and different silicon nanoantennas using a scanning-probe microscope. We then introduce a stochastic technique to experimentally determine the position and the fluorescence decay rate of single photo-activated molecules, with a localisation precision of the order of 10 nm. Finally, we use the Fisher information to estimate lower bounds on the standard errors on position and decay rate estimates performed in the context of single-molecule microscopy.
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Tuning of color and polarization of the fluorescence of nano-ribbons using laser microscopy and controlled self-assembly / Nano-rubans à fluorescence accordable en couleur et en polarisation par microscopie laser et auto-assemblage contrôléSchäfer, Philip Sudadyo 15 December 2016 (has links)
Des matériaux ayant des propriétés émissives spécifiques peuvent être obtenus par l'organisation contrôlée de fluorophores aux échelles moléculaire, nano- et micro-métrique. Dans ce travail, l'émission de lumière bleue polarisée est obtenue par l'auto-assemblage hautement anisotrope de n-acènes alcoxylés en nano-rubans. Des techniques de microscopie de fluorescence ont été utilisées pour déterminer le mécanisme de leur croissance et ont été combinées à la cristallographie aux rayons X pour déterminer l'empilement moléculaire dans les nano-objets. L'étude a révélé que la formation des nano-rubans est induite non seulement par le mécanisme de maturation d'Ostwald très commun, mais aussi par une croissance par attachement orienté rarement démontré dans des systèmes organiques. En plus des techniques plus courantes, la microscopie en polarisation de fluorescence de molécules uniques a contribué à caraxctériser l'emplilement moléculaire, bien que les nano-objets à haute densité en chromophore constituent des échantillons très difficiles à étudier. Dans ce travail, les propriétés des nano-rubans ont été contrôlées au niveau microscopique par les conditions de croissance, ainsi que par l'addition de dopants. Ainsi, en combinant différentes molécules et une réaction photochimique sous microscopie, des rubans à motifs colorés sub-micrométriques ont été obtenus. Par ailleurs, l'assemblage orthogonal a été exploité pour développer des réseaux interpénétrés. Ces derniers se distinguent par une émission à double couleur, un transfert d'énergie entre objets et une électroluminescence aux jonctions. / Materials with specific emissive properties can be obtained by the controlled organization of fluorophores at the molecular, nano- and microscales. In this work, polarized blue light emission is achieved by the highly anisotropic self-assembly of alkoxylated n-acenes into nano-ribbons. Fluorescence microscopy techniques were used to determine the growth mechanism and were combined to X-ray crystallography to determine the molecular packing in the nano-objects. The study revealed that the formation of the nano-ribbons is induced not only by the very common Ostwald ripening mechanism but also by an oriented attachnment growth, rarely observed with such evidence in organic systems. Besides more common techniques, single molecule fluorescence polarization microscopy contributed to characterize the molecular packing, although the nano-objects with high chromophore density represent very challenging samples. In this work, the properties of the nano-ribbons have been controlled at the microscopic level by the growth conditions, as well as by the addition of dopants Thereby, combining different molecules and photochemistry at the sub-micrometer scale under the microscope, colorful patterned ribbons could be obtained. In addition, orthogonal assembly was exploited to grow interpenetrated networks. The latter demonstrated dual color-emission, as well as inter-object energy transfer and electroluminescence at junctions.
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Light and single-molecule coupling in plasmonic nanogapsChikkaraddy, Rohit January 2018 (has links)
Plasmonic cavities confine optical fields at metal-dielectric interfaces via collective charge oscillations of free electrons within metals termed surface plasmon polaritons (SPPs). SPPs are confined in nanometre gaps formed between two metallic surfaces which creates an optical resonance. This optical resonance of the system is controlled by the geometry and the material of the nanogap. The focus of this work is to understand and utilize these confined optical modes to probe and manipulate the dynamics of single-molecules at room temperature. In this thesis, nanogap cavities are constructed by placing nanoparticles on top of a metal-film separated by molecular spacers. Such nanogaps act as cavities with confined optical fields in the gap. Precise position and orientation of single-molecules in the gap is obtained by supramolecular guest-host assembly and DNA origami breadboards. The interaction of light and single-molecules is studied in two different regimes of interaction strength. In the perturbative regime molecular light emission from electronic and vibrational states is strongly enhanced and therefore is used for the detection of single-molecules. In this regime the energy states remain unaltered, however profound effects emerge when the gap size is reduced to < 1 nm. New hybridized energy states which are half-light and half-matter are then formed. Dispersion of these energies is studied by tuning the cavity resonance across the molecular resonance, revealing the anti-crossing signature of a strongly coupled system. This dressing of molecules with light results in the modification of photochemistry and photophysics of single-molecules, opening up the exploration of complex natural processes such as photosynthesis and the possibility to manipulate chemical bonds.
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Single-molecule studies of bacterial DNA replication and translesion synthesisZhao, Gengjing January 2018 (has links)
Faithful replication of genomic DNA is crucial for the survival of a cell. In order to achieve high-level accuracy in copying its genome, all cells employ replicative DNA polymerases that have intrinsic high fidelity. When an error occurs on the template DNA strand, in the form of lesions caused by diverse chemicals, reactive oxygen species, or UV light, the high-fidelity replicative DNA polymerases are stalled. To bypass these replication blocks, cells harbor multiple specialized translesion DNA polymerases that are error-prone and therefore able to accommodate the lesions and continue DNA synthesis. As a result of their low fidelity, the translesion polymerases are associated with increased mutagenesis, drug resistance, and cancer. Therefore, the access of the translesion polymerases to DNA needs to be tightly controlled, but how this is achieved has been the subject of debate. This Thesis presents the development of a co-localization single-molecule spectroscopy (CoSMoS) method to directly visualize the loading of the Escherichia coli replicative polymerase on DNA, as well as the exchange between the replicative polymerase and the translesion polymerases Pol II and Pol IV. In contrast to the toolbelt model for the exchange between the polymerases, this work shows that the translesion polymerases Pol II and Pol IV do not form a stable complex with the replicative polymerase Pol IIIα on the β-clamp. Furthermore, we find that the sequential activities of the replication proteins: clamp loader, clamp, and Pol IIIα, are highly organized while the exchange with the translesion polymerases is disordered. This exchange is not determined by lesion-recognition but instead a concentration-dependent competition between the replicative and translesion polymerases for the hydrophobic groove on the surface of the β-clamp. Hence, our results provide a unique insight into the temporal organization of events in DNA replication and translesion synthesis.
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