Understanding the mechanisms by which proteins act on membrane surfaces is fundamental if we are to exploit their capabilities or halt the progression of the diseases they are associated with. Arguably, the best way to study these interactions is by using techniques that can obtain molecular-scale information, in real time and under physiologically relevant conditions. Studying supported lipid bilayer systems with high spatial resolution tools, such as atomic force microscopy (AFM), and high temporal resolution techniques, such as polarized total internal reflection fluorescence microscopy (pTIRFM), allows us to meet these requirements [1]. The goal of this project is to use methods that are currently available and further their applications and capabilities to provide insight into the mechanisms by which amyloidogenic and antimicrobial peptides act on membranes.

Understanding the mechanisms by which proteins act on membrane surfaces is fundamental if we are to exploit their capabilities or halt the progression of the diseases they are associated with. Arguably, the best way to study these interactions is by using techniques that can obtain molecular-scale information, in real time and under physiologically relevant conditions. Studying supported lipid bilayer systems with high spatial resolution tools, such as atomic force microscopy (AFM), and high temporal resolution techniques, such as polarized total internal reflection fluorescence microscopy (pTIRFM), allows us to meet these requirements [1]. The goal of this project is to use methods that are currently available and further their applications and capabilities to provide insight into the mechanisms by which amyloidogenic and antimicrobial peptides act on membranes.

In this effort a new measurement technique for the lateral Goos-Hanchen shift is developed, analyzed, and demonstrated. The new technique uses classical image formation methods fused with modern detection and analysis methods to achieve higher levels of sensitivity than obtained with prior practice. Central to the effort is a new mathematical model of the dispersion seen at a step shadow when the Goos-Hanchen effect occurs near critical angle for total internal reflection. Image processing techniques are applied to measure the intensity distribution transfer function of a new divergence model of the Goos-Hanchen phenomena providing verification of the model. This effort includes mathematical modeling techniques, analytical derivations of governing equations, numerical verification of models and sensitivities, optical design of apparatus, image processing

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.

In the past decade, single molecule imaging has advanced our understanding of processes at the molecular scale. Total internal reflection fluorescence (TIRF) microscopy is one implementation in particular that has been extensively applied in the study of protein adsorption to surfaces. The spatial and temporal resolution provided by TIRF has enabled dynamic measurements of individual proteins in solution, where previously only bulk measurements or static electron microscopy observations were possible. The ability to study individual proteins has revealed and sometimes clarified the complex interactions at their interfaces. Here, the utility of TIRF is expanded to introduce a new model of protein adsorption to the suface and to study the protein interface in contact with solution. Protein adsorption to surfaces has implications in surface biocompatibility, protein separation, and pharmaceutical nanoparticle development. For this reason, the phenomenon has been quantitatively by a variety of techniques, including single molecule imaging. The key data are the protein lifetimes on the surface, which have been shown to be broadly distributed and well-approximated by the sum of several exponential functions. The determined desorption rate constants are thought to reflect different interaction types between surface and protein, but the rates are not typically linked to a specific physical interaction. In the first part of this thesis, we establish appropriate imaging conditions and analysis methods for TIRF. A robust survival analysis technique is applied to capture the range of protein adsorption kinetics. In the second part, we utilize single molecule lifetime data from the adsorption of fibrinogen and bovine serum albumin (BSA) to glass surfaces and discover a heavy-tailed distribution: a very small fraction of proteins adsorbs effectively permanently, while the majority of proteins adsorb for a very short time. We then demonstrate that this characteristic power law behavior is well described by a model with a novel interpretation of the complex protein adsorption process. The second half of the thesis extends TIRF to study the solution-facing interface of the protein as opposed to the surface facing interface by establishing the parameters for a super-resolution imaging technique. Point accumulation for imaging nanoscale topography (PAINT) generates high-resolution images of the sample of interest through the positional tracking of many temporally-distinct instances of a fluorescent probe binding to the sample. Previously, this technique has been applied in the mapping of DNA nanostructures. Here, in the third part, we apply PAINT to the study of proteins. First, a workstream is established for a model system of Nile red and BSA. The kinetic parameters for the system are established to allow rational design of PAINT experiments with this system. The on-rate and off-rate for Nile red are determined. Additionally, the binding model between the two components is tested by studying how the presence of an inhibitor effects the parameters. In the final part, TIRF is used to study the protein-solution interface to examine the glycosylation of immunoglobulin A 1 (IgA1). Over 50% of eukaryotic proteins are glycosylated, and the glycan sequence is simultaneously difficult to study and crucial in the many functional roles proteins play. The glycosylation of IgA1, for example, plays a key role in the pathophysiology of IgA1 nephropathy. Lectins are proteins that bind to specifc glycan sequences and are often used to isolate glycosylated proteins. In this study, the appropriate surface conditions are established to allow specific binding between lectins and IgA1 glycans. The association and dissociation rate between lectins specific for the glycans on IgA1 are measured and affinity constants calculated. These efforts will help to rationally design experiments in the future to elucidate unknown glycan sequences on proteins.

In recent years, optical near-field has received an ever-increasing attention owing to its ability to localise optical signals beyond the diffraction limit. Optical near-field is a non-propagating field existing in the close vicinity of a matter within a range less than the wavelength of the illumination light and it carries the high spatial frequency information showing the fine details of the matter. An optical near-field can be generated by a near-field optical microscope with a nano-aperture or a metal-coated fibre tip. However, common difficulties associated with this approach, such as a fragile probe, a low throughput and signal-to-noise ratio, and a slow response of gap controlling between the probe and the sample, make it less applicable. Alternatively, optical near-field can be produced by total internal reflection (TIR) occurring at the interface of a prism, which is capable of localising the electromagnetic (EM) field in the close vicinity of the interface. However, in this geometry, no confinement of the field can be achieved in the transverse direction, whereas, in most applications such as optical trapping, micro-fabrication and optical data storage, a transverse confinement of the light field is essential. In order to achieve a transverse confinement of the light field, maintaining the high spatial resolution of the optical near-field, and at the same time eliminating the drawbacks associated with the conventional near-field optical microscope, a novel near-field probe based on a high numerical aperture (NA) TIR objective combined with annular illumination has been developed recently. In this arrangement, an obstruction disk is inserted at the back aperture of the objective to block the light with a convergence angle lower than the critical angle determined by the refractive indices of the two media, resulting in a pure focused evanescent field in the second medium. The evanescent field produced by this method provides a useful tool for studying light-matter interaction at the single molecule level not only because of its high resolution but also due to its inherent merits such as no distance regulation, no heating effect and simple experimental setup. But, the most significant advantage that makes this method unique and superior to the other approaches in terms of producing the optical near-field is that it allows the dynamic control of the focal field by simply modulating the phase or amplitude or even the polarisation state of the incident beam before it enters the objective so that complex illumination beams can be generated, whereas in other fibre probe based approaches this goal is extremely difficult to achieve. To make use of such a novel near-field probe, a thorough theoretical and experimental investigation is required. A complete knowledge of the focused evanescent field is a prerequisite for a wide range of applications including single molecule detection, Raman spectroscopy, near-field non-linear imaging and near-field trapping. Therefore, it is not only necessary but also urgent to exploit the focusing properties of a focused evanescent field under complex field illumination both experimentally and theoretically and this is the major aim of this thesis. The complex fields, which are of particular interest in this thesis, are the radially polarised beam and the Laguerre-Gaussian (LG) beam, because the former owns a more compact circularly symmetric field distribution in the focal region when focused by a high NA objective, while the latter is capable of rotating a trapped particle by transferring the orbital angular momentum. Combining them with the focused evanescent field is potentially able to induce novel functions in the near-field region, which cannot be fulfilled by other near-field approaches. In this thesis, in order to generate these two types of beams, a single liquid crystal spatial light modulator (LCSLM) is employed to produce useful phase modulation to the incident beam. Experimental characterisation of an evanescent focal spot is performed with scanning near-field optical microscopy (SNOM), which is capable of providing the direct mapping of the focused evanescent field not only because of its high spatial resolution and its ability to detect the near-field and far-field signals simultaneously, but also due to the motion of the piezzo-stage enables a three-dimensional characterisation of the evanescent focal spot. In this thesis, a SNOM system with an aluminum coated aperture probe is implemented. The field distributions at both the interface and parallel planes with a small distance away from the interface are obtained. To verify the applicability of SNOM as a characterisation methodology, the field distribution in the focal region of a high NA objective illuminated by a linearly polarised plane wave is measured first. A focus splitting along the direction of incident polarisation is observed threedimensionally near the interface under such a circumstance. It has been demonstrated that the depolarisation effect plays an important role in determining the coupling behaviour of the light into the fibre probe of SNOM. The good match between the experimental results and theoretical predications confirms the validity of SNOM. Theoretical investigation of a tightly focused radially polarised beam is undertaken based on the vectorial-Debye diffraction theory because under the tight focusing of a high NA objective, the vectorial nature of the highly localised field has to be carefully considered in order to represent the field distribution accurately. The calculations on the focusing properties of a radially polarised beam suggest that the longitudinal field component in the focal region plays a dominant role in determining the overall field distribution. Direct measurement of the focused evanescent radially polarised beam in a three-dimensional manner near the interface is performed with SNOM. A highly localised focal spot is achieved in the close vicinity of the coverglass. The measured intensity distributions from SNOM show that correction of the focal spot deformation associated with a linearly polarised beam is achieved by taking advantage of the radially symmetric focal spot of a radially polarised beam. A smaller focal spot is acquired due to the dominant longitudinal polarisation component in the focal region, which possesses a more compact focal intensity distribution than that of the overall field. The experimental results demonstrate a good agreement with the theoretical expectations. The fact that a radially polarised beam is capable of eliminating the focus deformation often presented in the focal region of a high NA objective when a linearly polarised beam is employed can be very useful in many applications, including microfabrication using two-photon photopolymerisation technique. The theoretical study on the two-photon point spread function (PSF) of a radially polarised beam indicates that the focus elongation and splitting associated with a linearly polarised beam are eliminated and the achievable lateral size of the focal spot is approximately a quarter of the illumination wavelength, which is less than half of that under the illumination of a linearly polarised beam. A further reductiont of the lateral size can be expected by using annular radial beam illumination. The investigation on the focusing properties of LG beams has also been one of the major tasks of this thesis. Theoretical investigations of a focused evanescent LG beam suggest that the phase shift induced by the boundary effect when a light beam passes the interface satisfying TIR condition plays a vital role in determining the overall shape of the total field distribution. A severe focal intensity deformation is predicted theoretically in the case of focused evanescent LG beam illumination, which might involve new physical phenomena when applied in the near-field trapping. Such a focal intensity deformation is evidenced experimentally by the direct mapping result obtained from the SNOM probe. A quantitative cross-section comparison with the theoretical predication is conducted, which demonstrates a good agreement. To achieve a controllable optical trap and rotation in the near-field region, complex optical fields such as LG beams carrying orbital angular momentum, have been induced for the manipulation of a polystyrene particle. The influence of the focal intensity deformation on a near-field trapping has been thoroughly investigated. Rotation motion of the particle is examined by mapping the two-dimensional (2D) transverse trapping efficiency of the particle. Theoretical investigation reveals that a significant tangential force component is generated on the particle when it is illuminated by a focused evanescent LG beam. Such findings may prove useful in introducing a rotation mechanism in near-field trapping. The research investigations and methodologies described in this thesis provide a new approach to characterise the near-field focal spot under complex field illumination. It enhances the understanding of the novel near-field probe, thus opening the pathway for numerous near-field applications including optical trapping, two-photon excitation (photopolymerisation) and spectroscopy. The focal field rotation phenomena demonstrated in this thesis may prove particularly beneficial in introducing a rotation mechanism in near-field trapping using a focused evanescent field.

The technique of single particle Total Internal Reflection Microscopy (TIRM) has been used to study the scattering intensity from levitated microspheres. TIRM can be used to monitor the separation between microscopic spheres immersed in liquid (water in our case) and a surface with nm resolution. In the technique, microspheres scatter light when the evanescent waves are incident upon them. The intensity of the scattered light is directly related to the height above the surface and allows determination of the height. From the separation distance histograms, the interaction between the microsphere and interface may be characterized with a force resolution in the range of 0.01 picoNewtons. Such a system can be applied to the measurement of biomolecular interactions biomolecules attached to the microsphere and the surface. The intensity and scattering pattern of this light has been modeled using a modified Mie theory which accounts for the evanescent nature of the incident light. Diffusing Colloidal Probe Microscopy (DCPM) is an extension of the TIRM technique that simultaneously monitors multiple microsphere probes. The use of multiple probes introduces the issue of probe polydispersity. When measured at the surface, a variation in scattered light intensity of nearly one order of magnitude has been observed from a purchased microsphere sample. Thus the polydisperse collection of microspheres adds significant complexity to the scattered light signal. It is hypothesized that the dependence of the total scattered light intensity on microsphere size accounts for the scattered intensity distribution in a polydisperse microsphere sample. Understanding this variation in the scattered light with microsphere size will allow improved characterization of the microsphere/surface separation. Additionally, larger microspheres have the ability to resonantly confine light and produce spectrally narrow Whispering Gallery Modes (WGMs). It is hypothesized that WGMs may be excited in microspheres with the DCPM system. These modes may be used as a refractometric biosensor with high sensitivity to local refractive index changes on the surface of the microsphere. This research involves modeling scattered intensity distributions for polydispersed collections of microspheres based on modified Mie theory. The theoretical results are compared to experimentally obtained results and found to qualitatively explain the scattered light intensity distribution in a multiple probe DCPM system. This is an important result suggesting that microsphere size variation plays a major role in determining the distribution of scattered intensity in multiple microsphere probe systems. This work also suggests that it may be possible to excite such WGMs in a DCPM system. The introduction of WGMs would enable refractometric biosensing in such evanescent mode systems.

Spatial and temporal tracking of passively diffusing functionalized colloids continues to be an improving and auspicious approach to measuring weak specific and non-specific biomolecular interactions. Evidence of this is given by the recent increase in published studies involving the development and implementation of these methods. The primary aim of the work presented in this dissertation was to modify and optimize video microscopy (VM) and total internal reflection microscopy (TIRM) methods to permit the collection of equilibrium binding and sampling data from interaction of surface-immobilized biomolecules. Supported lipid bilayers were utilized as model systems for functionalizing colloid and wall surfaces. Preliminary results measuring calcium-specific protein-protein interactions between surface immobilized cadherin fragments demonstrate the potential utility of this experimental system and these methods. Additionally, quantum dot-modified colloids were synthesized and evanescent wave-excited luminescence from these particles was used to construct potential energy profiles. Results from this work demonstrate that colloids can be used as ultra-sensitive probes of equilibrium interactions between biomolecules, and specialized probes, such as those modified with quantum dots, could be used in a spectral multiplexing mode to simultaneously monitor multiple interactions.

The technique of single particle Total Internal Reflection Microscopy (TIRM) has been used to study the scattering intensity from levitated microspheres. TIRM can be used to monitor the separation between microscopic spheres immersed in liquid (water in our case) and a surface with nm resolution. In the technique, microspheres scatter light when the evanescent waves are incident upon them. The intensity of the scattered light is directly related to the height above the surface and allows determination of the height. From the separation distance histograms, the interaction between the microsphere and interface may be characterized with a force resolution in the range of 0.01 picoNewtons. Such a system can be applied to the measurement of biomolecular interactions biomolecules attached to the microsphere and the surface. The intensity and scattering pattern of this light has been modeled using a modified Mie theory which accounts for the evanescent nature of the incident light. Diffusing Colloidal Probe Microscopy (DCPM) is an extension of the TIRM technique that simultaneously monitors multiple microsphere probes. The use of multiple probes introduces the issue of probe polydispersity. When measured at the surface, a variation in scattered light intensity of nearly one order of magnitude has been observed from a purchased microsphere sample. Thus the polydisperse collection of microspheres adds significant complexity to the scattered light signal. It is hypothesized that the dependence of the total scattered light intensity on microsphere size accounts for the scattered intensity distribution in a polydisperse microsphere sample. Understanding this variation in the scattered light with microsphere size will allow improved characterization of the microsphere/surface separation. Additionally, larger microspheres have the ability to resonantly confine light and produce spectrally narrow Whispering Gallery Modes (WGMs). It is hypothesized that WGMs may be excited in microspheres with the DCPM system. These modes may be used as a refractometric biosensor with high sensitivity to local refractive index changes on the surface of the microsphere. This research involves modeling scattered intensity distributions for polydispersed collections of microspheres based on modified Mie theory. The theoretical results are compared to experimentally obtained results and found to qualitatively explain the scattered light intensity distribution in a multiple probe DCPM system. This is an important result suggesting that microsphere size variation plays a major role in determining the distribution of scattered intensity in multiple microsphere probe systems. This work also suggests that it may be possible to excite such WGMs in a DCPM system. The introduction of WGMs would enable refractometric biosensing in such evanescent mode systems.

This thesis focuses on investigations of transient dark states of fluorescentmolecules using spectroscopic techniques. The main purpose is to show andconvince the reader that transient dark states are not always a nuisance, butalso represent an additional source of information. Several studies with fluorescencecorrelation spectroscopy were performed, all related to non-fluorescentstates such as triplet state or isomerized states.Photobleaching is one of the main problems in virtually all of the fluorescencetechniques. In this thesis, mechanisms that retard photobleaching arecharacterized. Several compounds, antioxidants and triplet state quenchers,which decrease photobleaching, are studied, and guidelines for achieving optimalfluorescence brightness using these compounds are presented.Triplet state quenching by several compounds was studied. Detailed investigationsof the fluorescence quencher potassium iodide demonstratedthat for some of fluorophores, except of quenching, there is fluorescence enhancementmechanism present. In agreement with the first publication inthis thesis, antioxidative properties were found to play an important role inthe fluorescence enhancement. Quenching of the triplet state is proposedas a tool for monitoring diffusion mediated reactions over a wide range offrequencies.Specially designed fluorophores combining high triplet yields with reasonablefluorescence brightness and photostability were characterized forpossible applications in novel super-resolution imaging techniques based onfluorescence photoswitching. Except of benefits for imaging techniques, photoinducedswitching to non-fluorescent states could be used for monitoringmolecular diffusion, which was also demonstrated in this thesis.Studies of the triplet state kinetics of fluorophores close to dielectric interfaceswere performed using fluorescence spectroscopy. The analysis of thetriplet state kinetic can provide information about the local microenvironmentand electrostatic interactions near dielectric interfaces. / QC 20100414