The electron transfer mechanisms of multiheme cytochromes were examined with scanning tunneling microscopy (STM). To simulate bacterial metal reduction mediated by proteins in direct contact with mineral surfaces, monolayers of purified decaheme cytochromes from the metal-reducing bacterium Shewanella oneidensis were prepared on Au(111) surfaces. Recombinant tetracysteine sequences were added to two outermembrane decaheme cytochromes (OmcA and MtrC) from S. oneidensis MR-1 to ensure chemical immobilization on Au(111). STM images of the cytochrome monolayers showed good coverage and their shapes/sizes matched that predicted by their respective molecular masses. Current-voltage (I-V) tunneling spectroscopy revealed that OmcA and MtrC exhibit characteristic tunneling spectra. Theoretical modeling of the single-molecule tunneling spectra revealed a distinct tunneling mechanism for each cytochrome: OmcA mediates tunneling current coherently whereas MtrC temporarily traps electrons via orbital-mediated tunneling. These mechanisms suggest a superexchange electron transfer mechanism for OmcA and a redox-specific (i.e. heme-mediated) electron transfer mechanism for MtrC at mineral surfaces during bacterial metal reduction. Additionally, a novel electrochemical STM configuration was designed to measure tunneling current from multiheme cytochromes to hematite (001) surfaces in various electrolyte solutions. Current-distance (I-s) profiles on hematite (001) reveal predictable electric double layer structure that changes with ionic strength. The addition of the small tetraheme cytochrome c (STC) from S. oneidensis on insulated Au tips resulted in modified tunneling profiles that suggest STC significantly modulates the double layer. This observation is relevant to understanding metal reduction in cases where terminal metal-reducing enzymes are unable to come in direct contact with reducible mineral surfaces. Electronic coupling to the mineral surface might therefore be mediated by a localized ion swarm specific to the mineral surface. / Ph. D.

As three-dimensional (3D) imaging and fluorescence techniques become standard in optical microscopy, novel approaches to 3D fluorescence microscopy are emerging. One such approach is based on the incoherent holography technique called optical scanning holography (OSH). The main advantage of OSH-based microscopy is that only a single two-dimensional (2D) scan is required to record 3D information, whereas most current 3D microscopes rely on sectioning techniques. To acquire a 3D representation of an object, current microscopes must physically scan the specimen in a series of 2D sections along the z-axis. In order to record holograms by OSH, the fluorescent specimen is scanned with an optically heterodyned laser field consisting of a Fresnel zone pattern. A unique acousto-optic modulator configuration is employed to generate a suitable heterodyne frequency for excitation of the fluorescent object. The optical response of a solution containing a high concentration of 15 um fluorescent latex beads to this type of excitation field has been recorded. In addition, holograms of the same beads have been recorded and reconstructed. To demonstrate the 3D imaging capability of the technique, the hologram includes beads with longitudinal separation of about 2 mm. A detailed comparison of 3D fluorescence microscopy by OSH and the confocal approach was conducted. Areas for comparison were 3D image acquisition time, resolution limits and photobleaching. The analysis shows that an optimized OSH-based fluorescence microscope can offer improved image acquisition time with equal lateral resolution, but with degraded longitudinal resolution when compared to confocal scanning optical microscopy (CSOM). For the photobleaching investigation, the parameter of concern is the fluence received by the specimen during excitation, which takes into account both the irradiance level and the time of illumination. Both peak and average fluence levels are addressed in the comparison. The analysis shows that during a 3D image acquisition, the OSH system will deliver lower peak fluence but higher average fluence levels to the specimen when compared to CSOM. / Ph. D.

The photothermal effect is a way in which chemical contrast can be measured as an optical pathlength or phase change. When a chemical species in a sample absorbs optical energy at a particular wavelength, this absorption raises the temperature at these points in the sample via the photothermal effect. This temperature change changes the local refractive index in the sample. Quantitative phase imaging is an interferometric technique for measuring the optical pathlength of sample features. Quantitative phase imaging is capable of detecting the photothermally-induced refractive index change, and is thus a powerful method for performing photothermal imaging. In this work, a thermal wave model is derived from Fourier's law of conduction in conjunction with a medium's heat capacity to derive the diffusion of temperature in a medium. This diffusion theory is transformed to a thermal wave model by applying a temporally modulated thermal source. Analytical expressions for the temperature field surrounding such a modulated thermal source are derived in multiple dimensions. The thermal wave equation is also simulated using a custom finite difference numerical method, and the simulated results are compared to the theoretical expressions with good agreement. The experimental apparatus for inducing such a thermal point source in a medium of water is described using the quantitative phase imaging system of spectral modulation interferometry. The spectral modulation interferometry system is aligned with a visible light pumping laser in two configurations for point source measurement and cell imaging. Label-free chemical imaging is then performed by pumping a field of cellular samples with wide-field illumination, and the resulting photothermal signal is detected by temporal analysis of the optical pathlength changes, generating the two-dimensional photothermal image. The measured photothermal cell image is qualitatively compared to predicted photothermal image based on the application of the thermal wave model in the spatial frequency domain. The chemical specificity of this technique is also verified by simultaneously pumping absorbing and non-absorbing biological cells in the same field-of-view. / Generating image contrast is a fundamental challenge in optical microscopy. Samples of interest in optical microscopy typically do not have visible absorption contrast without modification. A method of contrast that could provide information about a sample's absorption at different optical wavelengths would be useful for characterizing a sample's chemical content. The photothermal effect is an effect in which the small absorption of light by microscopic samples can be detected as a temperature change. With quantitative phase imaging, this temperature change can be measured by detecting the change in optical density of a sample due to its increase in temperature. Thus, quantitative phase imaging can be used to detect the small absorption of light by microscopic samples and generate two-dimensional images with chemical contrast. This work describes the theory of how thermal energy produced by optical absorption diffuses through a sample immersed in water. A thermal wave model is derived theoretically and compared to a custom simulation of the thermal wave physics with strong agreement. This thermal theory is verified with the quantitative phase imaging system used in this work to characterize the photothermal imaging technique. The photothermal imaging method is then applied to cellular samples, which are pumped with green light. The photothermal image is then generated and compared qualitatively to the image predicted by the thermal theory. The chemical imaging ability of the technique is then demonstrated by simultaneous imaging of absorbing and non-absorbing cells.

Natural and synthetic SnO₂ samples were studied using scanning tunneling microscopy (STM). The SnO₂ surface flattens considerably following high temperature treatments up to 1500 K. The conductivity of the synthetic SnO₂ surface is significantly reduced following annealing at temperatures of approximately 1200-1500 K, making tunneling impossible. A decrease in conductivity was not observed for the natural SnO₂ sample following similar high temperature treatments, most likely due to impurities which act as dopants. No atomic scale images were collected on the SnO₂ surface which provided information regarding atomic positions and point defects on the surface. Water adsorption was studied on the stoichiometric Cr₂O₃ (101̲2) surface, using thermal desorption spectroscopy (TDS). Water was the only desorption product observed during TDS. Adsorption is primarily dissociative following exposure to water at 163 K. Approximately, 0.12 monolayers of water dissociate on the clean, nearly stoichiometric Cr₂O₃ (101̲2) surface. The first order kinetics observed for the recombination of dissociated water are not well understood. One possible explanation is that the rate limiting step for desorption involves the breaking of a Cr-O bond resulting in a freely diffusing OH species. The exchange of halogen and oxygen was studied on Cr₂O₃ (101̲2) using Auger electron spectroscopy (AES) and TDS. The exchange of chlorine and oxygen is completely reversible. Chlorine is removed from the Cr₂O₃ (101̲2) surface following exposure to oxygen. Exposure of CFCl₂CH₂Cl reduces the surface oxygen concentration to that of the clean, nearly stoichiometric Cr₂O₃ (101̲2) surface. The exchange of chlorine with oxygen appears to involve only chemisorbed surface oxygen, not bulk lattice oxygen. / Master of Science

This study uses in situ atomic force microscopy (AFM) to directly observe the atomic scale effects of Sr on the monomolecular layer growth of abiotic calcite. These insights are coupled with quantitative measurements of the kinetics and thermodynamics of growth to determine the direction-specific effects of Sr on the positive and negative surface coordination environments that characterize calcite step edges. Low concentrations of strontium enhance calcite growth rate through changes in kinetics. A new conceptual model is introduced to explain this behavior. Higher concentrations of strontium inhibit and ultimately stop calcite growth by a step blocking mechanism. The critical supersaturation required to initiate growth (sigma*) increases with increasing levels of strontium. At higher supersaturations, strontium causes growth rates to increase to levels greater than those for the pure system. The step blocking model proposed by Cabrera and Vermilyea in 1958 does not predict the experimental data reported in this study because the dependence of sigma* upon strontium concentration is not the same for all supersaturations. Strontium inhibits calcite growth by different mechanisms for positive and negative step directions. Preliminary evidence indicates that strontium is preferentially incorporated into the positive step directions suggesting that impurity concentrations are not homogeneous throughout the crystal structure. Despite geochemical similarities, this study demonstrates that strontium and magnesium have different surface interaction mechanisms. The findings of this study demonstrate the importance of understanding microscopic processes and the significance of interpreting biominerals trace element signatures in the context of direction-specific interactions. / Master of Science

The ASTM C457 test has long been a standard used to obtain the air void parameters of concrete materials. These air void parameters provide valuable information that has been linked to the performance of concrete under conditions such as freezing and thawing cycles. The standard test procedure involves linearly traversing a cut and polished section of a concrete specimen while a technician observes it under a microscope. Chord lengths of material constituents that the technician observes along the linear traverse are recorded and later used to calculate air void parameters statistically. This procedure is long and tedious, which makes it susceptible to human error due to operator fatigue. This study proposes and implements a new test method for evaluating concrete air void parameters using an image analysis method. A polishing procedure along with a differential interference contrast microscope are used to obtain high contrast images of material constituents, which provide raw data for the image analysis method. Because of the high contrast that can be obtained, cement paste, air voids in the cement paste, and aggregate materials in the concrete can be distinguished from one another based on these images. An image analysis program has been written for this study which linearly traverses these images and records the chord lengths of material constituents in a similar way to the standard ASTM C457 test. The chord length data must be processed further, however, because features in the images can be truncated by the edge of the image. Correction calculations for this problem are implemented in the image analysis algorithm. Two specimens which have been previously tested using the standard ASTM C457 method by the Virginia Transportation Research Council, (VTRC), are used in this study. The air void parameters obtained using the new test are compared directly with the results obtained by VTRC for the two specimens. Statistical comparisons indicate that the results of the new test are indeed significant, showing the potential it has for practical implementation. There are drawbacks to the test including a long polishing procedure, but this process can be automated. The new test appears to have excellent potential for practical application, but it should be emphasized that the test has only been implemented using materials in two concrete specimens. Further study on a variety of other concrete materials would be required for implementation in a standard procedure. / Master of Science

Rotavirus (RV) is a viral pathogen that causes severe, watery diarrhea and vomiting in the young of humans and other animals. RV infections result in over 200,000 pediatric deaths around the world each year, especially in developing nations. Within the infected host cell, RV forms inclusion bodies, called viroplasms, where many stages of viral replication occur. The RV polymerase, known as VP1, must localize to viroplasms during infection where it replicates the virus' RNA genome. The work described in this dissertation focused on identifying region(s) of VP1 essential for its viroplasmic localization and its function as a polymerase. We found that a single amino acid change in a region of the polymerase called the N-terminal domain negatively impacted its capacity to localize to viroplasms during infection as well as its enzymatic activity in a test tube. Follow up studies using VP1 proteins from divergent strains and a mutant containing only the N-terminal domain of VP1 provided more insight into polymerase localization determinants. In total, our work suggests that the VP1 N-terminal domain plays an important role in localizing the polymerase to viroplasms via interactions with other viral proteins and supporting its function as a polymerase. / Ph. D. / Rotavirus (RV) is a viral pathogen that causes severe, watery diarrhea and vomiting in the young of humans and other animals. RV infections result in over 200,000 pediatric deaths around the world each year, especially in developing nations. Within the infected host cell, RV forms inclusion bodies, called viroplasms, where many stages of viral replication occur. The RV polymerase, known as VP1, must localize to viroplasms during infection where it replicates the virus’ RNA genome. The work described in this dissertation focused on identifying region(s) of VP1 essential for its viroplasmic localization and its function as a polymerase. We found that a single amino acid change in a region of the polymerase called the N-terminal domain negatively impacted its capacity to localize to viroplasms during infection as well as its enzymatic activity in a test tube. Follow up studies using VP1 proteins from divergent strains and a mutant containing only the N-terminal domain of VP1 provided more insight into polymerase localization determinants. In total, our work suggests that the VP1 N-terminal domain plays an important role in localizing the polymerase to viroplasms via interactions with other viral proteins and supporting its function as a polymerase.

The relationships between molecular parameters and microdomain formation of a variety of block- and graft-copolymers were studied by Transmission Electron Microscopy (TEM). Molecular variables included chemical composition: dimethyl-, fluoropropyl and diphenyl-siloxane , sulfone styrene, paramethylstyrene, t-butylstyrene, arylester and methyl methacrylate, as well as molecular weight and distribution. Effects of the kinetics of phase-separation were also determined . Thick (approximately lmm) films cast from solvent showed more complete phase separation than either thin (about 10nm) cast films or compression-molded specimens. Spherical domains formed in alternating poly(ester/siloxanes), and phase mixing seemed to correlate with the solubility parameters of the three siloxane types. Shear-stresses during molding changed domain shapes and eliminated short-range ordering. In the PMMA-graft-dimethyl siloxane system, SK, 10K and 20K <M_n> siloxanes were incorporated at 16% and 45% by weight. At 16%, spherical siloxane domains formed in both thick- and thin-cast films. The domain sizes and interdomain distances scaled with siloxane molecular weight and total block molecular weight respectively to a 2/3 power law in excellent agreement with theoretical predictions for di- and triblock copolymers. Thin films cast from the 45% siloxane graft copolymers also showed spherical domains with sizes dependent on molecular weight. However, the thick films showed phase transitions from disordered bicontinuous (M_n = 5K) to lamellar (M_n = 10K) to cylindrical <M_n = 20K). Qualitative TEM/EDX analysis of other systems was used to identify oligomers, homopolymers, and contaminants, thus monitoring the effects of novel reaction conditions and work-up procedures. / Master of Science

Twins are a pair of adjoining crystal grains related to each other by a special symmetry. They are frequently observed in bulk materials and nanomaterials. The formation of twins is an important topic in materials science and engineering because it affects material behaviors such as plastic deformation of metals, yield strength, and band gap energy in nanoscale semiconductors. Because of these unique phenomena and properties that the twinning can bring to the materials, it is of interest to investigate the formation of twins. Our primary objective in this dissertation is to study twinning in nanowires. Both gold and platinum <111> oriented nanowires were fabricated by similar solution-phase chemical synthesis methods. High-resolution transmission electron microscopy and electron diffraction patterns were carried out to analyze the structures of the nanowires. Nanodiffraction was used to demonstrate twinning is a general structural feature of the growth of gold nanowires growing in a <111> direction.  A model was proposed to explain the conditions under which twinning is energetically favored during nanowire growth. The model, which is based on a maximum rate hypothesis, considers the nanowire geometry and the relative surface and stacking fault energies and predicts twins should appear in gold nanowires but not in platinum nanowires, in agreement with experimental observations. During the structural characterization of gold nanowires, our interest is to resolve 3D structure of twinning. However, the structure of twinning in gold nanowires is very fine and the average spacing between twin boundaries is only 0.57nm (+/- 0.38 nm); therefore, regular 3D electron microscopy technique is unable to reconstruct these defected structures. Here we present a stereo vision technique to reconstruct 3D atomic non-periodic structures containing defects. The technique employs intrinsic atomic planes as epipolar planes to achieve the alignment accuracy needed to reconstruct a crystal with atomic resolution. We apply it to determine the 3D geometry and atomic arrangements of twinning in gold nanowire. In addition, an iterated cross-correlation algorithm was developed to analyze electron diffraction fully automatically to facilitate structural analysis of nanowires. A time-temperature-transformation diagram of platinum nanowires in chemical synthesis was determined to help optimize the fabrication process of the nanowires. / Ph. D.

Actin-based cell motility plays crucial roles throughout the lifetime of an organism. The dynamic rearrangement of the actin cytoskeleton triggers a plethora of cellular processes including cellular migration. Neural Wiskott Aldrich syndrome protein (N-WASP) is involved in transduction of signals from receptors on the cell surface to the actin cytoskeleton. N-WASP activated actin polymerization drives extension of invadopodia and podosomes into the basement layer. In addition to activating Arp2/3 complex, N-WASP binds actin filament barbed ends, and both N-WASP and barbed ends are tightly clustered in these invasive structures. We used nanofibers coated with N-WASP WWCA domains as model cell surfaces and single actin filament imaging to determine how clustered N-WASP affects Arp2/3-independent barbed end assembly. Individual barbed ends captured by WWCA domains of N-WASP grew at or below their diffusion limited assembly rate. At high filament densities, overlapping filaments formed buckles between their nanofiber tethers and myosin attachment points. These buckles grew 3.4-fold faster than the diffusion-limited rate of unattached barbed ends. N-WASP constructs with and without the native poly-proline (PP) region showed similar rate enhancements. Increasing polycationic Mg2+ or Spermine to enhance filament bundling increased the frequency of filament buckle formation, consistent with a requirement of accelerated assembly on barbed end bundling. Our preliminary data shows that tethered N-WASP construct containing one WH2 domain does not generate processive bundles or filament loops leading us to believe that tandem WH2 is required for processivity. We propose that this novel N-WASP assembly activity provides an Arp2/3-independent force that drives nascent filament bundles into the basement layer during cell invasion. Discovery of this bundle mediated unique pathway involved in invasion and metastasis will provide new targets for therapeutic development. / Ph. D.