Ziel dieser Arbeit ist es eine Toolbox zur Charakterisierung der anomalen Diffusion von Tracerpartikeln in dicht gepackten Systemen mit Fluoreszenz-Korrelationsspektroskopie (FCS) zur Verfügung zu stellen. Es wird gezeigt, dass die robusten Informationen über die Wahrscheinlichkeitsdichtefunktion (PDF) der Verschiebung des Tracers im asymptotischen Verhalten der FCS-Kurven auf langen, sowie auf kurzen Zeitskalen enthalten sind. So liefert die Analyse des Kurzzeitverhaltens zuverlässige Aussagen über die Werte des Exponenten der anomalen Diffusion, des Diffusionskoeffizienten und der niedrigeren Momente der PDF. Dies erlaubt es eine Gaußverteilung zu bestätigen oder zu widerlegen. Der Test auf Gaußverteilung könnte als Index verwendet werden, um die richtige Form der PDF aus einer Reihe von konkurrierenden Ergebnissen zu erraten. Darüber hinaus untersuchen wir die Konsequenz der nicht skalierenden PDF auf Ergebnis der FCS-Kurven. Wir berechnen die FCS für ein Continuous Time Random Walk Modell mit Wartezeiten gemäß einer Lévy-stabilen Verteilung mit exponentiellem cut-off. Die Ergebnisse zeigen, dass obwohl die Abweichungen vom Gauß’schen Verhalten bei der asymptotischen Analyse erkannt werden können, ihre Körper immer an Formen für die normale Diffusion perfekt angepasst werden können. Schließlich schlagen wir einen alternativen Ansatz für die Durchführung von Spot Variation FCS mit dem gewöhnlichen FCS-Setup vor. Wir führen eine nicht-lineare Transformation ein, die auf das mit Binning oder Kernel smoothing method geglättete Intensitätsprofil der detektierten Fluoreszenzphotonen angewendet wird. Ihre Autokorrelation imitiert die FCS-Kurven für die Größen des Laserspots, die im Experiment effektiv kleiner als die anfängliche Größe sind. Die erhaltenen FCS-Kurven werden verwendet, um künstliche dicht gepackte Systeme sowie lebende Zellen auf Nano-Domänen oder Barrieren hin zu untersuchen. / The objective of this thesis is to provide a toolbox for characterization of anomalous diffusion of tracer particle in crowded systems using fluorescence correlation spectroscopy (FCS). We discuss that the robust information about the probability density function (PDF) of the particle’s displacement is contained in the asymptotic behaviour of the FCS curves at long and short times. Thus, analysis of the short-time behaviour provides reliable values of exponent of anomalous, diffusion coefficient and lower moments of the PDF. This allows one to to confirm or reject its Gaussian nature. The Gaussianity test could be then used to guess the correct form of the PDF from a set of competing models. We show the applicability of the proposed analysis protocol in artificially crowded systems and in living cell experiments. Furthermore, we investigate the consequence of non-scaling PDF on the possible results of the FCS data. As an example of such processes, we calculate the FCS curve for a continues time random walk model with waiting times delivered from Lévy-stable distribution with an exponential cut-off in equilibrium. The results indicate that, although the deviations from Gaussian behaviour may be detected when analyzing the short- and long-time asymptotic of the corresponding curves, their bodies are still perfectly fitted by the fit form used for normal diffusion. Finally, we propose an alternative approach for performing spot variation FCS using an ordinary FCS set-up. We introduce a non-linear transformation which applies on the smoothed intensity profile of the detected fluorescence photons with binning or smoothing kernel method. Autocorrelation of the generated intensity profiles mimic the FCS curves for the sizes of laser spots which are effectively smaller than the initial one in the experiment. The obtained FCS curves are used to investigate the presence of nano-domains or barriers in artificially crowded systems and in living cells.

Le but initial de ce travail de thèse est de proposer une technique (basée sur la Fluorescence Correlation Spectroscopy, FCS) pour améliorer la sensibilité dans la discrimination de deux molécules ayant des constantes de diffusion proches. C'est dans ce contexte que nous avons étudié la FCCS (Fluorescence Cross-Correlation Spectroscopy). A défaut d'améliorer la sensibilité de la Spectroscopie à Correlation de Fluorescence nous avons proposé trois géométries de FCCS pour élargir le champ d'application de la corrélation de fluorescence.

Ziel dieser Arbeit ist es eine Toolbox zur Charakterisierung der anomalen Diffusion von Tracerpartikeln in dicht gepackten Systemen mit Fluoreszenz-Korrelationsspektroskopie (FCS) zur Verfügung zu stellen. Es wird gezeigt, dass die robusten Informationen über die Wahrscheinlichkeitsdichtefunktion (PDF) der Verschiebung des Tracers im asymptotischen Verhalten der FCS-Kurven auf langen, sowie auf kurzen Zeitskalen enthalten sind. So liefert die Analyse des Kurzzeitverhaltens zuverlässige Aussagen über die Werte des Exponenten der anomalen Diffusion, des Diffusionskoeffizienten und der niedrigeren Momente der PDF. Dies erlaubt es eine Gaußverteilung zu bestätigen oder zu widerlegen. Der Test auf Gaußverteilung könnte als Index verwendet werden, um die richtige Form der PDF aus einer Reihe von konkurrierenden Ergebnissen zu erraten. Darüber hinaus untersuchen wir die Konsequenz der nicht skalierenden PDF auf Ergebnis der FCS-Kurven. Wir berechnen die FCS für ein Continuous Time Random Walk Modell mit Wartezeiten gemäß einer Lévy-stabilen Verteilung mit exponentiellem cut-off. Die Ergebnisse zeigen, dass obwohl die Abweichungen vom Gauß’schen Verhalten bei der asymptotischen Analyse erkannt werden können, ihre Körper immer an Formen für die normale Diffusion perfekt angepasst werden können. Schließlich schlagen wir einen alternativen Ansatz für die Durchführung von Spot Variation FCS mit dem gewöhnlichen FCS-Setup vor. Wir führen eine nicht-lineare Transformation ein, die auf das mit Binning oder Kernel smoothing method geglättete Intensitätsprofil der detektierten Fluoreszenzphotonen angewendet wird. Ihre Autokorrelation imitiert die FCS-Kurven für die Größen des Laserspots, die im Experiment effektiv kleiner als die anfängliche Größe sind. Die erhaltenen FCS-Kurven werden verwendet, um künstliche dicht gepackte Systeme sowie lebende Zellen auf Nano-Domänen oder Barrieren hin zu untersuchen. / The objective of this thesis is to provide a toolbox for characterization of anomalous diffusion of tracer particle in crowded systems using fluorescence correlation spectroscopy (FCS). We discuss that the robust information about the probability density function (PDF) of the particle’s displacement is contained in the asymptotic behaviour of the FCS curves at long and short times. Thus, analysis of the short-time behaviour provides reliable values of exponent of anomalous, diffusion coefficient and lower moments of the PDF. This allows one to to confirm or reject its Gaussian nature. The Gaussianity test could be then used to guess the correct form of the PDF from a set of competing models. We show the applicability of the proposed analysis protocol in artificially crowded systems and in living cell experiments. Furthermore, we investigate the consequence of non-scaling PDF on the possible results of the FCS data. As an example of such processes, we calculate the FCS curve for a continues time random walk model with waiting times delivered from Lévy-stable distribution with an exponential cut-off in equilibrium. The results indicate that, although the deviations from Gaussian behaviour may be detected when analyzing the short- and long-time asymptotic of the corresponding curves, their bodies are still perfectly fitted by the fit form used for normal diffusion. Finally, we propose an alternative approach for performing spot variation FCS using an ordinary FCS set-up. We introduce a non-linear transformation which applies on the smoothed intensity profile of the detected fluorescence photons with binning or smoothing kernel method. Autocorrelation of the generated intensity profiles mimic the FCS curves for the sizes of laser spots which are effectively smaller than the initial one in the experiment. The obtained FCS curves are used to investigate the presence of nano-domains or barriers in artificially crowded systems and in living cells.

No description available.

Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / The preparation of mesoporous/macroporous silica materials and polarity gradient thin film are introduced in this thesis. These porous silica materials and gradient materials have the potential applications as stationary phases for chemical separations, as materials for combinatorial catalysis and as absorbent/adsorbent layers for use in chemical or biological sensors. Single molecule spectroscopy is used to probe the chemical interaction between single dye molecule and porous silica matrix. Bulk fluorescence spectroscopy is used to investigate the properties of gradient film. In Chapter one, the applications of single molecule spectroscopic methods to sol-gel silica materials are reviewed, which covers a subset of the recent literature in this area and provided salient examples of the new information that can be obtained by single molecule studies. In Chapter two, both the sample preparation and experiment setup are covered. In Chapter three, the preparation of mesoporous silica film is presented. Single molecule spectroscopy is used to probe the mass transport and molecule-matrix interactions in mesoporous thin-film systems. Three different dyes of varying size, charge, and hydrophilicity are used. Silica films with/without surfactant or containing different kind surfactant are studied. The results provide new information on mass transport through the films, evidence of reversible surface adsorption, and quantitative information on variations in these phenomena with film hydration. In Chapter four, a new model describing how to explore the actual dye concentration in single molecule experiment with considering the molecule orientation is presented, which is verified to be correct by both experimental and simulated data. In Chapter five, the growth process of Methylsilsesquioxane (MSQ) particle is studied by single molecule spectroscopy, in which, the MSQ particle is treated as “native” dye molecule. In Chapter six, silica films incorporating polarity gradients are produced by using “infusion-withdrawal dip-coating” method. The gradient film is characterized by bulk fluorescence spectroscopy, water contact angle and FTIR. In Chapter seven, a brief conclusion is drawn and future directions are presented.

A técnica de Espalhamento Dinâmico de Luz (do inglês Dynamic Light Scattering, ou DLS), também conhecida por técnica de Espectroscopia por Correlação de Fótons (do inglês Photon Correlation Spectroscopy, ou PCS), é amplamente utilizada na caraterização estrutural de sistemas coloidais, fornecendo informações importantes sobre tamanhos ca- ractersticos, tempos de correlação e efeitos de polarização no sistema. A facilidade de execução dos experimentos e praticidade das análises é uma das principais vantagens desta técnica. Especificamente para sistemas diludos compostos por partculas em solução, ela pode fornecer informações diretas sobre o raio hidrodinâmico das partculas do meio. No entanto, a obtenção desse parâmetro requer a utilização de métodos de ajuste e análise dos dados experimentais que assumem diversas caractersticas do sistema e possuem li- mitações intrnsecas devido à própria resolução do método frente presença de partculas de variados tamanhos e concentrações, polidispersões contnuas de tamanho, entre vários outros fatores. Existem casos, por exemplo, onde o mesmo conjunto experimental pode ser descrito por dois modelos diferentes. Neste projeto, é proposto um estudo sistemático das limitações dos métodos de análise frente a dados simulados e experimentais, de modo a permitir a aplicabilidade destes métodos a vários tipos de sistemas. Serão investigados casos de sistemas monodispersos e polidispersos, monomodais e multimodais, através da geração de dados simulados que mimetizem a presença desses grupos de partculas, em di- ferentes proporções e polidispersões. Como resultado, teremos indicações da precisão com que estes métodos de modelagem conseguem reproduzir os valores inseridos na simulação. Além disso, experimentos reais serão realizados utilizando amostras padrão de modo a aplicar os métodos de análise a estes dados bem como contrapor os resultados obtidos pelas simulações e pelos dados experimentais. / The Dynamic Light Scattering technique, also known as Photon Correlation Spectros- copy, is widely used for the structural characterization for colloidal systems, providing important information on characteristic length scales, correlation times and polarization effects. The relative simple experimental setup and easy-to-use modeling methods are one of the main advantages of this technique. Specifically for diluted systems of particles in solution, one can obtain direct information on the hydrodynamic for the particles in the system. However, in order to retrieve this parameter it is necessary to use modeling and analysis methods for the experimental methods which assumes intrinsic characteristics on the system and have intrinsic limitations due to the resolution of the method when particles with several sizes, concentrations, etc. In several cases, the same experimental data can be described by several different models. In this project it is proposed a systematic study on the limitations on the analysis methods upon simulated and experimental data in order to investigate the applicability of these methods for several system types. Monodisperse and polydisperse systems are investigated, either composed by one type of particles (monomo- dal) or several types of particles (multimodal). As a result, one can obtain indications on the accuracy that the modeling methods can reproduce the simulated parameters. Finally, real experiments were performed using standard samples in order to test the modeling methods and to calibrate the simulation procedures.

Thesis advisor: Mary F. Roberts / Thesis advisor: Steven D. Bruner / The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from <italic>Bacillus thuringiensis</italic> is specifically activated by low concentrations of a non-substrate lipid, phosphatidylcholine (PC), presented as an interface. However, if the PC concentration in the interface is too high relative to substrate, the enzyme exhibits surface dilution inhibition. Understanding this bacterial enzyme, which shares many kinetic features with the larger and more complex mammalian PI-PLC enzymes, requires elucidating the mechanism for PC activation and inhibition. Various techniques were applied to study the interaction of the protein with vesicles composed of both the activator lipid PC and the substrate lipid (or a nonhydrolyzable analogue). Fluorescence correlation spectroscopy (FCS), used to monitor bulk partitioning of the enzyme on vesicles, revealed that both the PC and the substrate analogue are required for the tightest binding of the PI-PLC to vesicles. Furthermore, the tightest binding occurred at low mole fractions of substrate-like phospholipids. Field cycling <super>31</super>P NMR (fc-P-NMR) spin-lattice relaxation studies provided information on how bound protein affects the lipid dynamics in mixed substrate analogue/PC vesicles. The combination of the two techniques could explain the enzyme kinetic profile for the PC activation and surface dilution inhibition: small amounts of PC in an interface enhanced PI-PLC binding to substrate-rich vesicles while high fractions of PC tended to sequester the enzyme from the bulk of its substrate leading to reduced specific activity. FCS binding profiles of mutant proteins were particularly useful in determining if a specific mutation affected a single or both phospholipid binding modes. In addition, an allosteric PC binding site was identified by fc-P-NMR and site directed spin labeling. A proposed model for PC activation suggested surface-induced dimerization of the protein. Experiments in support of the model used cysteine mutations to create covalent dimers of this PI-PLC. Two of these disulfide linked dimers, formed from W242C or S250C, exhibited higher specific activities and tighter binding to PC surfaces. In addition, single molecule total internal reflection fluorescence microscopy was used to monitor the off-rate of PI-PLC from surface tethered vesicles, providing us with a direct measure of off-rates of the protein from different composition vesicles. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

A espectroscopia de correlação de fluorescência (FCS) é uma das diferentes técnicas de análise por imagens de alta resolução espacial e temporal de biomoléculas em concentrações extremamente baixas. Ela se tornou uma técnica extremamente poderosa e sensível em áreas como bioquímica e biofísica. Como uma técnica bem estabelecida, ela é utilizada para medir concentrações locais de biomoléculas, através da marcação com moléculas fluorescentes. Coeficientes de difusão e constantes cinéticas também podem ser medidos através de FCS assim como detecção de molécula única. Ela também pode dar informação precisa sobre interações de antígeno-anticorpo, ácidos nucleicos e proteínas. Através de uma combinação de marcadores de alto rendimento quântico, fontes de luz estável (lasers), detecção ultrassensível e microscopia confocal, é possível realizar medidas de FCS em volumes de fentolitros (fL) e em concentrações de nanomolar (nM) em soluções aquosas ou em células vivas. Em contraste com outras técnicas de fluorescência, a sensibilidade da FCS aumenta com a diminuição da concentração do fluoróforo marcador, porque o parâmetro de interesse não é a intensidade de emissão de fluorescência, mas sim as flutuações espontâneas da fluorescência. Durante o tempo em que a partícula ou molécula atravessa o volume de medida pode ocorrer mudanças conformacionais e reações químicas e fotofísicas que alteram as características de emissão do fluoróforo e causam flutuações no sinal detectado. Estas flutuações são então monitoradas e transformadas em uma curva de autocorrelação, por intermédio de um software comercial que emprega um modelo físico apropriado para FCS. Em nosso estudo, utilizamos um marcador comercial (ALEXA 488&reg;) para marcar proteínas. Primeiramente utilizamos a técnica de FCS para medir concentrações extremamente baixas de marcadores fluorescentes. Também realizamos um experimento testando a influência da viscosidade do meio na difusão livre do fluoróforo, assim como as melhores condições em que temos um melhor sinal de FCS. Por fim, estudamos a difusão de proteínas marcadas (PUC II e IV) em meio aquoso (PBS) e no interior de células. / Fluorescence correlation spectroscopy (FCS) is one of the many different modes of high-resolution spatial and temporal analysis of extremely low concentrated biomolecules. It has become a powerful and sensitive tool in fields like biochemistry and biophysics. As a well established technique, it is used to measure local concentrations of fluorescently labeled biomolecules, diffusion coefficients, kinetic constants and single molecule studies. Through a combination of high quantum yield fluorescent dyes, stable light sources (lasers), ultrasensitive detection and confocal microscopy is possible to perform FCS measurements in femtoliters volumes and nanomolar concentrations in aquous solution or in live cells. Unlike with other fluorescence technics, its sensibility increases with the decrease of dye concentrarion, because the main factor is not the emission intensity itself. Instead this, spontaneous statistical fluctuation of fluorescence becomes the main factor in FCS analisys. During the time that the conjugated-dye cross the volume detection can occur conformational changes, chemical reaction and photophysical processes that can change the emission properties of the dye and, then, change the detected sinal. This fluctuations are tracked and changed into a autocorrelation curve, by a specific software, appropriate to perform FCS analisys. In our study, we use comercial dye (Alexa 488) to label proteins. Firstly, we applied FCS to measure extremally diluted concentrations of dyes (~1 nM). We have performed experiments testing the influence of the viscosity medium in the free difusion of the dyes and the optical apparatus and conditions that result in the best FCS signal. We also have studied protein diffusion (PUC II e IV) in aquous medium (PBS) and toward the inner of the cells.

A técnica de Espalhamento Dinâmico de Luz (do inglês Dynamic Light Scattering, ou DLS), também conhecida por técnica de Espectroscopia por Correlação de Fótons (do inglês Photon Correlation Spectroscopy, ou PCS), é amplamente utilizada na caraterização estrutural de sistemas coloidais, fornecendo informações importantes sobre tamanhos ca- ractersticos, tempos de correlação e efeitos de polarização no sistema. A facilidade de execução dos experimentos e praticidade das análises é uma das principais vantagens desta técnica. Especificamente para sistemas diludos compostos por partculas em solução, ela pode fornecer informações diretas sobre o raio hidrodinâmico das partculas do meio. No entanto, a obtenção desse parâmetro requer a utilização de métodos de ajuste e análise dos dados experimentais que assumem diversas caractersticas do sistema e possuem li- mitações intrnsecas devido à própria resolução do método frente presença de partculas de variados tamanhos e concentrações, polidispersões contnuas de tamanho, entre vários outros fatores. Existem casos, por exemplo, onde o mesmo conjunto experimental pode ser descrito por dois modelos diferentes. Neste projeto, é proposto um estudo sistemático das limitações dos métodos de análise frente a dados simulados e experimentais, de modo a permitir a aplicabilidade destes métodos a vários tipos de sistemas. Serão investigados casos de sistemas monodispersos e polidispersos, monomodais e multimodais, através da geração de dados simulados que mimetizem a presença desses grupos de partculas, em di- ferentes proporções e polidispersões. Como resultado, teremos indicações da precisão com que estes métodos de modelagem conseguem reproduzir os valores inseridos na simulação. Além disso, experimentos reais serão realizados utilizando amostras padrão de modo a aplicar os métodos de análise a estes dados bem como contrapor os resultados obtidos pelas simulações e pelos dados experimentais. / The Dynamic Light Scattering technique, also known as Photon Correlation Spectros- copy, is widely used for the structural characterization for colloidal systems, providing important information on characteristic length scales, correlation times and polarization effects. The relative simple experimental setup and easy-to-use modeling methods are one of the main advantages of this technique. Specifically for diluted systems of particles in solution, one can obtain direct information on the hydrodynamic for the particles in the system. However, in order to retrieve this parameter it is necessary to use modeling and analysis methods for the experimental methods which assumes intrinsic characteristics on the system and have intrinsic limitations due to the resolution of the method when particles with several sizes, concentrations, etc. In several cases, the same experimental data can be described by several different models. In this project it is proposed a systematic study on the limitations on the analysis methods upon simulated and experimental data in order to investigate the applicability of these methods for several system types. Monodisperse and polydisperse systems are investigated, either composed by one type of particles (monomo- dal) or several types of particles (multimodal). As a result, one can obtain indications on the accuracy that the modeling methods can reproduce the simulated parameters. Finally, real experiments were performed using standard samples in order to test the modeling methods and to calibrate the simulation procedures.

Scotch Whisky is a complex mixture comprising thousands of chemical species at a diverse range of concentrations. The identities, origins, and significance of many of these compounds is largely unknown. Routine characterisation of Scotch Whisky mostly utilises techniques such as gas or liquid chromatography (GC, LC) coupled to a FID, UV, or low-resolution mass spectrometry (MS) detector. In this thesis, advanced spectroscopic and spectrometric techniques are investigated as potential complementary means to unravel the chemical complexity of Scotch Whisky. Chemometric methods are applied to decipher the significance or potential origin of many of these compounds. Being predominantly (ca. 99 %) protonated 'solvent' - ethanol and water - 1H and 13C Nuclear Magnetic Resonance (NMR) required solvent suppression to be implemented into the acquisition of high resolution spectra. A 1D NOESY-presaturation sequence was modified and implemented in automation for this purpose. Furthermore, this solvent suppression was coupled with several 1D and 2D homo- and heterocorrelated NMR experiments for the analysis of Scotch Whisky. With limited sample preparation - only addition of buffer - an approximate limit of detection of 50 μm was achieved. The developed NMR methodology was subsequently used for structural elucidation of dozens of compounds in Scotch Whisky. Quantification of these compounds was hindered by variable chemical shifts, signal overlap, and for some compounds the existence of equilibria of different forms. Quantification of ethanol concentrations in model solutions and genuine Scotch Whisky samples was successful. A large set of Scotch Whisky samples were analysed by the solvent suppressed 1D 1H NMR methodology and various statistical techniques including statistical total correlation spectroscopy (STOCSY), independent and principal component analysis (ICA, PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA). Various parameters were modelled, and discrimination of 'malt' or 'blend' status was achieved, whilst maturation wood type discrimination was less successful. High resolution negative mode electrospray ionisation (ESI) Fourier transform ion cyclotron resonance (FTICR) MS was then used to examine a large set of Scotch Whisky samples. Thousands of unique molecular formulae were assigned within a 1 ppm threshold, representing an assignment rate of 72-88 % of the detected peaks in the spectra. Assignments were selectively confirmed by isotopic fine structure (IFS) analysis, and structural information obtained by both quadrupolar isolation and fragmentation, and in-cell isolation and fragmentation. Similar chemometric methods as applied to NMR data were used to model sample parameters, and identification of potential maturation wood marker compounds was achieved. Alternative ionisation sources - including atmospheric pressure chemical- and photo-ionisation (APCI, APPI), and laser desorption ionisation (LDI) - were compared for the analysis of Scotch Whisky by FTICR MS. The differing sources provide complementary compositional information on the sample set, with APCI and LDI being most different to ESI in terms of compounds ionised and spectral profiles. Positive mode ionisation was also successful, but molecular formula assignment was hindered by insufficient resolving power. Late experimentation pushed the achievable resolving power to 2.8 million at m/z 400, however the required approach is significantly more time consuming and prone to signal quality degradation. Formula assignment software, both commercial, open source, and in-house developed, were compared. The commercial and published open source software provided essentially identical results for Scotch Whisky, and whilst the in-house tools assigned fewer species (a subset of those assigned by the other tools), they did so with a smaller mean error of assignment. Various analysis and visualisation tools for MS data of complex mixtures were also developed.