Theses and Dissertations at Montana State University (MSU)

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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.
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. Cloninger
Cellular 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.
Single cell encapsulation, detection, and sorting of Pseudomonas syringae using drop-based microfluidics
(Montana State University - Bozeman, College of Engineering, 2023) Lindsay, Travis Carson; Chairperson, Graduate Committee: Abigail Richards; Connie Chang (co-chair)
Bacteria can survive antibiotic or bactericidal treatment through genetic mutations. Even within bacterial populations that are fully susceptible to treatment, a small proportion of cells can have enhanced survival capacity in a phenomenon called persistence. Traditional microbiology methods can fail to identify or isolate these persister cells present within the population. A novel method for high-throughput single cell analyses of microbial populations is that of drop-based microfluidics, in which individual cells can be isolated within picoliter-sized drops. In this work, fluorescent detection and dielectrophoresis-based sorting of drops was developed for isolating Pseudomonas syringae persister cells following antimicrobial treatment. We demonstrate: (1) the dielectrophoresis-based sorting of dye-filled 25 micron drops based upon two colors, (2) differences between laser-induced fluorescent detection of dyes compared to single bacterial cells, (3) single-cell isolation of P. syringae into 25 micron droplets with ~10% of droplets containing singlecells, and (4) the treatment, staining, and fluorescent characterization of P. syringae at 0.5x, 5x, and 50x the minimum inhibitory concentration of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an antibiotic which resulted in 6.2%, 10.2%, and 88.6% cell death of the population, respectively. These results provide the groundwork for studying antibiotic-treated P. syringae and the isolation of surviving cells that will lend insight into the molecular basis of persistence for preventing recurrent infections and decreasing the likelihood of antibiotic resistance.
Improving the two-photon absorption properties of fluorescent proteins for neuroscience
(Montana State University - Bozeman, College of Letters & Science, 2020) Molina, Rosana Sophia; Chairperson, Graduate Committee: Thomas Hughes; Yong Qian, Jiahui Wu, Yi Shen, Robert E. Campbell, Mikhail Drobizhev and Thomas E. Hughes were co-authors of the article, 'Understanding the fluorescence change in red genetically encoded calcium ion indicators' in the journal 'Biophysical Journal' which is contained within this dissertation.; Tam M. Tran, Robert E. Campbell, Gerard G. Lambert, Anya Salih, Nathan C. Shaner, Thomas E. Hughes and Mikhail Drobizhev were co-authors of the article, 'Blue-shifted green fluorescent protein homologues are brighter than enhanced green fluorescent protein under two-photon excitation' in the journal 'The Journal of physical chemistry letters' which is contained within this dissertation.; Jonathan King, Jacob Franklin, Nathan Clack, Christopher McRaven, Vasily Goncharov, Daniel Flickinger, Karel Svoboda, Mikhail Drobizhev, Thomas E. Hughes were co-authors of the article, 'An instrument to optimize fluorescent proteins for two-photon excitation' which is contained within this dissertation.
Untangling the intricacies of the brain requires innovative tools that power basic research. Fluorescent proteins, first discovered in jellyfish, provide a genetically encodable way to light up the brains of animal models such as mice and fruit flies. They have been made into biosensors that change fluorescence in response to markers of neural activity such as calcium ions (Ca 2+). To visualize them, neuroscientists take advantage of two-photon excitation microscopy, a specialized type of imaging that can reveal crisp fluorescence images deep in the brain. Fluorescent proteins behave differently under twophoton excitation compared to one-photon excitation. Their inherent two-photon properties, namely brightness and peak absorption wavelength, limit the scope of possible experiments to investigate the brain. This work aims to understand and improve these properties through three projects: characterizing a set of red fluorescent protein-based Ca 2+ indicators; finding two-photon brighter green fluorescent proteins; and developing an instrument to screen for improved fluorescent proteins for two-photon microscopy. Analyzing nine red Ca 2+ indicators shows that they can be separated into three classes based on how their properties change in a Ca 2+-dependent manner. In one of these classes, the relative changes in one-photon properties are different from the changes in two-photon properties. In addition to characterizing, identifying and directly improving fluorescent proteins for enhanced two-photon properties is important. Presented here is a physical model of the light-absorbing molecule within the green fluorescent protein (the chromophore). The model predicts that green fluorescent proteins absorbing at higher energy wavelengths will be brighter under two-photon excitation. This proves to be the case for 12 blueshifted green fluorescent proteins, which are up to 2.5 times brighter than the commonly used Enhanced Green Fluorescent Protein. A way to directly improve fluorescent proteins is through directed evolution, but screening under two-photon excitation is a challenge. An instrument, called the GIZMO, solves this challenge and can evolve fluorescent proteins expressed in E. coli colonies under two-photon excitation. These results pave the way for better two-photon fluorescent protein-based tools for neuroscience.
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.
Ultra high-throughput fluorescence detection for single cell applications in drop microfluidics
(Montana State University - Bozeman, College of Engineering, 2016) Schaefer, Robert Willman; Chairperson, Graduate Committee: Connie Chang
Conventional methods in microbiology can be limited by assay execution and analysis times, phenotypic dominance within bulk communities, reagent volumes, and single-use supply costs. These limitations can be overcome using drop-based microfluidics. In this discipline, pico-liter sized, water-in-oil emulsions serve as independent 'test tubes,' allowing for the compartmentalization of community constituents and interrogation at the single cell level. Furthermore, two-phase, continuous flow microfluidic devices enable drop populations to be manipulated and analyzed at kilohertz rates according to experimental needs. In this research, a fluorescence-based method for drop analysis and sorting was developed and applied, in conjunction with other microfluidic techniques, to perform assays in microbiology. The applications explored include cell dormancy within P. aeruginosa subpopulations, microalgae lipid accumulation for the production of biofuels, optimization of microbially-induced calcite precipitation (MICP), and human norovirus infectivity. Results from each application include: 1. The hibernation promoting factor (Hpf) was found to play a key role in the maintenance of P. aeruginosa viability during planktonic starvation. 2. Progress was made on a Nile Red based, ultra high-throughput, single cell algal lipid detection platform. 3. MICP was demonstrated at the single cell level. 4. A drop based human norovirus infection platform was attempted using human B cells as the viral host.
Long-term and over winter phytoplankton community dynamics in Lake Bonney, Antarctica
(Montana State University - Bozeman, College of Agriculture, 2017) Patriarche, Jeffrey Dennis; Chairperson, Graduate Committee: John C. Priscu
Lake Bonney is a hypersaline permanently ice-covered lake in the Taylor Valley, Antarctica that hosts simplified microbial food-webs. Studied since the 1960s, there are many aspects which are poorly understood. Logistical constraints have prevented sampling during the austral winter, a 4-month period of 24-hour darkness. Our knowledge of how the resident photosynthetic microorganisms respond during this period is limited. With inputs from ephemeral glacial-melt streams the lake level (stage) of Bonney has risen more than 3 m since 2004. With no outflow streams, the only known water loss is via ablation of the permanent ice-cover. A study of the spatial and temporal changes in the phytoplankton community structure during this period of rapid lake level rise is lacking. During the summers (November-January) from 2004-05 to 2014-15 an in situ submersible spectrofluorometer was deployed in Lake Bonney to quantify the chlorophyll-a concentrations (microgram L -1) of four functional groups of microalgae (green algae, brown/mixed algae, cryptophytes, cyanobacteria) using known excitation/emission spectra. During the 2013-14 field season this same instrument was mounted on autonomous cable-crawling profilers deployed in both east and west lobes of Lake Bonney, obtaining the first ever daily profiles of chlorophyll-a concentration at an annual scale. Following a summer of rapid lake level rise (2010-11), an increasing trend in depth integrated chlorophyll-a concentration was observed in Lake Bonney. During the same period, the nutrient poor surface water has become increasingly dominated by green algae. Dramatic shifts were also observed in the phytoplankton communities during the polar night. The highest concentrations of mean chlorophyll-a were measured during the 24-hour darkness. Algal spectral groups containing species capable of a mixotrophic metabolism (brown/mixed and cryptophytes) increased in concentration and relative abundance when photosynthetically active radiation was unavailable. This work provides valuable contributions to our knowledge of long-term and year-round phytoplankton community dynamics in Lake Bonney, and improves our understanding of the metabolic strategies employed by organisms in this high latitude permanently ice-covered lake.
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.
If you build it, they will come: engineering the next generation of optical tools to image neural activity deep within the living brain
(Montana State University - Bozeman, College of Letters & Science, 2017) Barnett, Lauren Marie; Chairperson, Graduate Committee: Thomas Hughes; Thomas E. Hughes and Mikhail Drobizhev were co-authors of the article, 'Deciphering the molecular mechanism responsible for GCAMP6M's Ca 2+ dependent change in fluorescence' in the journal 'PLoSONE' which is contained within this thesis.; Mikhail Drobizhev and Thomas E. Hughes were co-authors of the article, 'Making pKa-altering mutations in GCAMP6M changes the Ca 2+-dependent fluorescence response' submitted to the journal 'PLoSONE' which is contained within this thesis.; Jelena Platisa, Marko Popovic, Vincent A. Pieribone and Thomas Hughes were co-authors of the article, 'A fluorescent, genetically-encoded voltage probe capable of resolving action potentials' in the journal 'PLoSONE' which is contained within this thesis.; Lauren M. Barnett, Mikhail Drobizhev, Geoffrey Wicks, Alexander Mikhaylov, Thomas E. Hughes and Aleksander Rebane were co-authors of the article, 'Two-photon directed evolution of green fluorescent proteins' in the journal 'Nature Scientific Reports' which is contained within this thesis.
To see the activity of large, integrated neural circuits functioning in real-time inside of a living brain, neuroscientists will need multiple genetically-encoded fluorescent activity sensors that can be individually targeted to specific cell types, are fast enough to resolve multiple action potentials, can be distinguished from one another and imaged deep within the brain. The goal of this work is to better understand and improve upon the most recent generations of genetically-encoded Ca 2+ and voltage sensors, and to expand biosensor utility in two-photon excitation, which will be necessary to image neural activity deep within the brain. Genetically-encoded Ca 2+ sensors measure the intracellular Ca 2+ release that occurs downstream of an action potential. The GCaMP6 series are the best Ca 2+ sensors available, however little is known about how they work. Measurements of four different states in GCaMP6m reveal that its large Ca 2+-dependent change in 470 nm excited fluorescence is due to a redistribution of the chromophore protonation state, from a neutral form excited at ~400 nm to an anionic form excited at ~470 nm, via a change in pK a. Making pK a-altering mutations in GCaMP6m changes the Ca 2+-dependent fluorescence response. This highlights the importance of Delta pK a and identifies key amino acid positions that will be important for improving GCaMP6m and GCaMP-like biosensors. A direct readout of an action potential would be ideal for capturing complex signal transduction in the brain. This will require a bright, fast voltage sensor. ElectricPk is the first genetically-encoded voltage sensor with a fluorescence response fast enough to resolve multiple action potentials in mammalian neurons. This design indicates it is possible to couple a fluorescence change with a very fast (~1 ms) voltage-dependent movement in the Ciona intestinalis voltage-sensitive phosphatase protein. Whether imaging a downstream Ca 2+ signal or a direct change in membrane potential, to image neuronal activity in deep brain tissue biosensors will need to be brightly fluorescent in two-photon excitation. The two-photon directed evolution of green fluorescent proteins presented here is a proof-of-principle design that shows a high-throughput screen focused on improving the two-photon properties of a fluorescent protein is possible.
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. Cloninger
Fluorescent 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.

Theses and Dissertations at Montana State University (MSU)

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