Browsing by Subject "fluorescence"
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Item A Rationally-designed Fluorescence Competitive Binding Assay for Continuous Glucose Monitoring Applications(2014-03-18) Cummins, Brian MichaelImproved continuous glucose monitoring devices have the potential to increase patient compliance and improve the management of diabetes. One type of sensing chemistry that has been recognized as having significant amount of potential is based on the protein, Concanavalin A. However, to date, this assay has continually shown problems with sensitivity, stability, and reversibility in free solution. This work uses rational design to generate a new version of the competitive binding assay that can allow for its full potential to be shown. The first part of this work uses a glycosylated dendrimer as the competing ligand in the assay. This assay is optimized with regards to its fluorescence response and encapsulated within microporated microspheres to allow for the glucose-dependent aggregative response to occur. The exact mechanism of this assay was explored, and the stability issues were identified to be a direct result of the electrostatic interactions and multivalent presentation of sugar residues. The second part of this work generated a model for the unique recognition and transduction mechanisms of the ConA-based assay, in an attempt to generate maps to identify desirable qualities to generate an optimized assay. This model was validated with experimental data, and used to optimize a ConA-based assay to track glucose concentrations with anisotropy. They were also used to explain the problems with previous ConA-based approaches. The core trimannose of N-linked glycans was recognized as a high-affinity ligand that achieved the required affinities without leading to the aggregation that has caused previous assays to fail. A smartly-designed fluorescent ligand was generated based on this core trimannose to achieve the desired qualities as defined by the previous models, and it was used in a ConA-based assay to track glucose concentrations with anisotropy and F?rster Resonance Energy Transfer (FRET). Finally, in an attempt to generate a cost-effective smartly-designed fluorescent ligand that could be encapsulated with a size exclusion membrane, the glycated fraction of ovalbumin was fluorescently labeled and used in a ConA-based assay. This work proves that the smartly-designed fluorescent ligand concept can overcome the problems of previous ConA-based assays, and such an assay is expected to be capable of translation to practical applications.Item Advanced Laser Diagnostics Development for the Characterization of Gaseous High Speed Flows(2012-07-16) Sanchez-Gonzalez, RodrigoThe study of high-speed flows represents a challenging problem in the fluid dynamics field due to the presence of chemical reactions and non-equilibrium effects. Hypersonic flights, where speeds reach Mach 5 and above, are particularly influenced by these effects, resulting in a direct impact on the flow and consequently on the aerodynamic performance of a vehicle traveling at these speeds. The study of hypersonic flow conditions requires the experimental capability of determining local temperatures, pressures and velocities using non-intrusive techniques. Furthermore, the simultaneous measurement of two or more variables in a complex flow boosts the amount of information that is obtained since valuable correlations can be established. This research includes the design, construction and characterization of a hypersonic flow apparatus explicitly intended as a tool for advanced laser diagnostics development. This apparatus is characterized by its pulsed operation mode that translates into a significant reduction in mass flow rates and can be operated for long periods at Mach numbers ranging from 2.8 to 6.2. The flow conditions during the uniform flow time interval of each pulse vary by less than 1%, generating a flow of sufficient quality for quantitative measurements. The development of a laser diagnostic technique, the VENOM technique, which is a non-intrusive method to provide simultaneous 2-D measurements of the mean and instantaneous fluctuations in two-component velocity and temperature is also presented. This technique represents the first single diagnostic capable of instantaneous two-component velocimetry and thermometry in a gaseous flow field by combining two Nitric Oxide Planar Laser Induced Fluorescence methods: two-component Molecular Tagging Velocimetry and two-line thermometry, employing the nascent NO(v"=1) arising from the NO2 photodissociation as a molecular tracer. The VENOM technique is expected to be not only applicable to cold high-speed flows, which is the focus of the present work, but also to combustion and other reactive or high-enthalpy flow fields.Item Assaying protein import into mitochondria using fluorescence spectroscopy(Texas A&M University, 2006-08-16) Cargill, Holly BethMost proteins residing in the mitochondrial matrix are synthesized in the cytosol and post-translationally imported into the mitochondrial matrix. The matrix-targeted preproteins traverse the outer mitochondrial membrane (OM) via the Translocase of the Outer Membrane (TOM) complex, and the inner mitochondrial membrane (IM) via the Translocase of the Inner Membrane 23 (TIM23) complex. A novel system was set up to examine the import of matrix-targeted preproteins into mitochondria using fluorescence spectroscopy. The fluorescent probe 6-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminohexanoic acid (NBD) was site-specifically incorporated into different positions along the model matrix protein Su9-DHFR. The fluorescent-labeled polypeptides were either fully imported into isolated mitochondria or were arrested along the translocation pathway by the binding of methotrexate (MTX) to the DHFR moiety, creating NBDSu9- DHFR??MTX import intermediates. The NBD-Su9-DHFR polypeptides were able to be fully imported into the mitochondrial matrix in the absence of MTX, and were inaccessible to externally-added iodide ion quenchers. Treatment of the mitochondria with the pore-forming antibiotic alamethicin allowed the iodide ion quenchers access to the matrix through pores in the inner membrane (IM). After Alamethicin treatment the fully-imported NBD-Su9-DHFR polypeptides were accessible to the externally-added iodide ions. The extent of collisional quenching of the NBD fluorophores by the iodide ions was measured as the Stern-Volmer quenching constant, Ksv. Ksv values were obtained for the NBD-Su9-DHFR polypeptides in the presence of MTX (import intermediates) or in the absence of MTX (fully-imported). The Ksv values for NBD-Su9- DHFR import intermediates were similar, despite the location of the NBD probe along the translocation pathway. These Ksv values were similar to those obtained for the fullyimported NBD-Su9-DHFR polypeptides (-MTX). The locations of the varying probe positions along the import pathway were addressed using chemical crosslinking of Su9- DHFR Cys mutants. The use of fluorescence spectroscopy, in association with chemical crosslinking, to analyze the mitochondrial protein import pathways will prove a useful tool to probe the environment of the nascent chain as it is crossing the import pathway (the TOM, TIM23 complexes).Item Cancer diagnostics using dynamic near-infrared optical imaging and fluorescent contrast agents(Texas A&M University, 2006-04-12) Gurfinkel, MikhailA new optical imaging modality has been developed for small animal in vivo imaging of near-infrared fluorescence resulting from fluorescent contrast agents specifically targeted to molecular markers of cancer. The imaging system is comprised of an intensified charge-coupled device (ICCD) for the detection of ultra-low levels of re-emitted fluorescence following the delivery of an expanded beam of excitation light. The design of the ICCD detection system allows for both continuous wave (CW) and frequency-domain modes of operation. Since the accurate acquisition of frequency-domain photon migration (FDPM) data is important for tomographic imaging, the imaging system was also validated using experimentally obtained FDPM measurements of homogenous turbid media and diffusion theory to obtain estimates of the optical properties characteristic of the media. The experiments demonstrated that the absorption and reduced scattering coefficients are determined least accurately when relative rel measurements of average light intensity IDC are employed either alone or in a rel combination with relative modulation amplitude data IAC and/or relative phase shift data rel . However, when FDPM measurements of are employed either alone or in rel combination with IAC data, the absorption and reduced scattering coefficients may be found accurate to within 15% and 11%, respectively, of the values obtained from standard single-pixel measurements; a result that suggests that FDPM data obtained from an ICCD detection system may in fact be useful in tomographic imaging. Furthermore, intensified-detection allows for sub-second exposure times, permitting the acquisition of dynamic fluorescence images immediately following administration of the contrast agent. Experimental results demonstrate that when coupled with a suitable pharmacokinetic model describing targeted dye distribution throughout the body, dynamic fluorescence imaging may be used to discriminate spontaneous canine adenocarcinoma from normal mammary tissue. A separate experiment demonstrates that pharmacokinetic analysis of dynamic fluorescence images enables one to estimate the rate constant governing Kaposi's sarcoma tumor uptake of an integrin-targeted dye and integrin receptor turnover rate. The rate constant for uptake was calculated to be 0.16-sec-1 while the turnover rate of the integrin receptor was estimated to occur within 24-hours.Item Design and Verification of an Optical System to Interrogate Dermally-implanted Microparticle Sensors(2012-07-16) Long, RuiqiDiabetes mellitus affects 25.8 million Americans (8.3%) and over 300 million people worldwide. Clinical trials indicate that proper management of blood glucose levels is critical in preventing or delaying complications associated with diabetes. Thus, there is a common need to monitor and manage blood glucose properly for people with diabetes. However, the patients? compliance for recommended monitoring frequency is low due to the pain and inconvenience of current standard finger-pricking tests. To promote patient adherence to the recommended self-monitoring frequency, non-invasive/ minimally invasive glucose testing approaches are needed. Luminescent microparticle sensor is an attractive solution. For these sensors to be deployed in vivo, a matched optical system is needed to interrogate dermally-implanted sensors. This research project investigated the light propagation in skin and the interaction with implants using Monte Carlo modeling. The results of the modeling were used to design an optical system with high interrogation and collection efficiency (40~300 times improvement). The optical system was then constructed and evaluated experimentally. A stable skin phantom mimicking the optical properties of human skin was developed as a permanent evaluation medium to minimize the use of animals. The optical properties of the skin phantom matched the maximum published values of human skin in scattering and absorption over the spectral range of 540~700nm in order to avoid overestimation of the capability of the system. The significant photon loss observed at the connection between the designed system and a commercial spectrometer was overcome using two optimized designs: a two-detector system and a customized low-resolution spectrometer system. Both optimization approaches effectively address the photon loss problem and each showed good SNR (>100) while maintaining a sufficient system resolution for use with fluorescent materials. Both systems are suitable for luminescence measurement, because broad bands of the luminescent spectrum are of interest. In the future, either system can be easily modified into a more compact system (e.g. handheld), and it can be directly coupled to an analog-to-digital converter and integrated circuits offering potential for a single compact and portable device for field use with luminescent diagnostic systems as well as implanted sensors.Item Design of novel dyes towards the near-infrared(2009-05-15) Loudet, AuroreA series of seven functionalized near-infrared aza-BODIPY dyes have been synthesized and their spectroscopic properties measured. Their fluorescence emissions could be tuned by altering the electronic substituents on the aryl-groups. A through-bond energy transfer cassette featuring two fluorescein units as donor, and an aza-BODIPY dye as acceptor, was then synthesized and its preliminary spectroscopic properties examined. This cassette exhibited absorption and fluorescence characteristics that were highly dependent on the pH and the solvent polarity. Furthermore, no energy transfer was observed upon excitation of the donor. Novel near-infrared aza-BODIPY were also synthesized via a one-pot, two step reaction. Upon demethylation and intermolecular cyclization onto the B-atom, a ~ 100 nm red?shift of both the absorption and fluorescence emission maxima could be observed. Through-bond energy transfer cassettes based on squaraines have also been synthesized and their spectroscopic properties studied. These cassettes exhibited fast and efficient energy transfer from the donor to the acceptor. In depth experiments have also been realized to correlate the rate of energy transfer and structure on 3 different sets of through-bond energy transfer cassettes. No correlations could be made between the rate of the energy transfer and the nature of the acceptor, and the distance between the donor and acceptor. Finally, the use of DPP (diketo-pyrrolopyrolle) pigment as a potential donor for through-bond energy transfer cassettes was investigated. Three water-soluble DPPs dyes were prepared and studied. They all displayed weak fluorescence in water.Item Dissolved organic matter discharge in the six largest arctic rivers-chemical composition and seasonal variability(2009-05-15) Rinehart, Amanda J.The vulnerability of the Arctic to climate change has been realized due to disproportionately large increases in surface air temperatures which are not uniformly distributed over the seasonal cycle. Effects of this temperature shift are widespread in the Arctic but likely include changes to the hydrological cycle and permafrost thaw, which have implications for the mobilization of organic carbon into rivers. The focus of this research was to describe the seasonal variability of the chemical composition of dissolved organic matter (DOM) in the six largest Arctic rivers (Yukon, Mackenzie, Ob, Yenisei, Lena and Kolyma) using optical properties (UV-Vis Absorbance and Fluorescence) and lignin phenol analysis. We also investigated differences between rivers and how watershed characteristics influence DOM composition. Dissolved organic carbon (DOC) concentrations followed the hydrograph with highest concentrations measured during peak river flow. The chemical composition of peak-flow DOM indicates a dominance of freshly leached material with elevated aromaticity, larger molecular weight, and elevated lignin yields relative to base-flow DOM. During peak flow, soils in the watershed are still frozen and snowmelt water follows a lateral flow path to the river channels. As the soils thaw, surface water penetrates deeper into the soil horizons leading to lower DOC concentrations and likely altered composition of DOM due to sorption and microbial degradation processes. The six rivers studied here shared a similar seasonal pattern and chemical composition. There were, however, large differences between rivers in terms of total carbon discharge reflecting the differences in watershed characteristics such as climate, catchment size, river discharge, soil types, and permafrost distribution. The large rivers (Lena, Yenisei), with a greater proportion of permafrost, exported the greatest amount of carbon. The Kolyma and Mackenzie exported the smallest amount of carbon annually, however, the discharge weighted mean DOC concentration was almost 2-fold higher in the Kolyma, again, indicating the importance of continuous permafrost. The quality and quantity of DOM mobilized into Arctic rivers appears to depend on the relative importance of surface run-off and extent of soil percolation. The relative importance of these is ultimately determined by watershed characteristics.Item Dissolved oxygen and pH monitoring within cell culture media using a hydrogel microarray sensor(2009-05-15) Lee, Seung JoonProlonged exposure of humans and experimental animals to microgravity is known to be associated with a variety of physiological and cellular disturbances. With advancements in aerospace technology and prolonged space flights, both organism and cellular level understanding of the effects of microgravity on cells will become increasingly important in order to ensure the safety of prolonged space travel. To understand these effects at the cellular level, on-line sensor technology for the measurement and control of cell culture processes is required. To do this measurement, multiple sensors must be implemented to monitor various parameters of the cell culture medium. The model analytes used in this study were pH and dissolved oxygen which have physiological importance in a bioreactor environment. In most bioprocesses, pH and dissolved oxygen need to be monitored and controlled to maintain ionic strength and avoid hypoxia or hyperoxia. Current techniques used to monitor the value of these parameters within cell culture media are invasive and cannot be used to make on-line measurements in a closed-loop system. In this research, a microfabricated hydrogel microarray sensor was developed to monitor each anlyte. Either a pH or an oxygen sensitive fluorescent agent was immobilized into a hydrogel structure via a soft lithography technique and the intensity image of the sensor varied from the target analyte concentration. A compact detection system was developed to quantify concentration of each analyte based on the fluorescence image of the sensor. The system included a blue LED as an illumination source, coupling optics, interference filters and a compact moisture resistant CCD camera. Various tests were performed for the sensor (sensitivity, reversibility, and temporal/spatial uniformity) and the detection system (temporal/spatial stability for the light source and the detector). The detection system and the sensor were tested with a buffer solution and cell culture media off-line. The standard error of prediction for oxygen and pH detection was 0.7% and 0.1, respectively, and comparable to that of commercial probes, well within the range necessary for cell culture monitoring. Lastly, the system was coupled to a bioreactor and tested over 2 weeks. The sensitivity and stability of the system was affordable to monitor pH and dissolved oxygen and shows potential to be used for monitoring those analytes in cell culture media noninvasively.Item Evaluation of chlorophyll fluorescence as a tool for the identification of drought tolerance in upland cotton(2009-05-15) Longenberger, Polly SuzanneA novel bioassay for the evaluation of plant water status was developed by Burke (2007). The research reported herein was designed to evaluate this new protocol as a tool for use in cotton breeding programs for the identification of drought tolerant genotypes. Twenty genotypes were selected to represent diverse germplasm pools for a two-year field evaluation. Replicated tests were performed in Lubbock, TX and College Station, TX in 2005, 2006, and 2007. Dryland and irrigated treatments were administered in a split plot arrangement of a randomized complete block design. Fluorescence measurements were taken at mid-bloom and late bloom growth stages of growth. Source leaf tissue was harvested at predawn and subjected to high temperature incubation with fluorescence measurements subsequently taken hourly for five hours. Drought stressed plants had not mobilized their carbohydrate reserves from their source leaves overnight and thus maintained cell viability and therefore higher chlorophyll fluorescence values throughout the incubation with the opposite being true for nonstressed plants. Fiber lint yield and fiber properties were measured at the conclusion of the 2005 season in College Station and the 2006 season in College Station and Lubbock for comparison with the fluorescence data. Five genotypes, ?Acala 1517-99?, ?Deltapine 491? (PVP no. 200100159), ?Tamcot CAMD-E?, ?Tamcot 22? and TAM 89E-51, an unreleased breeding line, were selected based on field evaluation results in a preliminary study in 2005 to be included in a diallel analysis to determine the heritability of fluorescence measurements. Genotype x treatment effects complicated the classification of genotypic responses to drought. Few and inconsistent correlations were found among fluorescence values and lint yield or fiber properties. The diallel analysis did not identify general combining ability or specific combining ability effects for chlorophyll fluorescence measurements. Thus this procedure provides little potential in selecting plants for drought tolerance when plants are grown under field culture. Selection among Tamcot 22 and TAM 89E-51 plants for high and low genotypes according to fluorescence values did not yield progeny different from unselected Tamcot 22 and TAM 89E-51.Item Improving Fluorescence Lifetime Imaging Microscopy Deconvolution Using Constrained Laguerre Basis Functions(2014-04-25) Khatkhatay, Mohammed MFluorescence lifetime imaging microscopy (FLIM) is a noninvasive invasive optical imaging modality which is finding new applications in medical imaging. In FLIM, the fluorescence time decay is measured at a pixel. The fluorescence impulse response function (IRF) is then estimated using a deconvolution of the instrument response and the measured fluorescence time decay. Two of the challenges facing FLIM are speed of the deconvolution and the accuracy of the IRFs. The linear expansion of the fluorescence decays based on the orthonormal Laguerre basis functions (LBFs) is among the fastest methods for estimating the IRFs. The automated implementation to optimize the Laguerre parameter improves the speed of the deconvolution using the LBFs but uses a global optimization. Therefore, the IRFs do not necessarily mimic exponential time decays, or monotonically decreasing functions. On the other hand, applying a constraint to the LBFs using the Active Set Nonnegative Least Squares (NNLS) method improves the IRF estimation. The estimation of the Laguerre parameter using the NNLS method, however, is about 10-15x slower. By combining these two deconvolution techniques, we found that the deconvolution time is similar to the automated global Laguerre parameter deconvolution while the IRF estimation always results in a monotonically decreasing function.Item Kinetics and dynamics study on the allosteric pathway of phosphofructokinase from Escherichia coli(Texas A&M University, 2008-10-10) Tie, CuijuanPhosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated by MgADP and phosphoenolpyruvate (PEP), which act to activate or inhibit, respectively, by changing the substrate (Fru-6-P) affinity of the enzyme. Both ligands bind to the same allosteric site in EcPFK. Therefore, the questions we want to address are how these two molecules regulate EcPFK and how the allosteric signal is propagated throughout the enzyme. EcPFK has 28 potential site-site interactions. These interactions in turn derive from multiple copies of 6 potentially unique homotropic interactions and 4 potentially unique heterotropic interactions. Making hybrid tetramer of EcPFK is used to isolate a single heterotropic interaction. To improve the yield of the 1:3 hybrid, the in vivo hybrid formation method was developed. Four heterotropic interactions were isolated by this manner and re-evaluated. The same kinetics characteristics were obtained for each 1:3 hybrid from both the in vivo and in vitro method. To address the question of how the allosteric signal is transmitted throughout EcPFK, we identified residues (G184, Asp59 and S157) that are important for the allosteric regulation for both PEP inhibition and MgADP activation. The impact of each mutation on individual interaction is unique and also suggests that the structural basis for PEP inhibition is different from that for MgADP activation. Most importantly, since the sum of each heterotropic interaction with a modification in only one subunit is equal to the total heterotropic interaction with a modification in all four subunits, this result indicates that the heterotropic allosteric signal transmission is realized in a single subunit. The 23? heterotropic interaction, which contributes the most to the PEP inhibition, was chosen to study the dynamic properties. Fluorescence was used to study the dynamic perturbations of the 23? interaction upon ligand binding. Taking advantage of the hybrid formation strategy and the tryptophan-shift mutagenesis method, a tryptophan residue can be placed at different individual locations throughout the native subunit containing the 23? heterotropic interaction. The steady-state anisotropy and lifetime measurement at each tryptophan position indicate that the 23? allosteric interaction involves the perturbation of side-chain dynamics both near and quite far away from the respective ligand binding sites.Item Probing the denatured state ensemble with fluorescence(Texas A&M University, 2004-09-30) Alston, Roy WillisTo understand protein stability and the mechanism of protein folding, it is essential that we gain a better understanding of the ensemble of conformations that make up the denatured state of a protein. The primary goal of the research described here was to see what we might learn about the denatured state using fluorescence. To this end, tryptophan was introduced at five sites in Ribonuclease Sa (RNase Sa): D1W, Y52W, Y55W, T76W, and Y81W. The fluorescent properties of the denatured states of these five proteins were studied and compared to the fluorescent properties of eight model compounds: N-acetyl-tryptophan-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and five pentapeptides based on the sequence around the original tryptophan substitutions in RNase Sa. Regardless of the denaturant, ?max for the proteins and model compounds differed very little, 349.3 ? 1.2 nm. However, significant differences were observed in the fluorescence intensity at ?max (IF), suggesting that IF is more sensitive to the immediate environment than ?max. The differences in IF are due in part to quenching by neighboring side chains. More importantly, IF was always significantly greater in the protein than in its corresponding pentapeptide, indicating that the protein exerts an effect on the tryptophan, which cannot be mimicked by the pentapeptide models. Acrylamide and iodide quenching experiments were also performed on the model compounds and proteins. Significant differences in the Stern-Volmer quenching constant (KSV) were also observed between the proteins and between the proteins and their corresponding pentapeptides. Importantly, the KSV for the protein was always less than in its corresponding pentapeptide. These data along with the IF data show that non-local structure in the unfolded state influences tryptophan fluorescence and accessibility. In summary, these and our other studies show that fluorescence can be used to gain a better understanding of the denatured states of proteins.Item Reflectance and Fluorescence Confocal Microscope for Imaging of the Mouse Colon(2012-02-14) Saldua, Meagan AlyssaMany Americans are afflicted with inflammation of the colon. They are also at a higher risk of developing colon cancer. Confocal microscopy of bulk epithelial tissue has the potential to provide information on tissue structural properties that may be lost in the fixation and slicing procedures required for histopathology. Optical sectioning provides images in three dimensions capturing the organizational structure of cells and colon crypts throughout the entire colon. I have constructed a custom built fluorescence and reflectance confocal microscope for imaging molecular and morphological changes associated with development of inflammation in a mouse model. A confocal microscope is a point scanning system that removes out of focus light by placing a pinhole aperture in the conjugate image plane located in front of the detector. We have two sources, 488 nm and 811 nm, for fluorescence and reflectance imaging, respectively. A polygon scanning mirror and a galvanometer scanning mirror allow for a variable scan rate between 8 and 15 fps. The lateral resolution of the system is approximately 3 ?m with an axial resolution of 6 ?m and 4 ?m for reflectance and fluorescence mode, respectively. As colon tissue becomes inflamed, there is a distinct change in the structure and architecture of the tissue. The colon crypts are no longer uniform in size or distribution throughout the tissue. Having a large field of view of 1mm2 allows for many colon crypts to be visualized within a single frame. Histology was performed on the same tissue imaged for the inflammatory study confirming the constructed confocal microscope?s ability to characterize inflamed tissue and the potential use for guided biopsy. Mosaicing, or image tiling, is an imaging technique that stitches single frames together to produce a much larger field of view. An extended frame with 1 mm x 2 cm field of view is achieved within seconds. This extended frame would allow mosaicing of the entire mouse colon much faster than conventional methods without loss of resolution. The acquired confocal images of colon tissue demonstrate the microscope?s ability to resolve cell nuclei lining the colon crypts within a relatively large field of view.Item Spectroscopic investigations of the vibrational potential energy surfaces in electronic ground and excited states(Texas A&M University, 2007-09-17) Yang, JuanThe vibrational potential energy surfaces in electronic ground and excited states of several ring molecules were investigated using several different spectroscopic methods, including far-infrared (IR), Raman, ultraviolet (UV) absorption, fluorescence excitation (FES), and single vibronic level fluorescence (SVLF) spectroscopies. Based on new information obtained from SVLF and millimeter wave spectra, the far-IR spectra of coumaran were reassigned and the one-dimensional ring-puckering potential energy functions for several vibrational states in the S0 ground state were determined. The barrier was found to be 154 cm-1 and the puckering angles to be ???? 25????, in good agreement with the millimeter wave barrier of 152 cm-1 and puckering angles of ???? 23????. Moreover, the UV absorption and FES spectra of coumaran allowed the one-dimensional ring-puckering potential energy functions in the S1 excited state to be determined. The puckering barrier is 34 cm-1 for the excited state and the puckering angles are ???? 14????. Several calculations with different basis sets have been carried out to better understand the unusual vibrational frequencies of cyclopropenone. It was shown that there is strong interaction between the C=O and symmetric C-C stretching vibrations. These results differ quantitatively from a previous normal coordinate calculation and interpretation. The vapor-phase Raman spectrum of 3,7-dioxabicyclo[3.3.0]oct-1,5-ene was analyzed and compared to the predicted spectrum from DFT calculations. The spectrum further shows it has D2h symmetry, in which the skeletons of both rings are planar. The infrared and Raman spectra of vapor-phase and liquid-phase 1,4-benzodioxan and 1,2,3,4-tetrahydronaphthalene were collected and the complete vibrational assignments for both molecules were made. Theoretical calculations predicted the barriers to planarity to be 4809 cm-1 for 1,2,3,4-tetrahydonaphthalene and 4095 cm-1 for 1,4-benzodioxan. The UV absorption, FES, and SVLF spectra of both molecules were recorded and assigned. Both one and two-dimensional potential energy functions of 1,4-benzodioxan for the ring-twisting and ring-bending vibrations were carried out for the S0 and S1(????,????*) states, and these were consistent with the high barriers calculated for both states. The low-frequency spectra of 1,2,3,4-tetrahydronaphthalene in both S0 and S1(????,????*) states were also analyzed.Item Studies in biological surface science: microfluidics, photopatterning and artificial bilayers(Texas A&M University, 2004-09-30) Holden, Matthew AlexanderHerein is presented the collective experimental record of research performed in the Laboratory for Biological Surface Science. These investigations are generally classified under the category of bioanalytical surface science and include the following projects. Chapters III and IV describe the creation of a microfluidic device capable of generating fixed arrays of concentration gradients. Experimental results were matched with computational fluid dynamics simulations to predict analyte distributions in these systems. Chapters V and VI demonstrate the discovery and utility of photobleaching fluorophores for micropatterning applications. Bleached fluorophores were found to rapidly attach to electron rich surfaces and this property was used to pattern enzymes inside microfluidic channels in situ. Finally, Chapter VII exhibits a method by which solid supported lipid bilayers can be dried and preserved by specifically bound proteins. The intrinsic property of lateral lipid mobility was maintained during this process and a mechanism by which the protein protects the bilayer was suggested.Item The subunit exchange rate of the cyanobacterial circadian clock component kaic is independent of phosphorylation state(2009-05-15) Ihms, Elihu CarlThe study of the in vitro circadian oscillator of the cyanobacterium Synechococcus elongatus has uncovered a complex interplay of its three protein components. Synchronization of the clock's central oscillatory component, KaiC, has been thought to be achieved through subunit shuffling at specific intervals during the clock?s period. By utilizing an established fluorescence-based analysis on completely phosphorylated and dephosphorylated mutants as well as wild-type KaiC, this study has shown that shuffling rates are largely unaffected by phosphorylation state. These findings conflict with previous reports and hence revise our understanding of this oscillator.Item Yield Optimization of Nitrogen Vacancy Centers in Diamond(2012-10-19) Chen, JesonTo fully exploit the capability of NV centers in diamond as magnetic sensors and quantum bits, the optimum production recipe as well as the method to enhance its optical performance has been studied in this work. The NV centers in bulk diamond were prepared by ion implantation and electron irradiation, and the optimum dose and temperature are found by comparing its optical and magnetic performance both experimentally and theoretically. In addition, the enhancement of optical performance and size characterization of NV centers in nanodiamonds will be discussed in this work.