PART I. A modified Hadamard transform has been developed and applied to the analysis of biologically active species using capillary electrophoresis. Hadamard transformations, a matrix based multiplexing technique, when coupled with a capillary electrophoresis instrument capable of rapid sample injection, provides a means to semi-continuously inject samples. The multiple injections separate, interpenetrate, and are detected as the summation of the multiple injections. Deconvolution of the multiplexed signal by multiplication with the inverse of the injection matrix yields a single injection electropherogram that exhibits improved S/N. In modified Hadamard transform capillary electrophoresis (mHTCE), an injection sequence of half the length as conventional HTCE (cHTCE) is utilized. Modifying the manner in which the raw data is manipulated before deconvolution facilitates the reduced injection sequence. When coupled with software, mHTCE can reduce the collection time for a Hadamard sequence by up to 48%. The substantial time reduction afforded by mHTCE is utilized to demonstrate the first time-resolved application of Hadamard transformations for the analysis of neurotransmitters. Additionally, mHTCE has been demonstrated as a means to improve the sensitivity for analysis of amino acids and proteins including gamma-aminobutyric acid, dopamine, and enhanced green fluorescent protein (EGFP) with picomolar detection limits.Part II. Two-photon excitation provides a means to activate chemical and physical processes with high spatial resolution and improved depth penetration compared to one-photon excitation. When combined with three-dimensional lithographic microfabrication (3DLM), these advantages provide a means to fabricate complex structures through radical and cationic two-photon induced polymerization (TPIP). A strategy for realizing high-fidelity microstructures is reported that considers the inherent structural limitations of acrylate monomers. Utilizing this strategy, a series of high-fidelity microstructures is reported for application in microfluidic devices, microelectromechanical systems (MEMS), and microoptical devices such as photonic bandgap (PBG) crystals. Improved periodicity is reported here for f.c.c. PBG crystals compared to earlier examples through addition of micromechanical supports that provide increased strength to the high-aspect ratio crystals. To extend TPIP to cationic polymerization, a series of two-photon activatable photoacid generators has been developed. The new PAGs exhibit one to two orders of magnitude lower polymerization threshold intensities than conventional ultraviolet-sensitive initiators.

This project explores optimization of two-photon excited fluorescence (TPEF) enhancement between tunable narrowband and un-tuned broadband femtosecond (fs) laser pulse excitations for two-photon microscopy (TPM). The research is conducted preliminarily in time domain and comprehensively in frequency domain to understand the physics behind TPEF enhancement by un-tuned sub-10 fs nearly transform-limited pulse (TLP) versus tunable 140 fs pulse. The preliminary study on inverse proportionality of TPEF yield to fs-pulse duration delimits a general lower-bound to narrowband fs-pulse regime (pulse duration > 40 fs) with assumption of dye-molecule frequency invariant response. Deviations from this inverse proportionality in broadband fs-pulse regime (pulse duration < 40 fs) highlights dye-molecule frequency variant response, necessity of group delay dispersion (GDD) compensation, and broadband TLP for TPEF enhancement. The follow-up comparative study is made on un-tuned sub-10 fs TLP versus tunable 140 fs pulse excitations using three dye-phantoms (Indo-1, FITC, and TRITC) representative of fluorescent probes with similar TPEF characteristics. The integrated experimental system, with custom-designed GDD compensation, dispersion-less laser-beam expanding and focusing, and compound-lens for efficient fluorescence collection with good spectral resolution, ensures accurate TPEF measurements. Differentiated TPEF enhancements of Indo-1 (1.6), FITC (6.7), and TRITC (5.2) proportionally agree with calculated ones due to the overlap of fs-pulse second harmonic (SH) power spectrum with dye-molecule two-photon excitation (TPE) spectrum. Physically speaking, with broadband sub-10 fs TLP readily involved in both degenerate (v1 = v2) and non-degenerate (v1 ≠ v2) two-photon absorption (TPA), this un-tuned ultrashort fs-pulse excitation simultaneously allows for more accessibility to TPA-associated final states and diversely promotes population of thus excited dye-molecules with the three dye-phantoms. Under environmental influences (mutual quenching through one-photon absorption(s) and solvent effect), multicolor TPEF enhancement observed from a mixture of the three dyes shows promise of sub-10 fs TLP as simultaneous excitation for multiple-dye labeled samples in contrast to compromised excitation with narrowband fs-pulse tuning. Both single- and multicolor TPEF enhancements clarify tradeoff between tunability of narrowband fs-pulse and un-tuned broadband fs-pulse excitations, being instructive to further considerations on optimization of TPEF enhancement by strategic utilization of broadband fs-pulse for better performance of TPM.

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).

The spatial arrangement of neuronal responses in primary auditory cortex (A1) has so far been investigated by using microelectrode recording techniques or imaging of the intrinsic signal, which led to controversial results, at present still discussed. On the other hand, two-photon calcium imaging allows us to investigate the cortical functions at an unprecedented level of spatial detail, and has recently offered new insight into the fine-scale organization of frequency responses in A1. In this thesis, I used two-photon calcium imaging to compare, for the first time, the fine-scale cortical representation of sound frequency to that of two other sound features, crucial for survival and communication in all mammals: differences in intensity between the two ears (interaural level differences; ILDs), and frequency modulation (FM). I found that most neurons in layers II-III of the mouse A1 were tuned to ILDs favouring the contralateral ear, but midline and ipsilateral tuning were present too. Binaural preferences were heterogeneously distributed in space, both on the fine scale (within &Tilde; 200 &mu;m) and on the global one (up to &Tilde; 1 mm). Moreover, A1 neurons were mostly tuned to slow FM sweeps within the range of those used in species-specific calls. Cells activated by similar rates tended to be spatially proximal, indicating a level of local organization similar to the one I found for frequency tuning, and higher than that of ILD responses. Finally, I set the groundwork for two-photon studies of the A1 of the ferret, by presenting the first evidence of the microscopic organization of the tonotopic map in this species. My results shed light on some long-held questions about the response properties of A1, and confirm two-photon imaging as a powerful tool for investigating the processing of sensory signals in the cortex of both small and large mammals.

No description available.

Proteins are of primary importance to the structure and function of all living cells. Study of proteins relies on the ability to separate a complex mixture so that individual proteins can be more easily processed by other techniques. Since protein samples often exist at low concentration in a small volume, the trend in chemical analysis is toward micro total analysis systems (µTAS) or lab-on-a-chip devices. Among µTAS separation methods, the relatively new electric field gradient focusing (EFGF) technique has shown potential. It focuses and separates analytes based on their electrophoretic migration in an opposing hydrodynamic flow. The detection principles that are compatible with µTAS separation may not always scale down. This thesis represents the development of laser-induced two-photon fluorescence detection on a microchip separation device. This detection is based on excitation of native fluorescence of aromatic amino acids by simultaneous absorption of two photons. First, a compact two-photon prototype detector was investigated. Its sensitivity was improved after discovery of the source of the background and subsequent reduction of background levels. Simple CE separation on a square capillary was coupled to this detector to demonstrate its ability for micro-scale detection. However, this detector did not provide a way to view the location illuminated by a laser and was difficult to use for on-microchip detection. A two-photon microscope was constructed on the frame of a commercial Olympus microscope to solve this problem. The eyepiece of this microscope enabled viewing of the detection volume, and the removal of a glass compensator from the trinocular head allowed for UV detection. This detection system was carefully aligned and optimized before coupling to microchip CE. Two microchip substrates including poly (methyl methacrylate) (PMMA) and glass, and two chip layouts were explored for their compatibilities with the microscope detector. It was found that the PMMA chip with conventional chip layout was not suitable for two-photon detection; therefore, a novel chip layout on PMMA was designed. Through testing the new design, it was concluded that precise focusing of the laser was essential to successful detection on microchips. Although the precise focusing of the laser inside microchip channels was not achieved completely in the limited research period, it is believed that this new design should be an appropriate solution to coupling PMMA chips with the two-photon microscope. Finally, glass chips were employed to successfully demonstrate the detection of amino acids.

Over the past several decades the phenomenon of luminescence (divided into fluorescence and phosphorescence) has received great attention in the field of biological science. This quest has motivated scientists for a variety of applications, including fluorescence imaging. Fluorescence microscopy techniques that provide unique advantages, such as high spatial resolution and superior sensitivity, have been regarded as attractive tools in biophotonics. With the progress of ultrafast laser sources, two-photon absorption (2PA), in which a molecule absorbs two photons simultaneously, has opened possibilities of using it for various applications. Two-photon fluorescence microscopy (2PFM), which affords deeper tissue penetration and excellent three-dimensional (3D) images, is now being widely employed for bioimaging. This dissertation focuses on the design, synthesis, and photophysical characterization of new fluorophores, as well as desirable applications. Chapter 1 gives an account of a brief introduction of luminescence and 2PA, as well as their utilities in biological applications. In chapter 2, a series of new BODIPY derivatives are presented along with their comprehensive linear and nonlinear characteristics. They exhibited excellent photophysical properties including large extinction coefficients, high fluorescence quantum yields, good photostability, and reasonable two-photon absorption cross sections. Two promising compounds were further evaluated as NIR fluorescent probes in one-photon and two-photon fluorescence imaging. Chapter 3 provides the design, synthesis, and photophysical characterization of two BODIPY dyes. In order to assess the potential of using the dye as a fluorescent probe, Lysotracker Red, a commercial lysosomal marker, was investigated for comparison purposes. The results indicate that figure of merit of both compounds were three orders of magnitude higher than that of Lysotracker Red. With an eye towards applications, one of the compounds was encapsulated in silica-based nanoparticles for in vitro and ex vivo one-photon and two-photon fluorescence imaging, in which the surface of the nanoparticle was modified with RGD peptides for specific targeting. The nanoprobe exhibited good biocompatibility and highly selective RGD-mediated uptake in ?V?3 integrin-overexpressing cancers, while maintaining efficient fluorescence quantum yield and high photostability. In chapter 4, the synthesis and photophysical properties of a novel photosensitizer with heavy atoms (halogen) were presented. The dye exhibited low fluorescence quantum yield, resulting in high singlet oxygen generation quantum yield. In vitro photodynamic studies demonstrated that photosensitization of the agent can induce cellular damage, subsequently leading to cell death by a necrotic cell death mechanism, supporting the therapeutic potential of using the agent for photodynamic therapy.

Molecules that are non-superimposable on their mirror image are named chiral or optically active compound. Over the years, molecular chirality has played an essential role in the understanding of fundamental aspects associated the origin of life, drug and food technologies and, asymmetric catalysis, among others. Moreover, most of the groundbreaking discoveries and advances made in this field have happened due to the development of spectroscopic techniques based on the natural asymmetry of the enantiomers and their ability to preferentially absorb right or left polarized light. For instance, circular dichroism (CD), which measures the difference in absorption between these two states of polarized light, has emerged as one of the most useful spectroscopic methods to identify and characterize chiral compounds. Unfortunately, CD is based on linear absorption which, in most common organic molecules, takes place in the UV region of the spectrum where the majority of organic solvents absorb as well. This certainly imposes limitations in the indiscriminated applicability of this technique to the study of chiral chromophores of biological interest in non-aqueous solutions. Consequently, a systematic and comprehensive characterization of the electronic and optical properties of such molecular entities still remains a major issue to be addressed. On this regard, nonlinear optics offers new alternatives to overcome some of the shortcomings of the standard linear CD-based spectroscopy. In order to surmount the existent limitations in this field and deepen in the fundamental understanding of chiral systems, we have mainly directed the attention of our research to the experimental and theoretical study of the polarization dependent two-photon absorption (2PA) of several chiral azo-compounds and binaphthol derivatives in solution. The first part of this dissertation (Chapters I-IV) covers a full characterization of the linear and nonlinear optical properties of a series of non-chiral and chiral azo derivatives. The combination of experimental techniques such as absorption, fluorescence, excitation anisotropy, circular dichroism, two-photon absorption and two-photon absorption circular-linear dichroism in combination with density functional theory calculations allowed us to unambiguously distinguish and assign the spectral position of the main electronic transitions (n-[pi]* and [pi]-[pi]*) in azobenzene derivatives. Our results represent a major contribution to the understanding of the electronic structure of these organic chromophores which have been reported of potential interest in the design of optoelectronic devices. Then, Chapter V describes the development of a novel experimental technique called the synchronized double L-scan for the study of polarization dependent multiphoton absorption in chiral samples. The high sensitivity of this technique resides in the use of "twin" pulses to account for energy and mode fluctuations of the excitation pulse when determining absorption nonlinearities as a function of the light polarization. The robustness of this method was validated by measuring the first ever reported two-photon absorption circular dichroism (2PA-CD) spectrum on a chiral binaphthol derivative in solution. Finally, Chapters VI and VII compile an ample experimental and theoretical investigation of the chirality-dependent 2PA of axial enantiomers in solution. We combined the use of the synchronized double L-scan technique with state-of-the-art density functional theory calculations to provide a precise and reliable description of the contribution of the different electronic excited states to the 2PA-CD and 2PA-CLD spectra. Our findings are foreseen to have a tremendous impact in the comprehension of some of the most fundamental aspects of chiral phenomena.

<p>Two-photon excited fluorescence microscopy (TPM) has become a standard biological imaging tool due to its simplicity and versatility. The fundamental contrast mechanism is derived from fluorescence of intrinsic or extrinsic markers via simultaneous two-photon absorption which provides inherent optical sectioning capabilities. The NIR-II wavelength window (1000–1350 nm), a new biological imaging window, is promising for TPM because tissue components scatter and absorb less at longer wavelengths, resulting in deeper imaging depths and better contrasts, compared to the conventional NIR-I imaging window (700–1000 nm). However, the further enhancement of TPM has been hindered by a lack of good two-photon fluorescent imaging markers in the NIR-II. </p><p>In this dissertation, we design and characterize novel two-photon imaging markers, optimized for NIR-II excitation. More specifically, the work in this dissertation includes the investigation of two-photon excited fluorescence of various highly conjugated porphyrin arrays in the NIR-II excitation window and the utilization of nanoscale polymersomes that disperse these highly conjugated porphyrin arrays in their hydrophobic layer in aqueous environment. The NIR-emissive polymersomes, highly conjugated porphyrins-dispersed polymersomes, possess superb two-photon excited brightness. The synthetic nature of polymersomes enables us to formulate fully biodegradable, non-toxic and surface-functionalized polymersomes of varying diameters, making them a promising and fully customizable multimodal diagnostic nano-structured soft-material for deep tissue imaging at high resolutions. We demonstrated key proof-of-principle experiments using NIR-emissive polymersomes for in vivo two-photon excited fluorescence imaging in mice, allowing visualization of blood vessel structure and identification of localized tumor tissue. In addition to spectroscopic characterization of the two-photon imaging agents and their imaging capabilities/applications, the effect of the laser setup (e.g., repetition rate of the laser, peak intensity, system geometry) on two-photon excited fluorescence measurements is explored to accurately measure two-photon absorption (TPA) cross-sections. A simple pulse train shaping technique is demonstrated to separate pure nonlinear processes from linear background signals, which hinders accurate quantification of TPA cross-sections.</p> / Dissertation