Thesis (Ph.D)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009. / Committee Chair: Brédas, Jean-Luc; Committee Member: Janata, Jiri; Committee Member: Kippelen, Bernard; Committee Member: Marder, Seth; Committee Member: Sherrill, David. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 143-146).

Thesis (M.S.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2006. / Includes bibliographical references.

Thesis (PhD)--Montana State University--Bozeman, 2010. / Typescript. Chairperson, Graduate Committee: Aleksander Rebane. Includes bibliographical references (leaves 126-144).

As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed [1]. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 113-123).

Two-photon absorption and upconverted fluorescence has been utilized in a variety of applications in pure science and engineering. Multiphoton-based techniques were used in this research in order to understand photophysical and chemical characteristics of several fluorescent dyes and to demonstrate some of their key applications. Two-photon fluorescence microscopy (2PFM) has become a powerful technique in bio-photonics for non-invasive imaging in the near-infrared (NIR) region (700~1000 nm) that often results in less photobleaching. In Chapter 1, there is a brief introduction to fluorescence, examples of fluorescence materials, and a discussion of the advantages of two-photon absorption. 2PFM imaging was utilized in Chapters 2 to 4 for various applications. In Chapter 2, a new squaraine dye is introduced and its linear and nonlinear photophysical properties are characterized. This compound has very high two-photon absorption (2PA) cross sections and high photostability both in an organic solvent and when encapsulated in micelles. Based on these properties, this dye was demonstrated as a near-infrared (NIR) probe in in vitro 2PFM imaging with excitation over 800 nm wavelength. In Chapter 3, new profluorescent nitroxides are introduced. Nitroxide radicals are utilized for electron paramagnetic resonance (EPR) spectroscopy and in biological systems as some are known, in some manner, to mimic the behavior of superoxide dismutase (SOD) that detoxifies or mitigates oxidative stress by trapping free radicals. Here, two profluorescent nitroxides investigated for use as a two-photon fluorescent oxidative stress indicator in in vitro two-photon fluorescence microscopy (2PFM) imaging. In Chapter 4, two-photon excited (2PE) fluorescence of a conjugated polyelectrolyte (CPE), PPESO3, was studied in methanol and in water. The results of CPE quenching studies were comparable under both one-photon excitation conditions and two-photon excitation. CPE coated silica nanoparticles were incubated in HeLa cells and 2PFM imaging was demonstrated for this new class of fluorescent probe. Supramolecular structures based on organized assemblies/aggregation of chromophores have attracted widespread interest as molecular devices with potential applications in molecular electronics, artificial light harvesting, and pharmacology. In Chapter 5, J-aggregate formation was investigated for two porphyrin-based dyes, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS, 4) and an amino tris-sulfonate analog (5) in water via UV-vis, fluorescence, and lifetime decay studies. The effect of aggregation on two-photon absorption properties was also investigated. A functionalized norbornene-based homopolymer, synthesized by the ring opening metathesis polymerization technique was used as a J-aggregation enhancement template and had a role of polymer-templating to facilitate porphyrin aggregation and modulate 2PA. In Chapter 6, squaraine dye aggregates templated with single wall carbon nanotubes (SWCNTs) that were atomically clean were studied by using optical absorption spectroscopy, atomic force microscopy (AFM), and photoconductivity measurements. SWCNTs selectively promote the formation of squaraine dye aggregates with a head-to-head stacking arrangement, and these dye aggregates effectively photosensitize SWCNTs, demonstrating that this novel approach can yield highly photosensitized devices.

Two-photon fluorescence microscopy (2PFM) has become a powerful technique for bioimaging in non-invasive cancer diagnosis and also investigating the mechanization and original of a variety of diseases by tracking various biological processes. Because the fluorescence emission by two photon absorbing (2PA) is directly proportional to the square of the intensity of excitation light, this intrinsic property of 2PA provides 2PFM great advantages over traditional one-photon fluorescence microscopy (1PFM), including high 3D spatial localization, less photodamage and interference from biological tissue because of using longer wavelength excitation (700-1300 nm). However, most 2PA probes are hydrophobic and their photostabilities are questionable, severely limiting their biological and medical applications. In addition, probes with significant specificity for certain organelles for tracking cellular processes or metal ions for monitoring neural transmission are somewhat rare. Moreover, it is also very significant to deliver the probes to specific disease sites for early cancer diagnosis. In order to increase the water solubility of probes, polyethylene glycol (PEG) was introduced to a fluorene-based 2PA probe LT1 for lysosomal 2PFM cell imaging. The 2PFM bioimaging application of the novel two-photon absorbing fluorene derivative LT1, selective for the lysosomes of HCT 116 cancer cells is described in Chapter II. Linear and nonlinear photophysical and photochemical properties of the probe were investigated to evaluate the potential of the probe for 2PFM lysosomal imaging. After the investigation of the cytotoxicity of this new probe, colocalization studies of the probe with commercial lysosomal probe Lysotracker Red in HCT 116 cells were conducted. A high colocalization coefficient (0.96) was achieved and demonstrated the specific localization of the probe in lysosomes. A figure of merit, F[subscript M], was introduced by which all fluorescent probes for 2PFM can be compared. LT1 was demonstrated to have a number of properties that far exceed those of commercial lysotracker probes, including much higher 2PA cross sections, good fluorescence quantum yield, and, importantly, high photostability, all resulting in a superior figure of merit. Consequently, 2PFM was used to demonstrate lysosomal tracking with LT1. In addition to lysosomes, it is also very significant to investigate the physiological roles of free metal ions in biological processes, especially Zn²⁺, because Zn²⁺ normally serves either as the catalytic elements in enzymatic activity centers or as structural elements in enzymes and transcription factors. However, biocompatible and effective Zn²⁺ probes for 2PFM bioimaging are infrequent. In Chapter III, 2PFM bioimaging with a hydrophilic 2PA Zn²⁺ sensing fluorescent probe, bis(1,2,3-triazolyl)fluorene derivative, is described. 2PFM bioimaging of the probe in living HeLa cancer cells was demonstrated. The results revealed a significant fluorescence increase upon introduction of Zn²⁺ into the cancer cells, and a reversible Zn²⁺ binding to the probe was also demonstrated, providing a robust probe for two-photon fluorescence zinc ion sensing. Early cancer diagnosis is another critical application for 2PFM, but there are still huge challenges for this new technique in clinical areas. Most 2PA probes with large two-photon absorbing cross sections and fluorescence quantum efficiency are synthetically more accessible in hydrophobic forms. In order to increase the efficiency of the probes and minimize the effect of the probe on the human body, delivery of the probe specifically to cancer sites is desired. The synthesis and characterization of narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, are reported in Chapter IV. The surface of the SiNPs was functionalized with folic acid to specifically deliver the probe to folate receptor (FR) over-expressing HeLa cells, making these folate 2PA dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing HeLa cells demonstrated specific cellular uptake of the functionalized nanoparticles. However, when the concentration of the dye in SiNPs increased for higher signal output, the fluorescence quantum efficiency of a probe normally decreases because of self-quenching. In Chapter V, a near-infrared (NIR) emitting probe is reported to overcome this limitation through both aggregate-enhanced fluorescence emission and aggregate enhanced two-photon absorption. The dye was encapsulated in SiNPs and the surface of the nanoparticles was functionalized with PEG followed by a folic acid derivative to specifically target folate receptors. NIR emission is important for deep tissue imaging. In vitro studies using HeLa cells that upregulate folate receptors indicated specific cellular uptake of the folic acid functionalized SiNP nanoprobe. Meanwhile, the probe was also investigated for live animal imaging by employing mice bearing HeLa tumors for in vivo studies. Ex vivo 2PFM tumor imaging was then conducted to achieve high quality 3D thick tissue tumor images.

The aim of this dissertation is to gain a better understanding of the unique electronic structure of lead salt quantum dots (QDs) and its influences on the nonlinear optical (NLO) properties as well as the time dynamics of the photogenerated charge carriers. A variety of optical techniques such as Z-scan, two-photon excited fluorescence and time-resolved pump probe spectroscopy are used to measure these properties. The one-photon as well as the degenerate and nondegenerate two-photon absorption (2PA) spectra are measured and the electronic wave functions from a four-band envelope function formalism are used to model the results. We observe local maxima in the 2PA spectra for QD samples of many different sizes at energies where only 1PA is predicted by the model. This is similar to the previously measured transitions in the 1PA spectra which are not predicted by the model but accrue at the energies of the two-photon allowed transitions. Most importantly we observe 2PA peaks for all samples at the energy of the first one-photon allowed transition. This result can only be understood in terms of symmetry breaking and therefore is strong evidence that other transitions, not predicted by the model if the selection rules are left intact, also have the origin in the lifted spatial symmetry of the wave functions. On the other hand, the uniquely symmetric eigenenergies of these quantum-confined energy states in the conduction and valance bands explain the observed trend toward larger two-photon cross-sections as the quantum confinement is increased in smaller QDs. Moreover, this unique feature is shown to reduce the possible relaxation channels for photoexcited carriers, which is confirmed experimentally by the reduced carrier relaxation rate as compared to CdSe QDs which lack this symmetry. Carrier multiplication (CM), a process in which several electrons are excited by the iv absorption of a single photon is studied in PbS QDs. We show that for PbS QDs with radius smaller than 2.5 nm the parameters of CM get very close to the theoretical optimum. Nextgeneration solar cells operating under these ideal conditions could potentially have conversion efficiency of up to 42%. This compares favorably to the 30% efficiency limit of a single junction silicon solar cell.

Structure-property relationships for two-photon absorption (2PA) in branched organic chromophores is a topic of current interest, as is the design of chromophores with advantageous properties for two-photon laser scanning microscopy (2PLSM). The main goals of this dissertation were to study and explain the one-photon absorption (1PA) and 2PA properties of cruciform chromophores based on 1,4-distyryl-2,5-bis(phenylethynyl)benzene with varying electron donor (D) and acceptor (A) groups, and to characterize the 2PLSM-relevant response of some of these chromophores and a set of dipolar chromophores to binding with zinc ions. The compounds were studied by 1PA, fluorescence and 2PA spectroscopy. A ππ* exciton model was developed to explain the spectral properties of the 1,4-distyryl-2,5-bis(phenylethynyl)benzene cruciform with no D or A groups or with four identical D groups at the termini of the linear arms of the chromophore. This model indicated that there is some coupling and mixing of the lowest excited states e of the linear arms, leading to splitting of the 1PA spectrum of the cruciform. There was little coupling or mixing of the higher excited states e′ accessed in 2PA, leading to a two-band 2PA spectrum for the chromophore, in contrast to cruciform compounds in the literature with identical conjugated arms, which have one visible 2PA band. For cruciforms with D groups on the styryl arm and A character on the terminal phenyls of the phenylethynyl arms (D/A cruciforms), the ππ* exciton model was complemented with a charge-transfer (CT) exciton model describing interactions of charge-transfer pathways between the D and A groups. This model explained the broadness of the 1PA band of D/A cruciforms as well as the two 2PA bands observed for these chromophores. The fluorescence and 2PA spectral responses to binding of Zn²⁺ ions to the D or A groups of some cruciform compounds were also assessed, to provide insight into the design of new analyte-sensing cruciforms for 2PLSM that take advantage of enhancement or reduction of D/A character upon analyte binding. It was found that canceling charge donation from the D groups in differing D/A cruciforms resulted in fluorescence and 2PA spectra nearly indistinguishable from each other, suggesting that turn-off of D groups is not an optimal modality of 2PLSM analyte sensing in cruciforms. Binding Zn²⁺ to A groups was shown to result in an increase in the D/A character of the cruciform, with fluorescence peak energies that changed depending on the location of the A group. It is suggested that the use of non-binding donors and analyte-binding A groups in differing patterns on the arms could be a valuable design motif to achieve 2PLSM sensor compounds based on this cruciform structure. The 2PA spectra of a set of dipolar Zn²⁺ sensing dyes designed for ratiometric imaging in 2PLSM were also studied. These dyes had moderate 2PA strength, with redshifts of fluorescence 2PA spectra on Zn²⁺ binding. The isosbestic point of 2PA of most chromophores was within the range of 2PLSM excitation sources commonly used, rendering these dyes good candidates for use in ratiometric sensing in 2PLSM.