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- All Subjects: Fluorescence spectroscopy
- Creators: Ciuba, Monika A
- Creators: Nye, Joshua
Description
Fluorescence spectroscopy is a powerful tool for biophysical studies due to its high sensitivity and broad availability. It is possible to detect fluorescence from single molecules allowing researchers to see the behavior of subpopulations whose presence is obscured by “bulk” collection methods. The fluorescent probes used in these experiments are affected by the solution and macromolecular environments they are in. A misunderstanding of a probe’s photophysics can lead researchers to assign observed behavior to biomolecules, when in fact the probe is responsible. On the other hand, a probe’s photophysical behavior is a signature of the environment surrounding it; it can be exploited to learn about the biomolecule(s) under study. A thorough examination of a probe’s photophysics is critical to data interpretation in both cases and is the focus of this work. This dissertation investigates the photophysical behavior of symmetric and asymmetric cyanines in a variety of solution and biomolecular environments. Using fluorescent techniques—such as time-correlated single photon counting (TCSPC) and fluorescence correlation spectroscopy (FCS)—it was found that cyanines are influenced by the local environment. In the first project, the symmetric cyanines are found to be susceptible to paramagnetic species, such as manganese(II), that enhance the intersystem crossing (ISC) rate increasing triplet blinking and accelerating photobleaching. Another project found the increase in fluorescence of Cy3 in the protein induced fluorescence enhancement (PIFE) technique is due to reduced photoisomerization caused by the proximity of protein to Cy3. The third project focused on asymmetric cyanines; their photophysical behavior has not been previously characterized. Dy630 as a free dye behaves like Cy3; it has a short lifetime and can deactivate via photoisomerization. Preliminary experiments on Dy dyes conjugated to DNA show these dyes do not photoisomerize, and do not show PIFE potential. Further research will explore other conjugation strategies, with the goal of optimizing conditions in which Dy630 can be used as the red-absorbing analogue of Cy3 for PIFE applications. In summary, this dissertation focused on photophysical investigations, the understanding of which forms the backbone of rigorous fluorescent studies and is vital to the development of the fluorescence field.
ContributorsCiuba, Monika A (Author) / Levitus, Marcia (Thesis advisor) / Liu, Yan (Committee member) / Vaiana, Sara (Committee member) / Arizona State University (Publisher)
Created2017
Description
I present for the first time a broad-scale assessment of dissolved organic matter in the continental hot springs of Yellowstone National Park. The concentration of dissolved organic carbon in hot springs is highly variable, but demonstrates distinct trends with the geochemical composition of springs. The dissolved organic carbon concentrations are lowest in the hottest, most deeply sourced hot springs. Mixing of hydrothermal fluids with surface waters or reaction with buried sedimentary organic matter is typically indicated by increased dissolved organic carbon concentrations. I assessed the bulk composition of organic matter through fluorescence analysis that demonstrated different fluorescent components associated with terrestrial organic matter, microbial organic matter, and several novel fluorescent signatures unique to hot springs. One novel fluorescence signature is observed exclusively in acidic hot springs, and it is likely an end product of thermally-altered sedimentary organic matter. This acid-spring component precipitates out of solution under neutral or alkaline conditions and characterization of the precipitate revealed evidence for a highly condensed aromatic structure. This acid-spring component serves as a reliable tracer of acidic, hot water that has cycled through the subsurface. Overall, dissolved organic carbon concentrations and fluorescent features correlate with the inorganic indicators traditionally used to infer spring fluid mixing in the subsurface. Further, the fluorescence information reveals subtle differences in mixing between fluid phases that are not distinguishable through classic inorganic indicator species. My work assessing dissolved organic carbon in the Yellowstone National Park hot springs reveals that the organic matter in hydrothermal systems is different from that found in surface waters, and that the concentration and composition of hot spring dissolved organic matter reflects the subsurface geochemical and hydrological environment.
ContributorsNye, Joshua (Author) / Hartnett, Hilairy E (Thesis advisor) / Shock, Everett L (Committee member) / Jones, Anne K (Committee member) / Arizona State University (Publisher)
Created2020