Browsing by Subject "Fluorescence"
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Item Biochemical mechanisms for selectivity governing RNA and DNA replication(2021-04-07) Dangerfield, Tyler Lane; Johnson, Kenneth A (Kenneth Allen); Ellington, Andrew; Vasquez, Karen; Zhang, YanEnzymes that catalyze DNA and RNA replication are biologically important and provide examples to understand enzyme specificity as the correct substrate and alternate substrates are well known. The DNA polymerase from bacteriophage T7 has been a model system for understanding high-fidelity DNA replication, yet outstanding questions remain about the controversial nucleotide induced conformational change. I developed methods to directly measure the conformational change by site specifically incorporating a fluorescent unnatural amino acid into the enzyme at a position that gives a fluorescence signal upon nucleotide binding. In addition, I developed high throughput methods to analyze kinetic samples by capillary electrophoresis to assist in the kinetic analysis of DNA replication. Kinetic studies show that the conformational change is the primary determinant of specificity at the polymerase active site. I also characterized the enzyme’s 3’--5’ proofreading exonuclease activity and showed that the enzyme has multiple opportunities to correct mistakes during replication. I also used molecular dynamics simulation methods to propose a structure of the proofreading exonuclease editing complex. While finishing studies on the T7 DNA polymerase, the SARS-CoV-2 pandemic devastated the world. I used techniques developed in my previous studies to characterize the RNA dependent RNA polymerase (RdRp) from SARS-CoV-2, a prime target for antiviral drugs to stop replication of the virus. I developed methods to express the tag free complex in E. coli and subsequent purification to give highly active enzyme, which for the first time met standards for physiologically relevant activity. I characterized the pre-steady state kinetics of UTP and ATP incorporation and found that the enzyme catalyzes nucleotide incorporation at a fast rate, sufficient to replicate the 30 kb viral genome in less than 2 minutes. The ATP analog Remdesivir triphosphate, the only FDA approved treatment for COVID-19, was then studied to calculate the discrimination relative to ATP. We found that Remdesivir is incorporated more efficiently than ATP and acts as a delayed chain terminator, although this termination can be partially overcome by incubation with high nucleotide concentrations. Cryo-EM was used to determine the structure of the Remdesivir stalled RdRp complex and we found that the translocation step was inhibited after incorporation of 3 Remdesivirs due to a steric block between Remdesivir and the enzyme.Item Computational modeling of tumor cell growth as a function of nutrient dynamics guided by time-resolved microscopy(2021-12-03) Yang, Jianchen (Ph. D. in biomedical engineering); Yankeelov, Thomas E.; Brock, Amy; Dunn, Andrew K; Virostko, JackThe varying and extreme nutrient conditions found in the tumor microenvironment force reprogramming of metabolism in tumor cells. This metabolic reprogramming has been identified as a hallmark of cancer. This dissertation focuses on the development and validation of an experimental-mathematical approach that predicts how the dynamics of glucose and lactate influence tumor metabolism and development. Firstly, we developed a baseline model that predicts tumor cell growth as a function of glucose availability. We employed time-resolved microscopy to track the temporal change in the number of live and dead tumor cells under different initial conditions and seeding densities. A family of mathematical models that describes the overall tumor cell growth in response to the initial glucose and confluence was constructed. The most parsimonious model selected from the family using the Akaike Information Criteria was calibrated and validated in two breast cancer cell lines (BT-474 and MDA-MB-231) and demonstrated accuracy in predicting tumor growth. Secondly, we developed noninvasive imaging of nutrient dynamics with a stable transfection of two FRET reporters, one assaying glucose concentration and one assaying lactate concentration, in the MDA-MB-231 breast cancer cell line. The FRET ratio from both reporters was found to increase with increasing concentration of the corresponding ligand and decrease over time for high initial concentration of the ligand. Significant differences in the FRET ratio corresponding to metabolic inhibition were found when cells were treated with glucose/lactate transporter inhibitors. The FRET reporters enabled us to track intracellular glucose and lactate dynamics, providing insight into tumor metabolism and response to therapy over time. Finally, we compared mechanism-based and machine learning models for predicting tumor cells growth when we introduced an inhibitor of glucose uptake as a potential treatment. We extended the baseline model to account for glucose uptake inhibition, considering both the real glucose level in the system and the glucose level accessible to tumor cells. The random forest model provided the best prediction while the mechanism-based model presented a comparable predictive capability.Item Coral Fluorescent Proteins as Antioxidants(Public Library of Science, 2009-10-06) Palmer, Caroline V.; Modi, Chintan K.; Mydlarz, Laura D.Background -- A wide array of fluorescent proteins (FP) is present in anthozoans, although their biochemical characteristics and function in host tissue remain to be determined. Upregulation of FP's frequently occurs in injured or compromised coral tissue, suggesting a potential role of coral FPs in host stress responses. Methodology/Principal Findings -- The presence of FPs was determined and quantified for a subsample of seven healthy Caribbean coral species using spectral emission analysis of tissue extracts. FP concentration was correlated with the in vivo antioxidant potential of the tissue extracts by quantifying the hydrogen peroxide (H2O2) scavenging rates. FPs of the seven species varied in both type and abundance and demonstrated a positive correlation between H2O2 scavenging rate and FP concentration. To validate this data, the H2O2 scavenging rates of four pure scleractinian FPs, cyan (CFP), green (GFP), red (RFP) and chromoprotein (CP), and their mutant counterparts (without chromophores), were investigated. In vitro, each FP scavenged H2O2 with the most efficient being CP followed by equivalent activity of CFP and RFP. Scavenging was significantly higher in all mutant counterparts. Conclusions/Significance -- Both naturally occurring and pure coral FPs have significant H2O2 scavenging activity. The higher scavenging rate of RFP and the CP in vitro is consistent with observed increases of these specific FPs in areas of compromised coral tissue. However, the greater scavenging ability of the mutant counterparts suggests additional roles of scleractinian FPs, potentially pertaining to their color. This study documents H2O2 scavenging of scleractinian FPs, a novel biochemical characteristic, both in vivo across multiple species and in vitro with purified proteins. These data support a role for FPs in coral stress and immune responses and highlights the multi-functionality of these conspicuous proteins.Item Diversity and Evolution of Coral Fluorescent Proteins(Public Library of Science, 2008-07-16) Alieva, Naila O.; Konzen, Karen A.; Field, Steven F.; Meleshkevitch, Ella A.; Hunt, Marguerite E.; Beltran-Ramirez, Victor; Miller, David J.; Wiedenmann, Jörg; Salih, Anya; Matz, Mikhail V.GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a “chromo-red” color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.Item Exploration of the iron chelator deferasirox : a multifaceted platform for the development of antineoplastic agents and water-soluble sensors(2022-10-06) Steinbrueck, Axel; Sessler, Jonathan L.; Anslyn, Eric V.; Liu, Hung-Wen; Fast, Walter L.; Page, Zachariah A.Chelators have gained considerable interest due to the wide range of potential applications for this class of molecules, including as therapeutics and chemosensors. Over recent decades, a number of metal chelators have successfully demonstrated cytotoxicity and targeted activity against cancer in vitro, in vivo and in preliminary clinical trials. Considering these promises, during my dissertation I have explored the strategic derivatization of the clinical iron chelator deferasirox with focus on the preparation of chelators that show enhanced activity against cancer cells. In addition, I have investigated the optimization of deferasirox as chemosensor for Fe³⁺ in water samples. In chapter 1, an overview on the recent progress in the use of transition metal chelators, pro-chelators, and ionophores as potential cancer chemotherapeutics is provided. This chapter focuses on the reported agents that are able to coordinate iron, copper, and zinc. Chapter 2 describes the design, synthesis, and biological evaluation of strategically functionalized derivatives of the FDA approved iron chelator deferasirox. Preparative efforts focused on derivatives containing organelle-targeting moieties and their in vitro activity against A549 lung cancer cells is discussed. In contrast to the parent chelator deferasirox, several of the new derivatives could be traced inside cells through fluorescent cell imaging. One of the derivatives exerted improved antiproliferative activity relative to deferasirox and was revealed to preferentially localises within the lysosome. Chapter 3 discusses the evaluation of deferasirox as colorimetric chemosensor for the detection and quantification of Fe³⁺ in aqueous samples. The optimization of deferasirox and the water-soluble, non-toxic derivative ExSO₃H are discussed. In addition, ExSO₃H was identified as potential chelator for uranyl (UO₂²⁺) in water and the interaction between these two species was investigated. Chapter 4 discusses the observation that a fluorescent turn-on response was produced when a water-soluble deferasirox derivative was allowed to interact with human serum albumin (HSA). This fluorescence was quenched in the presence of Fe³⁺, thus permitting the monitoring of the presence of this biologically important metal cation with a protein-bound chelator.Item Fluorescent coatings for corrosion detection in steel and aluminum alloys(2010-05) Liu, Guangjuan; Wheat, Harovel Grays; Goodenough, John B.; Kovar, Desiderio; Taleff, Eric M.; Juenger, MariaCoatings are often used as a means of protecting aluminum alloy and steel structures in industry. The assessment of corrosion under these coatings can be challenging. Corrosion sensing coatings can exhibit properties that allow undercoating corrosion to be identified before it can be seen with the naked eye. This would be very advantageous and could potentially result in tremendous savings in time and money when structures undergo routine maintenance. Our work involved the study of corrosion sensing coatings with incorporated fluorescent indicators that can be used to sense the undercoating corrosion on metal substrates. The fluorescent indicator in the coated-aluminum system was a negative indicator, i.e. the indicator in the coating was initially fluorescent and subsequently non-fluorescent due to the reduced pH at the anodic sites of corrosion. The fluorescent indicator in coated-steel system was positive, in the sense that the coating changed from non-fluorescent to fluorescent over the cathodic areas due to increased pH. The corrosion sensing coating was composed of commercial epoxy-polyamide and the indicator: 7-amino-4-methylcoumarin (7-AMC) for the coated-aluminum alloy system and 7-diethylamino-4-methylcoumarin (7-DMC) for the coated-steel system. The feasibility of using 7-AMC for sensing early undercoating corrosion was demonstrated by using fluorescent observations, Electrochemical Impedance Spectroscopy (EIS), Scanning Electron Microscope (SEM) and Energy Dispersive Spectroscopy (EDS) tests. EIS results estimated that with continuous immersion the undercoating corrosion occurred within 24 hours after immersion in the salt solution. When corrosion occurred, the corrosion was invisible under natural light. However, small spots appeared in the fluorescent image, changing from initially fluorescent to non-fluorescent where the anodic sites were identified by SEM and EDS. In other words, the fluorescent indicator could sense the early undercoating corrosion, although blistering can be a competing mechanism associated with corrosion under some conditions. The sensitivity of the 7-AMC corrosion detection system was tested by applying anodic current to the metal and measuring the charge at which fluorescence quenching was detected. The critical charge for a detectable pit under the coating was approximately 2x10⁻⁵ C, which implied a critical radius of a single corrosion spot or set of spots of approximately 10 [mu]m. The fluorescent properties of 7-AMC, its effect on the protectiveness, its sensitivity to pH and its concentration in the coating are explored as well. Fourier transform spectroscopy (FTIR) was used to characterize the structure of the coating with and without 7-AMC. The results suggested that there is no structure change occurring after adding 7-AMC into the coating. Fluorescence behavior, electrochemical behavior, microscopic evidence, and visual observations of coated steel specimens with 7-DMC are compared based on exposure to saltwater conditions. Some of the challenges associated with the use of these types of coatings will be presented. This includes the interference from the increased production of ferrous and ferric ions. All of this information is aimed at the development of corrosion sensing coatings that can reveal undercoating corrosion before it is visible to the naked eye.Item A Green Fluorescent Protein with Photoswitchable Emission from the Deep Sea(Public Library of Science, 2008-11-19) Vogt, Alexander; D'Angelo, Cecilia; Oswald, Franz; Denzel, Andrea; Mazel, Charles H.; Matz, Mikhail V.; Ivanchenko, Sergey; Nienhaus, G. Ulrich; Wiedenmann, JörgA colorful variety of fluorescent proteins (FPs) from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP)-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that ~15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37°C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.Item Hybrid systems of plasmonic nanostructures and functional materials for light-matter interactions and active plasmonic devices(2018-08-15) Wang, Mingsong; Zheng, Yuebing; Ben-Yakar, Adela; Milliron, Delia; Li, WeiAdvances in nanofabrication and characterization of nanomaterials enable the development of plasmonic nanostructures with unique optical properties. Plasmonic nanostructures have been extensively studied for their potential applications in optical sensing, photothermal therapy, photovoltaics, and photocatalysis. In this dissertation, we present studies of light-matter interactions in hybrid systems consisting of plasmonic nanostructures and functional materials. These studies are focused on four major types of light-matter interactions in plasmonic nanostructures: (1) plasmon-induced resonance energy transfer (PIRET); (2) plasmon-enhanced spontaneous emission; (3) Fano interference between plasmonic nanostructures and emitters; and (4) strong plasmon-exciton coupling. We also achieved the tuning of light-matter interactions by modifying the physical properties of functional materials or plasmonic nanostructures. In addition, the active control of light-matter interactions was demonstrated by integrating plasmonic nanostructures with switchable materials, such as photochromic dyes. Specifically, we first demonstrated the blue-shifted PIRET from a single gold nanorod (AuNR) to dye molecules. AuNRs enable the energy transfer from plasmonic donors to dye acceptors with light having a longer wavelength and lower intensity, compared to dye donors. Secondly, we studied the tuning of plasmon-trion and plasmon-exciton resonance energy transfer from a single gold nanotriangle (AuNT) to monolayer MoS₂. We achieved these phenomena by the combination of rationally designed monolayer MoS₂-plasmonic nanoparticle hybrid systems and single-nanoparticle measurements. Thirdly, we realized the large modulation of hybrid plasmonic waveguide mode (HPWM) in single hybrid molecule-plasmon nanostructures through the strong molecule-plasmon coupling. The HPWM features both the capacity of plasmonic nanostructures to manipulate light at the nanoscale and the low loss of dielectric waveguides. Fourthly, we demonstrated the photoswitchable plasmon-induced fluorescence enhancement. This large switchable modulation of fluorescence was derived from the large near-field enhancement at the subnanometer gap between Au nanoparticles and switchable intersystem crossing as a nonradiative decay channel in photochromic dyes. Finally, we achieved tunable Fano resonances and plasmon-exciton coupling in two-dimensional (2D) WS₂-AuNT hybrid structures at room temperature. The tuning of Fano resonances and plasmon-exciton coupling were achieved by the active control of the WS₂ exciton binding energy and dipole-dipole interaction through controlling the dielectric constant of the surrounding medium.Item Localized surface plasmon resonance spectroscopy of gold and silver nanoparticles and plasmon enhanced fluorescence(2011-12) Vokac, Elizabeth Anne; Willets, Katherine A.; Brodbelt, Jennifer S.This thesis presents spectroscopic studies of metallic nanoparticle localized surface plasmons and plasmon enhanced fluorescence. We investigated the dielectric sensitivity of silver nanoprisms to an external electric field and gold nanorods to the formation of a self-assembled surface monolayer. Dark field microscopy was used to image plasmonic scattering from single nanoparticles, and a liquid crystal tunable filter was used to construct corresponding spectra. The plasmon resonances of silver nanoprisms displayed both reversible red shifts and irreversible blue shifts along with drastic intensity changes upon exposure to an applied bias. The plasmon resonances of gold nanorods showed sensitivity to the presence of alkanethiol molecules adhered to the particle surface by a moderate red shift. An increase in the effective external dielectric caused a shift toward longer wavelengths. We imaged plasmon enhanced fluorescence in order to optimize experimental parameters for a developing project that can characterize nanoparticle structure on sub-wavelength dimensions. Preliminary controls were performed to account for the effect of O₂ plasma treatment, solvent and alkanethiol monolayer formation on surface plasmon resonances. We found that O₂ plasma treatment for different time intervals did not result in a plasmon shift compared to untreated nanoparticles exposed to N₂; however when exposed to solvent the surface plasmons of the treated particles shifted five times as far toward the red. Interestingly, the solvent effect only resulted in a plasmon shift when the particles were N₂ dried after solvent incubation. Gold nanorods incubated in ethanol showed no wavelength maximum shift in pure solvent over time, but shifted moderately to the red after incubation in a solution of alkanethiol molecules. Conditions for the plasmon enhanced fluorescence study were optimized using a dye conjugate of the same alkanethiol molecule used previously by formation from solution in a monolayer on the gold nanorod surface. The appropriate synthesis for dye functionalization, molecular concentrations, solvents and optical settings were determined.Item Matrix metalloproteinase and carbonic anhydrase detection based on ¹⁹F MRI and fluorescence(2019-02-06) Beyer, Sophia Katherine; Que, EmilyZinc metalloproteins are heavily involved in a wide variety of human diseases,¹ such as rheumatoid arthritis, periodontitis, and cancer. Matrix metalloproteinase (MMP) and carbonic anhydrase (CA) are two zinc containing enzymes that are seen as attractive biomarkers for a multitude of pathological conditions, due to their overexpression in these diseases as well as their role in the spread and metastasis of cancer cells. Therefore, we focused on creating probes designed to track the location and relative concentration of matrix metalloproteinase and carbonic anhydrase through fluorescence and ¹⁹F MRI. In the first project, an inhibitor and a set of fluorophores were synthesized to bind to MMPs and display a ‘turn on’ signal. In the second project, a probe was designed to detect MMP activity through ¹⁹F MRI. Finally, in the third project, a probe was created to detect CA activity via ¹⁹F MRI. Here, we describe the synthesis and characterization of these probes and report preliminary UV and fluorescence response along with ¹⁹F NMR T₁ and T₂ relaxivity.Item Microfluidics for bioanalytical research : transitioning into point-of-care diagnostics(2014-12) Scida, Karen; Crooks, Richard M. (Richard McConnell)In this dissertation, three different microfluidic devices with bioanalytical applications are presented. From chapter to chapter, the bioanalytical focus will gradually become the development of a point-of-care sensor platform able to yield a reliable and quantitative response in the presence of the desired target. The first device consists of photolithographically-patterned gold on glass bipolar electrodes and PDMS Y-shaped microchannels for the controlled enrichment, separation from a mixture, and delivery of two charged dyes into separate receiving microchannels. The principle for the permanent separation of these dyes is based on the concept of bipolar electrochemistry and depended on the balancing/unbalancing of convective and electromigrating forces caused by the application of a potential bias, as well as the activation/deactivation of the bipolar electrodes. Two different bipolar electrode configurations are described and fluorescence is used to optimize their efficiency, speed, and cleanliness of delivery. The second device is a DNA sensor fabricated on paper by wax printing and folding to form 3D channels. DNA is detected by strand-displacement induced fluorescence of a single-stranded DNA. A multiplexed version of this sensor is also shown where the experiment results in “OR” and “AND” Boolean logic gate operations. In addition, the nonspecific adsorption of the reagents to cellulose is studied, demonstrating that significant reduction of nonspecific adsorption and increased sensitivity can be achieved by pre-treating the substrate with bovine serum albumin and by preparing all analyte solutions with spectator DNA. The third device, also made of paper, has a novel design and uses a versatile electrochemical detection method for the indirect detection of analytes via the direct detection of AgNP labels. A proof-of-concept experiment is shown where streptavidin-coated magnetic microbeads and biotin-coated AgNPs are used to form a composite model analyte. The paper device, called oSlip, and electrochemical method used are easily coupled so the resulting sensor has a simple user-device interface. LODs of 767 fM are achieved while retaining high reproducibility and efficiency. The fourth device is the updated version of the oSlip. In this case, the objective is to show the current progress and limitations in the detection of real analytes using the oSlip device. A sandwich-type immunoassay approach is used to detect human chorionic gonadotrophin (pregnancy hormone) present in human urine. Various optimization steps are performed to obtain the ideal reagent concentrations and incubation time necessary to form the immunocomposite in one step, that is, by mixing all reagents at the same time in the oSlip. Additionally, improvements to the electrochemical detection step are demonstrated.Item Proving 2-aminobiphenyl nitric oxide probes in cells and designing sequence-defined oligomers for sequencing(2021-05-06) Escamilla, Pedro Rogelio; Anslyn, Eric V., 1960-; Shear, Jason B; Liu, Hung-wen; Humphrey, Simon M; Sessler, Jonathan LThe simple diatom radical nitric oxide (NO) has numerous roles in biology. In organisms, healthy nanomolar NO levels are highly regulated. Deficiencies in NO can lead to atherosclerosis, diabetes, glaucoma, and many other conditions. Yet organisms also purposefully increase NO concentrations to micromolar levels in combatting pathogens. However, these high levels can backfire and damage cells and tissues, as found for Parkinson’s disease and ischemia, among others. Fluorescent NO probes enable live cell, tissue, and sometimes whole-animal imaging with minimal perturbation of their biological environment. 2-aminobiphenyl-based probe NO550 brought about a novel mechanism of detection in which NO-surrogate nitrosonium cation is assimilated into the nascent cinnoline fluorophore, resulting in low background and increased sensitivity. A family of second-generation 2-aminobiphenyl based probes were designed, producing four top performers in abiotic conditions. Chapter 1 describes the synthesis of these top candidates and their evaluation in cells. Two emerged as promising options for NO researchers. Chapter 2 describes the design and synthesis of sequence-defined non-natural oligomers that are sequenceable. Biological polymers peptides and DNA/RNA’s exquisitely complex properties are dictated by their sequence; changing the sequence sufficiently may have detrimental effects on their performance. Both their synthesis and sequencing continue being optimized; a human genome that once required thirteen years to sequence now takes one day. Not as developed but growing rapidly is the field of non-natural sequence-defined polymers. Without the constraints of biology, limitless options exist for backbone structures and sidechains. Such diversity discourages the focused development of sequencing technologies on par with those for biological polymers. Consequently, tandem MS is the favored form of analysis. We sought to change the approach to polymer design by factoring in the sequencing of the polymer in addition to its synthesis. Oligourethanes based on β-aminoalcohols were designed so that the terminal unit could be derivatized to sequence by cyclizing upon itself and thereby releasing the remainder of the oligomer. Several derivatizations were investigated, to find that the oligomer sequenced itself without any terminal unit transformation, rather by self-immolation under conditions that slowed the “unzipping” of the polymer sufficiently to be able to detect the intermediate sequences.Item Quantitative measurements of ablation-products transport in supersonic turbulent flows using planar laser-induced fluorescence(2015-08) Combs, Christopher Stanley; Clemens, Noel T.; Danehy, Paul M; Ezekoye, Ofodike A; Raja, Laxminarayan; Varghese, Philip LA recently-developed experimental technique based on the sublimation of naphthalene, which enables imaging of the dispersion of a passive scalar using planar laser-induced fluorescence (PLIF), is applied to a Mach 5 turbulent boundary layer and a NASA Orion capsule flowfield. To enable the quantification of naphthalene PLIF images, quantitative fluorescence and quenching measurements were made in a temperature- and pressure-regulated test cell. The test cell measurements were of the naphthalene fluorescence lifetime and integrated fluorescence signal over the temperature range of 100 K to 525 K and pressure range of 1 kPa to 40 kPa in air. These data enabled the calculation of naphthalene fluorescence yield and absorption cross section over the range of temperatures and pressures tested, which were then fit to simple functional forms for use in the calibration of the PLIF images. Quantitative naphthalene PLIF images in the Mach 5 boundary layer revealed large-scale naphthalene vapor structures that were regularly ejected out to wall distances of approximately y/δ = 0.6 for a field of view that spanned 3δ to 5δ downstream of the trailing edge of the naphthalene insert. The magnitude of the calculated naphthalene mole fraction in these structures at y/δ = 0.2 ranged from approximately 1-6% of the saturation mole fraction at the wind tunnel recovery temperature and static pressure. An uncertainty analysis showed that the uncertainty in the inferred naphthalene mole fraction measurements was ± 20%. Mean mole fraction profiles collected at different streamwise locations were normalized by the mole fraction measured at the wall and a characteristic height of the scalar boundary layer, causing the profiles to collapse into one “universal” mole fraction profile. Two-dimensional fields of naphthalene mole fraction were also obtained simultaneously with velocity by using particle image velocimetry (PIV) and PLIF. The images show large-scale naphthalene vapor structures that coincide with regions of relatively low streamwise velocity. The covariance of naphthalene mole fraction with velocity indicates that an ejection mechanism is transporting low-momentum, high-scalar-concentration fluid away from the wall, resulting in the protrusions of naphthalene vapor evident in the instantaneous PLIF images. Lastly, naphthalene PLIF was used to visualize the dispersion of gas-phase ablation products on a scaled Orion capsule model at four different angles of attack at Mach 5. High concentrations of scalar were imaged in the capsule recirculation region. Additionally, intermittent turbulent structures were visualized on the heat shield surface, particularly for the 12° and 52° AoA cases.Item Technique for imaging ablation-products transported in high-speed boundary layers by using naphthalene planar laser-induced fluorescence(2010-08) Lochman, Bryan John; Clemens, Noel T.; Raman, VenkatramananA new technique is developed that uses planar laser-induced fluorescence (PLIF) imaging of sublimated naphthalene to image the transport of ablation products in a hypersonic boundary layer. The primary motivation for this work is to understand scalar transport in hypersonic boundary layers and to develop a database for validation of computational models. The naphthalene is molded into a rectangular insert that is mounted flush with the floor of a Mach 5 wind tunnel. The distribution of naphthalene in the boundary layer is imaged by using PLIF, where the laser excitation is at 266 nm and the fluorescence is collected in the range of 320 to 380 nm. To investigate the use of naphthalene PLIF as a quantitative diagnostic technique, a series of experiments is conducted to determine the linearity of the fluorescence signal with laser fluence, as well as the temperature and pressure dependencies of the signal. The naphthalene fluorescence at 297 K is determined to be linear for laser fluence that is less than about 200 J/m². The temperature dependence of the naphthalene fluorescence signal is found at atmospheric pressure over the temperature range of 297K to 525K. A monotonic increase in the fluorescence is observed with increasing temperature. Naphthalene fluorescence lifetime measurements were also made in pure-air and nitrogen environments at 300 K over the range 1 kPa to 40 kPa. The results in air show the expected Stern-Volmer behavior with decreasing lifetimes at increasing pressure, whereas nitrogen exhibits the opposite trend. Preliminary PLIF images of the sublimated naphthalene are acquired in a Mach 5 turbulent boundary layer. Relatively low signal-to-noise-ratio images were obtained at a stagnation temperature of 345 K, but much higher quality images were obtained at a stagnation temperature of 380 K. The initial results indicate that PLIF of sublimating naphthalene may be an effective tool for studying scalar transport in hypersonic flows.Item Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami(Public Library of Science, 2010-07-14) Hunt, Marguerite E.; Scherrer, Michael P.; Ferrari, Frank D.; Matz, Mikhail V.Background -- Fluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development. Results -- Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M−1 cm−) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness. Conclusions -- The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.