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. / 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.

Cette thèse se concentre sur le design et la synthèse de molécules fluorescentes contenant un motif triazole et un squelette benzothiadiazole (BTD), coumarine, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacène (BODIPY) ou dicyanométhylène-4H-pyran (DCM) par chimie "click" et l’étude de leurs propriétés et applications en biologie et en chimie analytique. Dans le but de synthétiser des peptides fluorescents et d'étudier leurs applications, des acides aminés fluorescents contenant BTD, coumarine et BODIPY ont été préparés par réaction "click", et incorporés dans la somatostatine par synthèse peptidique en phase solide. Les peptides fluorescents synthétisés pourront être utilisés pour le développement d'un test de "binding" des analogues de la somatostatine. Des dérivés de BTD et BODIPY ont également été conçus et synthétisés pour servir de mimes de coudes beta- qui conduisent à des peptides courts qui pourraient être facilement détectés et étudiés en utilisant des techniques de fluorescence. La capacité des composés obtenus à former des liaisons hydrogène intramoléculaires a été étudiée par spectroscopie infrarouge. En outre, une série de macrocycles à base de BODIPY contenant un C-glucopyranoside conjugué ou non à des acides aminés tels que glycine, acid aspartique ou méthionine ont été synthétisés avec succès en utilisant une réaction "click" comme étape de macrocyclisation. Certains des composés synthétisés présentent des propriétés de reconnaissance sélective vers Cu2+, Fe3+, F- et CN- dans l'acétonitrile. Enfin, un nouveau capteur fluorescent, qui est capable de reconnaître les cations et anions d'une manière coopérative, a été conçu et synthétisé par chimie "click". Ce composé est très sensible à des combinaisons de Cu2+, F- et / ou Br- d’une manière séquence- et halogénure-dépendante. / This thesis is focused on the design and synthesis of triazole-containing fluorescent molecules based on benzothiadiazole (BTD), coumarin, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) or dicyanomethylene-4H-pyran (DCM) by “click” chemistry and investigation of their properties and applications in biology and analytical chemistry. In the aim to synthesize fluorescent peptides and investigate their applications, fluorescent amino acids containing BTD, coumarin and BODIPY were prepared by “click” reaction, and incorporated into somatostatin through solid phase peptide synthesis. The resulting fluorescent peptides could be used for the development of a binding assay for somatostatin analogues. BTD and BODIPY derivatives have also been designed and synthesized to act as beta-turn mimics which lead to short conformationally restricted peptides that could be easily detected and studied using fluorescence techniques. The ability of the synthesized compounds to form intramolecular hydrogen bond was studied by infrared spectroscopy. Moreover, a series of BODIPY-based macrocycles containing a C-glucopyranoside conjugated or not with various amino acids such as glycine, aspartic acid or methionine have been successfully synthesized by using “click” reaction as the macrocyclization step. Some of the synthesized compounds exhibited selective recognition properties towards Cu2+, Fe3+, F- and CN- in acetonitrile. Finally, a new fluorescent sensor, which has the ability to recognize cations and anions in a cooperative way, was designed and synthesized by “click” chemistry. This compound was highly sensitive to combinations of Cu2+, F– and/or Br– in a sequence- and halide-dependent way.

Several pyrene-labeled polymers and polypeptides were synthesized and their chain dynamics were characterized using steady-state and time-resolved fluorescence techniques. Firstly, four series of pyrene-labeled polystyrenes (Py-PS) were synthesized to determine the effect that the method used for pyrene attachment, the linker connecting pyrene to the backbone, and the distribution of pyrene along the backbone all have on excimer formation. It was found that the amount of excimer formed was different in each case. The differences were described by utilizing the fluorescence blob model (FBM) analysis of the time-resolved monomer and excimer fluorescence decays. Secondly, two Py-PS series were studied in several different solvents with viscosities ranging from 0.36 to 1.19 mPa.s to demonstrate the effect of viscosity on the FBM parameters. The rate constant for excimer formation within a blob, kblob, was found to remain constant with viscosity while the number of monomer units per blob, Nblob, increased with decreasing viscosity. Thirdly, in a technical note, the inherent analogy existing between the aggregation number of surfactant micelles, Nagg, and Nblob, is taken advantage of to establish a “model-free” procedure to determine Nblob. This procedure was validated through the analysis of the time-resolved fluorescence decays of five different pyrene-labeled polymers in seven different solvents. Fourthly, the side-chain dynamics of alpha-helical pyrene-labeled poly(glutamic acid) were investigated. A longer linker connecting pyrene to the backbone resulted in an increase in Nblob which agreed quantitatively with predictions made by molecular modeling. Decreasing the lifetime of the excited pyrene from 215 ns to 50 ns with a quencher increased kblob significantly, while Nblob decreased slightly. Finally, the backbone dynamics of pyrene-labeled poly(aspartic acid) (Py-PAA) were investigated and quantified using the FBM. It was determined that excimer formation was controlled by the very rigid polypeptide backbone and not by the solvent viscosity, contrary to previous results obtained with other pyrene-labeled vinyl polymers.

Several pyrene-labeled polymers and polypeptides were synthesized and their chain dynamics were characterized using steady-state and time-resolved fluorescence techniques. Firstly, four series of pyrene-labeled polystyrenes (Py-PS) were synthesized to determine the effect that the method used for pyrene attachment, the linker connecting pyrene to the backbone, and the distribution of pyrene along the backbone all have on excimer formation. It was found that the amount of excimer formed was different in each case. The differences were described by utilizing the fluorescence blob model (FBM) analysis of the time-resolved monomer and excimer fluorescence decays. Secondly, two Py-PS series were studied in several different solvents with viscosities ranging from 0.36 to 1.19 mPa.s to demonstrate the effect of viscosity on the FBM parameters. The rate constant for excimer formation within a blob, kblob, was found to remain constant with viscosity while the number of monomer units per blob, Nblob, increased with decreasing viscosity. Thirdly, in a technical note, the inherent analogy existing between the aggregation number of surfactant micelles, Nagg, and Nblob, is taken advantage of to establish a “model-free” procedure to determine Nblob. This procedure was validated through the analysis of the time-resolved fluorescence decays of five different pyrene-labeled polymers in seven different solvents. Fourthly, the side-chain dynamics of alpha-helical pyrene-labeled poly(glutamic acid) were investigated. A longer linker connecting pyrene to the backbone resulted in an increase in Nblob which agreed quantitatively with predictions made by molecular modeling. Decreasing the lifetime of the excited pyrene from 215 ns to 50 ns with a quencher increased kblob significantly, while Nblob decreased slightly. Finally, the backbone dynamics of pyrene-labeled poly(aspartic acid) (Py-PAA) were investigated and quantified using the FBM. It was determined that excimer formation was controlled by the very rigid polypeptide backbone and not by the solvent viscosity, contrary to previous results obtained with other pyrene-labeled vinyl polymers.

We have constructed a single molecule detection system with the capability to simultaneously measure many parameters, including transient fluorescence intensity, fluorescence lifetime, and photon anti-bunching behavior via the Hanbury Brown-Twiss photon-correlation technique. In addition, we apply the system to study the single DiI (1, 1 '- dioctadecyl- 3, 3 , 3 ', 3 ' - tetramethylindocarbocyanine perchlorate) molecule, to characterize the photo-physical behaviors. Cyanine dyes are the molecules that constitute of two nitrogen centers, one of which is positive charged, and is linked by a conjugated chain with odd number of carbon atoms to the other nitrogen center. Cyanine dyes are interested in the photo sensitization, optical recording media, nonlinear optics, laser dyes, and many interesting photophysical and photochemical behaviors. Among them, DiI plays an important role in single molecule fluorescence investigations. The high photo-stability, good QE, and low inter-system crossing rates, make it a pioneer for the widely investigations in single molecule studies. Our experimental goal is to understand the characteristic of the monitored single molecule by the measuring photo-physical parameters. Our results include the typical behaviors in DiI molecules: clear on-off blinking, fluorescence anti-bunching, one-step photo-bleaching, and consistent fluorescence polarization orientation. In addition, we also observed some change during measurement, which indicates the corresponding change of structure. Few molecules also exhibit non-zero probability around the zero delay time, which indicates the simultaneous existence of more than one quantum emitters in the detected region. These results demonstrate that the parameters are essential for understanding and characterizing the observed molecules in single molecule level.

The motion of dye molecules in the solution is highly influenced by the Brownian motion caused by the stochastic collisions with the solvents, and it results the fluorescence intensity fluctuation. The thesis study the fluorescence intensity fluctuation of dilute dye molecule (Rhodamine B) in methanol solution ( - ), under confocal and total internal reflection (TIR) microscopy configurations. Five parameters are used to probe the fluorescence characteristics: (1) the difference between confocal and the TIR configurations. The configuration influences the laser focusing area and consequently the intensity distribution. The effective focusing area in confocal configuration is an ellipsoid shape, while that of TIR configuration is a disk shape around the interface with depth 100-200 nm. It results the TIR configuration less background and higher concentration capability. (2) concentration. We control the concentration from much less than one molecule to more than one molecule in the effective focusing area, and we observe the change of burst intensity distribution. (3) the focus position. By changing the focusing position, we study the effective focusing region changes. (4) excited intensity, and (5) fluorescence correlation spectroscopy (FCS). Our results indicate that TIR configuration exhibits lower background, and is suitable to higher concentration solution. In addition, when the dye concentration in the focusing area is much less than 1, the FCS amplitude is no longer follow 1/N, but rather be proportional to N, where N is the concentration.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010. / Committee Chair: Griffin, Paul; Committee Member: Butera, Robert; Committee Member: Halpern, Michael; Committee Member: Nichols, Richard; Committee Member: Vidakovic, Brani. Part of the SMARTech Electronic Thesis and Dissertation Collection.

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The processes involved in the enhancement of the fluorescence profile of dipicolinic acid (DPA) were measured and analysed, with particular emphasis on their potential application to the rapid identification of suspicious powders. The research was conducted in contribution to the anthrax detector currently under development at this department. Using the enhancement of fluorescence as a method of determining whether a sample contains spores shows great potential because DPA is not found in most powders that do not contain spores. Thus, its detection is a good indication of the presence of spores. The research presented in this thesis primarily focuses on the optimisation of measurement and enhancement techniques. Both DPA and milk powder (containing spores) were used as anthrax simulants. We found that 210 nm light was the optimal wavelength for the enhancement of DPA; however, as most light sources have a higher intensity at longer wavelengths, the use of 270 nm light may be more effective. At low concentrations, there is a linear relationship between detected fluorescence intensity and the quantity of DPA present. A linear response was also found to the enhancement-light exposure time.

The object of this thesis is to present two novel applications of Spatiotemporal Image Correlation Spectroscopy (STICS) to biological systems. STICS is a technique which uses the correlations in pixel intensity fluctuations of an image time series, captured under fluorescence microscopy, to measure the speed and direction of a flowing population of fluorescently labeled molecules. The method was first applied to measure the dynamics of transport vesicles inside growing pollen tubes of lily flowers. The measured vector maps allowed to confirm the presence of actin filaments along the periphery of the tubes, as well as the presence of a reverse-fountain pattern in the apical region. In a second set of experiments, STICS was used to measure the retrograde flow of filamentous actin in migrating chick DRG neuronal growth cones. These results serve as proof of principle that STICS can be used to probe the response of the growth cone cytoskeleton to external chemical cues.