The present work utilizes single molecule methods and analysis to investigate soft and hard substrates. First, the effect of charged hard surfaces on charged probes were evaluated, as the soft surfaces were built upon such a structure. Then, selected polymers were selected according to their importance in smart surface technology. The extent of interaction of the selected probes with the array of soft surfaces gives insight to the potential tunability of these surfaces. The three distinct polymers range from ubiquitous usage to advancements in current technology. The studies presented here are needed to characterize, on the nanoscale, the Coulombic interactions of these polymers.

Luminescent conjugated polymers are widely used in organic optoelectronics. The device is fabricated by spin coating the polymer solutions into thin films. It is important to understand the chain conformation in the solution phase, which is mainly determined by the solubility properties of the solutes and the solvents. The purpose of this study is focused on the aggregate structures of MDMO-PPV polymer in the solution mixing of toluene, heptanes, and decahydronaphthalene. Compared to the polymer in toluene solution, the absorption and fluorescence spectra in the mixing solutions are red-shifted, which indicates the aggregation between polymer chains. In order to identify the aggregation is inter-chain or intra-chain effect, we perform concentration dependent measurements of the fluorescence spectra down to 10-10 M. Our results suggest that inter-chain aggregation is the major influence in the concentration. Since the intra-chain aggregation is strongly influenced by polymer concentration, we carry out the experiments to identify how the inter-chain effect influences at even lower concentrations. Fluorescence correlation spectroscopy (FCS) is used to determine the particle size at 10-12M concentration, which relates directly to the aggregation size. The results show that particle size in the poor solution is larger than that in the good solution. Therefore, we conclude that the change of the fluorescence spectra is caused by the inter-chain aggregation even at the concentration to 10-12M.

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.

Research in the field of non-viral gene delivery has demonstrated that a deeper understanding of the fundamental processes of nanoparticle assembly is required in order to improve their efficacy. While gemini nanoparticles (gemini NPs) and other non-viral delivery systems have been vigorously characterized using several techniques, our knowledge is still incomplete. The first objective of this study was the development of new methodology using fluorescence correlation spectroscopy (FCS) to investigate the stages of gemini NPs assembly. It was demonstrated that by labeling the plasmid, different stages of gemini NP assembly from the gemini-plasmid pre-complex (GP) to the final gemini nanoparticle (or gemini-plasmid-lipid complex; GPL), could be studied. Based on diffusion coefficients and particle numbers extrapolated from the autocorrelation function (ACF), FCS was able to determine that each phase of assembly had distinct characteristics. The FCS study using 12-3-12 gemini surfactant showed that both the diffusion coefficient and particle number of GPs (0.98??0.31 x 10-12 m2/s) was significantly lower than the final GPL (3.11??0.41 x 10-12 m2/s). Based on the Stokes-Einstein equation the particle size was calculated to be 300-500 nm for GP and 200-300 nm for GPLs. The raw intensity histograms showed that both GPs and GPLs are composed of multiple plasmids. Furthermore the study showed that the final GPLs contain fewer plasmids compared to the intermediate GP. FCS results were validated by using existing characterization methods including dynamic light scattering (DLS), zeta potential and dye exclusion assays. The second objective involved the detailed characterization of gemini NP. Nine different gemini surfactants and two different phospholipids were used in a systematic study to assess the effect of gemini surfactant and lipid structure on the final morphology of gemini NP. The study revealed that gemini surfactant structure had a strong effect on structure of GP intermediates, but addition of phospholipids resulted in the formation of uniform gemini NPs. Based on the results of this study a new model for GP and GPL assembly is proposed based on the formation of supramolecular aggregates of gemini-plasmids, governed by gemini surfactant chemical structure, and dispersed by phospholipids to form GPLs.

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.

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.

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.

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.

This thesis explores the benefits of intensity modulation for the purpose of extending the range of applications of fluorescence spectroscopy and imaging in cellular and molecular biology and medicine. Long-lived transient states of fluorescent molecules can, because of their long lifetimes, be used to detect subtle changes in the microenvironment of the molecule. A method for determining the kinetic rates for transitions to and from such states by registration of changes in the average fluorescence intensity related to different modulation of the excitation source is introduced. It combines the detection sensitivity of fluorescence with the environmental sensitivity of the long-lived transient states and allows the use of slow detectors such as CCD cameras, making parallelization and wide-field imaging possible developments. An extension of this method, generating image contrast based on triplet state population using a standard laser scanning microscope, is also shown. A strategy to combine fluorescence correlation spectroscopy (FCS) with modulated excitation, in a way that allows extraction of correlation data for all correlation times, is presented. This enables the use of modulation to optimize measurement conditions with respect to photophysical properties of the dyes used. FCS with modulated excitation will probably prove useful in future studies involving multiple kinetic processes occurring in overlapping time ranges. One of the ideas from this project also constitutes a powerful method for generating artifact free correlation curves from data sets where sections have been removed. This is potentially very useful in biological studies where spikes in the measurements often cause problems. In the final project, cross-correlation and alternating excitation are combined in measurements on a pH-sensitive ratiometric dye to clearly distinguish the protonation–deprotonation dynamics from other processes. The presented approach makes the protonation related fluctuations manifest themselves as a very distinct anti-correlating component in the correlation curve. This enables robust data analysis using a simple model. / QC 20100805