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Item Illuminating dynamic phenomena within organic microstructures with time resolved broadband microscopies(Montana State University - Bozeman, College of Letters & Science, 2024) Hollinbeck, Skyler Robert; Chairperson, Graduate Committee: Erik Grumstrup; This is a manuscript style paper that includes co-authored chapters.Materials derived from organic chromophore subunits are currently at the forefront of academic and industrial interest. This strong interest is driven in part by the tunability of their extant properties through engineering of both the intra-molecular and inter-molecular structure. The structure of organic materials affects optoelectronic properties because organic chromophores are sensitive to dipole-dipole and charge-transfer coupling interactions. This sensitivity presents both opportunities for tuning functional properties through designing specific packing geometries, and liabilities arising from the disruptive effects of structural disorder. Many organic materials are built from weak noncovalent interactions between chromophores, leading solid-state deposition, and crystallization to be susceptible to microscopic variations in environmental conditions. Structural heterogeneity is regularly intrinsic to organic materials, and even self-assembled systems of covalently linked chromophores exhibit defects. Ergo, in order to disentangle the effects of structural heterogeneity from the inherent properties of the material, the study of organic materials must be anchored with techniques that are capable of correlating optoelectronic properties and excited state evolution with microscale morphological characteristics. We have employed broadband pump-probe microscopies, in conjunction with steady-state and time resolved fluorescence techniques, to examine the effects of structure and coupling on excited state dynamics in solid-state organic microstructures. The study of perylene diimide (PDI) materials revealed that kinetically trapping PDI (KT-PDI) enhanced long-range ordering and led to distinct excited state evolution, delocalized charge-transfer states and long-lived charge separated species. In the MOF PCN-222, excitation energy dependent excited state behavior was observed. Pumping the first excited state (Q-band) led to immobile excited states that were relatively unaffected by local defect densities, whereas pumping the second excited state (Soret-band) led to mobile subdiffusive excited state species whose lifetimes are significantly impacted by morphologically correlated defect sites. Finally, we present progress made toward the construction and utilization of a frequency modulated-femtosecond stimulated Raman microscope, yielding spectra that resolve the location of photoinduced anion formation in KT-PDI. The work presented herein highlights broadband time-resolved microscopy as a potent tool for studying the structure-function relationship and photophysical behavior in molecular solids, deepening our understanding of how structural characteristics influence excited state evolution.Item The synthesis and characterization of fluorescently labeled, lactose-functionalized poly(amidoamine) (PAMAM) dendrimers(Montana State University - Bozeman, College of Letters & Science, 2024) Frometa, Magalee Rose; Chairperson, Graduate Committee: Mary J. CloningerCellular uptake of lactose-functionalized poly(amidoamine) dendrimers (PAMAM) has yet to be fully understood and deeply studied. Before sufficient cellular uptake studies can be made, optimization of the synthesis of the lactoside, and the coupling and purification of dye-tagged lactose-functionalized PAMAM had to be completed, as reported here. The synthesis of the requisite lactoside derivative for dendrimer functionalization was optimized. The coupling of the dye, Alexa Fluor 647, to the lactoside-functionalized PAMAM was performed in the presence of a sodium acetate buffer and utilized size separation methods to ensure purity. The structures of the lactoside derivatives and of lactose functionalized PAMAMs were confirmed via nuclear magnetic resonance (NMR) spectroscopy. The purity and degree of labeling (DOL) of the dye labeled, lactose-functionalized PAMAMs were determined with UV-vis. Results show high success of yield and purity resulting from the optimized procedure described in this study.Item Fluorescence quenching in 2-aminopurine-labeled model DNA systems(Montana State University - Bozeman, College of Letters & Science, 2019) Remington, Jacob Michael; Chairperson, Graduate Committee: Patrik R. Callis; Abbey M. Philip, Mahesh Hariharan and Bern Kohler were co-authors of the article, 'On the origin of multiexponential fluorescence decays from 2-aminopurine labeled dinucleotides' in the journal 'The journal of chemical physics' which is contained within this thesis.; Martin McCullagh and Bern Kohler were co-authors of the article, 'Molecular dynamics simulations of 2-aminopurine-labeled dinucleoside monophosphates reveal multiscale stacking kinetics' in the journal 'Journal of physical chemistry B' which is contained within this thesis.For the last 50 years changes to the fluorescence properties of 2-aminopurine have been used to probe the structure and dynamics of DNA. 2-Aminopurine's utility has arisen from the quenching of its emission when pi-stacked with neighboring nucleobases. In the time-domain, the emission decay profile of 2-aminopurine requires multiple exponential decay components to model. Despite its extensive usage, the microscopic origin of the decay heterogeneity is not clear. In this thesis, steady-state absorption, fluorescence, and time-resolved fluorescence results are compared to multiple microsecond molecular dynamics simulations of 2-aminopurine-labeled adenine containing single-stranded DNA oligomers of varying length and position of the 2-aminopurine probe. First, previous reports of ultrafast electron transfer in pi-stacked adenine oligomers are used to build a new model for quenching of 2-aminopurine that is pi-stacked with adenine. For dinucleotides, a static distribution of unstacked structures combined with a distance dependent electron transfer mechanism is posited to explain the disperse emission decay timescales. Investigating the dinucleotides with molecular dynamics simulations analyzed with Markov state models quantify the structural heterogeneity of the dinucleotides. At least seven structures are sampled that could alter the quenching of 2-aminopurines's fluorescence. The Markov state models also demonstrate the timescales for transitions between these structures range from 1.6 to 25 ns, suggesting 2-aminopurine, with its monomer-like lifetime of 10 ns, is sensitive to the conformational dynamics of the dinucleotides as well. This dual fluorescence quenching and molecular dynamics simulation approach is extended to 2-aminopurine labeled trinucleotides and 15 base oligomers to interrogate the position dependent structural heterogeneity and conformational dynamics in these systems. Both shifts in the experimental absorption spectra, and molecular dynamics simulations agree that the interior base is more likely to be stacked than the exterior bases. Time-resolved emission experiments reveal emission from 2-aminopurine is quenched faster on the 5' end relative to the 3' end, in agreement with the faster stacking kinetics observed for bases on the 5' end relative to the 3' end obtained from molecular dynamics simulation. These results suggest that the time-resolved emission from 2-aminopurine may serve as an experimental observable for calibration of the dynamical properties predicted by molecular dynamics simulation.Item Temperature dependent solvation in phospholipid membranes(Montana State University - Bozeman, College of Letters & Science, 2017) Gobrogge, Christine Ann; Chairperson, Graduate Committee: Robert Walker; Victoria A. Kong and Robert A. Walker were co-authors of the article, 'Temperature dependent solvation and partitioning of coumarin 152 in phospholipid membranes' in the journal 'Journal of physical chemistry B' which is contained within this thesis.; Heather S. Blanchard and Robert A. Walker were co-authors of the article, 'Temperature dependent partitioning of C152 in phosphatidylcholine lipid bilayers' in the journal 'Journal of physical chemistry B' which is contained within this thesis.; Robert A. Walker was a co-author of the article, 'Quantifying solute partitioning in phosphatidylcholine membranes' submitted to the journal 'The journal of analytical chemistry' which is contained within this thesis.Experiments described in this dissertation were designed to systematically investigate solute partitioning in phospholipid bilayers as a function of phospholipid identity, solute identity, membrane phase, and membrane composition. Experiments use time-resolved fluorescence, steady-state fluorescence, dynamic light scattering, and differential scanning calorimetry to experimentally quantify solute partitioning in three specific regions of a model membrane, as well as track how solutes migrate into and out of lipid bilayers as a function of temperature. Phosphatidylcholine vesicles were comprised of DLPC (12:0 PC), DMPC (14:0 PC), and DPPC (16:0 PC). In all three lipid systems, coumarin 152 (C152) showed partitioning behavior that was qualitatively similar but quantitatively different. Partitioning into a gel phase membrane was slightly exothermic and slightly entropically unfavorable. Partitioning of C152 near the lipid membrane melting temperature was entropically driven and endothermic. Well above the melting temperature, exsolvation of C152 from the membrane back into the aqueous buffer was enthalpically driven but entropically unfavorable. Regardless of solution temperature, relatively little (<20%) C152 partitioned into the hydrophobic core of the membrane. The magnitudes of the thermodynamic forces driving C152 partitioning systematically increased with alkyl chain length (DLPC < DMPC < DPPC). C152 and C461 differ solely in the 4-position where C152 has a trifluoro methyl group in place of C461's -CH3 group. Fluorescence amplitudes were used to calculate absolute partition coefficients and average number of solutes per DPPC vesicle. C152 shows a ~10-fold greater affinity than C461 for lipid bilayers, despite both solutes having similar log P values. Differential scanning calorimetry traces of vesicles composed of binary mixtures of lipids show moderate miscibility between DLPC and DMPC and low miscibility between DMPC and DMPE. Time-resolved fluorescence decays indicate C152 partitioning into mixed PC membranes is nearly ideal; that is, even if mixed PC vesicles do form single lipid domains, C152 partitioning behavior is largely unaffected. Time-resolved fluorescence decays show C152 partitioning behavior into PC/PE membranes is distinctly non-ideal, but the cause of this non-ideal behavior requires further studies.Item Design and synthesis of novel chromophores for aptamer based imaging(Montana State University - Bozeman, College of Letters & Science, 2015) Robison, Jacob Michael; Chairperson, Graduate Committee: Mary J. CloningerFluorescent proteins are an incredibly versatile tool in biological imaging. Unfortunately, fluorescent proteins cannot be used to track small metabolites in vivo. The purpose of the work described herein was to create novel red-shifted RNA aptamer based probes for use in molecular imaging. All potential dyes were prescreened using molecular modeling and only the dyes that absorbed wavelengths longer than 500 nm were synthesized. The first aminothiophene based imidazolinone dyes (ATI-1 and ATI-2) were synthesized and their electronic properties were evaluated. SELEX was performed on ATI-2 to find several random RNA sequences that were capable of binding the chromophore and activating ATI-2 fluorescence. It is proposed that fluorescence activated cell sorting can be used to separate and isolate the sequences that form the brightest complexes with ATI-2.Item Computational prediction and experimental measurement of time resolved fluorescence properties of tryptophan and 5-fluoro-tryptophan dipeptides(Montana State University - Bozeman, College of Letters & Science, 2016) Fahlstrom, Carl Ashley; Chairperson, Graduate Committee: Patrik R. Callis; Lee Spangler (co-chair)The widely exploited high sensitivity of the amino acid tryptophan (Trp) fluorescence wavelength and quantum yield on local environment in proteins results in multiexponential decay from two mechanisms: quenching rate heterogeneity and/or spectral relaxation. 5-uorotryptophan (5FTrp) reduces quenching rate heterogeneity by suppression of electron transfer quenching. A comparison of fluorescence properties of Trp and 5FTrp provides a means of differentiating between relaxation and heterogeneity. Four observations concerning the fluorescence of Trp dipeptides required further explanation: decay components of approximately 20 ps, a sub 300 fs 10% loss of quantum yield, the quantum yield for Gly-Trp being significantly lower than Trp-Gly, and the fluorescence wavelength of Trp-X being 10 nm shorter than X-Trp at pH 5. With the goal of distinguishing between electron and proton transfer quenching mechanisms, the time resolved fluorescence--with time resolution of 0.5 ns--for dipeptides was measured in the X-Trp and Trp-X configurations(where X=Leu, Gly, Asp, Arg, Met), with 5FTrp substitution, at pH 5 and pH 10, and in water and D 2O solvent--resulting in 84 distinct species. Molecular dynamics simulations were performed on Gly-Trp and Trp-Gly dipeptides with ground and excited state charges employing multiple force fields. The methods developed by Callis and coworkers were used to calculate the instantaneous rates of electron transfer rates, quantum yield, and fluorescence wavelength. Experimentally three types of multiexponential decay were observed: quenching rate heterogeneity with no relaxation, relaxation only, and combinations thereof. The substitution of 5FTrp for Trp reduced quenching rate heterogeneity, resulting in reduction of short lifetime components, allowing for the observation of relaxation components that were most likely masked in the Trp case. Calculated electron transfer rates support lifetimes of approximately 20 ps, but not those less than 300 fs, and predict a lower quantum yield for Trp-Gly than Gly-Trp. Calculated fluorescence wavelengths reproduce the observed shorter fluorescence wavelength of Trp-X zwitterions. Failure to predict quantum yields may be caused by the inability of the molecular dynamics force fields to reproduce the conformational populations. Results support both relaxation and heterogeneity in Trp dipeptides. 5FTrp is a useful tool in distinguishing between heterogeneity and relaxation.Item Polarized fluorescence of the dinucleotides(Montana State University - Bozeman, College of Letters & Science, 1974) Wilson, Robert WilfredItem Spectroscopic evaluation and fluorescence dynamics of several erbium doped laser materials(Montana State University - Bozeman, College of Letters & Science, 2001) Farris, BruceItem The room temperature fluorescence of DNA(Montana State University - Bozeman, College of Letters & Science, 1982) Aoki, Timothy IsamuItem One and two photon excitation properties of selected biologically important molecules(Montana State University - Bozeman, College of Letters & Science, 1983) Anderson, Bruce Eldon