Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 58-60).

Thesis (M.S.)--Oregon Graduate Center, 1979.

Thesis (Ph. D.)--Oregon Graduate Center, 1980.

This dissertation focuses on laser-based noninvasive photoacoustic microscopy of subsurface structures in vivo. Photoacoustic microscopy is a hybrid imaging modality that combines the high-resolution advantage of ultrasonic imaging in deep tissue with the high-contrast advantage of optical imaging. It detects the short-pulsed laser-induced photoacoustic waves, whose amplitudes reflect the localized laser energy absorption, to image the internal optical absorption distributions. The spatial resolution is determined by the ultrasonic focal ability and the ultrasonic bandwidth. The imaging depth is primarily limited by the acoustic attenuation within the reach of diffuse photons. The ratio of maximum imaging depth to axial resolution in photoacoustic microscopy is greater than 100, which is comparable to that of modern high-resolution optical imaging modalities, such as confocal microscopy, two-photon microscopy, and optical coherence tomography. However, the maximum imaging depth has been much enlarged by taking advantages of absorbed diffuse photons. Based on the intrinsic optical contrast, we have achieved in vivo volumetric imaging of subcutaneous microvasculature, skin melanoma, and acute thermal injuries in high spatial resolution. We have imaged physiological parameters in subcutaneous microvessels, such as total hemoglobin concentration and hemoglobin oxygen saturation, on a single vessel basis in small animals in vivo. We have also monitored changes of hemoglobin oxygen concentration between different systemic physiological states on a vessel-by-vessel basis. Moreover, we have demonstrated the feasibility of human imaging using photoacoustic microscopy by imaging finger tips and subcutaneous palm vessels.

Single-molecule (SM) fluorescence spectroscopy has become a useful and important experimental approach for investigating the optical properties of chemical systems. In this thesis, four subprojects in the field of SM fluorescence spectroscopy are presented in which SM spectroscopy has provided invaluable experimental insight into the systems of interest.<p> In the first project, the photophysical properties of Calcium-Green 1 (CG-1), a calcium-ion indicator, were studied at both the ensemble and SM levels. CG-1 is non-fluorescent in the absence of Ca2+ and becomes strongly fluorescent when bound to Ca2+. In the ensemble measurements, the absorption and fluorescence spectra were collected under various Ca2+ concentrations. In addition, the fluorescence lifetime of CG-2 was also studied as a function of [Ca2+]. From SM measurements, the photobleaching time and fluorescence intensity distributions of CG-1 were studied both in the presence and in absence of Ca2+. The results were compared with those obtained for the dual-fluorophoric variant, Calcium-Green 2 (CG-2), whose photophysical properties have been investigated by previous researchers. The experimental results reveal that CG-1 can exist in two different forms: a highly-quenched form due to the occurrence of photoinduced electron transfer (PET) in the absence of Ca2+, and a strongly fluorescent form when bound to Ca2+.<p> The second project is a continuation of a previous study on CG-2. In the dual-chromophore CG-2 system, energy transfer between chromophores is controlled by the orientation and spatial separation between chromophores. Dual polarization fluorescence microscopy was used to determine the relative conformation of the two fluorophores in the emissive form of CG-2. Distributions of fluorescence polarization of individual CG-2 molecules were collected for both Ca2+-free and Ca2+-saturated conditions. The experimental polarization results were compared to those calculated from a simple geometric model based on randomly-orientated fluorescent dimers. The results show good agreement with previous calculations of the molecular conformation of CG-2. This indicates that the dual polarization imaging approach has significant potential as a general tool for characterizing chromophore orientation in coupled-fluorophore systems.<p> In the third project, Nile Red (NR), a solvatochromic lipid stain, was incorporated into phase separated Langmuir-Blodgett (LB) films composed of arachidic acid (AA) and perfluorotetradecanoic acid (PA). According to previous studies by atomic force microscopy (AFM), two types of separated domains are formed in the LB films: micron-sized hexagonal discontinuous domains that are exclusively comprised of AA, and the surrounding continuous domains which are enriched in PA. The photophysical properties of NR were characterized in the two physically and chemically distinct domains via bulk and SM fluorescence measurements. In addition to fluorescence microscopy, fluorescence confocal spectromicroscopy was also applied in the ensemble measurements to determine the spectral properties of NR in different sub-environments. Experimental results indicated that a small sub-population of dye molecules localize on the perfluorinated regions of the sample, but this sub-population is spectroscopically indistinguishable from that associated with the hydrogenated domains. Contrast in images was primarily due to preferential accumulation of the hydrophobic dye on the hydrophobic regions of the LB films.<p> In the final project, the fluorescence quenching behavior of a strongly fluorescent probe Alexa Fluor 514 (AF514) was investigated when it was covalently bound to gold metal protected clusters (AuMPC) with negligible plasmon bands. The fluorescence emission of the dye-AuMPCs system was characterized at different dye/Au MPC loading ratios with a combination of steady state and time-resolved ensemble spectroscopic measurements. It was found that the extent of fluorescence quenching in the system was small. After correction of inner filter effects, the results from bulk measurement demonstrate that the weak quenching is due to static quenching of the dye by the AuMPCs. SM measurements provided further support for the bulk measurements, with the fluorescence intensity of coupled AF514 molecules being comparable with that of unconjugated molecules. The photobleaching of the dye-AuMPC conjugates took place as a series of consecutive photobleaching events, without additional blinking dynamics within the time resolution of the experiment. These results suggest that the fluorophores on the AuMPCs are either entirely quenched, or remaining unquenched, as is consistent with the ensemble measurements.

Single-molecule (SM) fluorescence spectroscopy has become a useful and important experimental approach for investigating the optical properties of chemical systems. In this thesis, four subprojects in the field of SM fluorescence spectroscopy are presented in which SM spectroscopy has provided invaluable experimental insight into the systems of interest.<p> In the first project, the photophysical properties of Calcium-Green 1 (CG-1), a calcium-ion indicator, were studied at both the ensemble and SM levels. CG-1 is non-fluorescent in the absence of Ca2+ and becomes strongly fluorescent when bound to Ca2+. In the ensemble measurements, the absorption and fluorescence spectra were collected under various Ca2+ concentrations. In addition, the fluorescence lifetime of CG-2 was also studied as a function of [Ca2+]. From SM measurements, the photobleaching time and fluorescence intensity distributions of CG-1 were studied both in the presence and in absence of Ca2+. The results were compared with those obtained for the dual-fluorophoric variant, Calcium-Green 2 (CG-2), whose photophysical properties have been investigated by previous researchers. The experimental results reveal that CG-1 can exist in two different forms: a highly-quenched form due to the occurrence of photoinduced electron transfer (PET) in the absence of Ca2+, and a strongly fluorescent form when bound to Ca2+.<p> The second project is a continuation of a previous study on CG-2. In the dual-chromophore CG-2 system, energy transfer between chromophores is controlled by the orientation and spatial separation between chromophores. Dual polarization fluorescence microscopy was used to determine the relative conformation of the two fluorophores in the emissive form of CG-2. Distributions of fluorescence polarization of individual CG-2 molecules were collected for both Ca2+-free and Ca2+-saturated conditions. The experimental polarization results were compared to those calculated from a simple geometric model based on randomly-orientated fluorescent dimers. The results show good agreement with previous calculations of the molecular conformation of CG-2. This indicates that the dual polarization imaging approach has significant potential as a general tool for characterizing chromophore orientation in coupled-fluorophore systems.<p> In the third project, Nile Red (NR), a solvatochromic lipid stain, was incorporated into phase separated Langmuir-Blodgett (LB) films composed of arachidic acid (AA) and perfluorotetradecanoic acid (PA). According to previous studies by atomic force microscopy (AFM), two types of separated domains are formed in the LB films: micron-sized hexagonal discontinuous domains that are exclusively comprised of AA, and the surrounding continuous domains which are enriched in PA. The photophysical properties of NR were characterized in the two physically and chemically distinct domains via bulk and SM fluorescence measurements. In addition to fluorescence microscopy, fluorescence confocal spectromicroscopy was also applied in the ensemble measurements to determine the spectral properties of NR in different sub-environments. Experimental results indicated that a small sub-population of dye molecules localize on the perfluorinated regions of the sample, but this sub-population is spectroscopically indistinguishable from that associated with the hydrogenated domains. Contrast in images was primarily due to preferential accumulation of the hydrophobic dye on the hydrophobic regions of the LB films.<p> In the final project, the fluorescence quenching behavior of a strongly fluorescent probe Alexa Fluor 514 (AF514) was investigated when it was covalently bound to gold metal protected clusters (AuMPC) with negligible plasmon bands. The fluorescence emission of the dye-AuMPCs system was characterized at different dye/Au MPC loading ratios with a combination of steady state and time-resolved ensemble spectroscopic measurements. It was found that the extent of fluorescence quenching in the system was small. After correction of inner filter effects, the results from bulk measurement demonstrate that the weak quenching is due to static quenching of the dye by the AuMPCs. SM measurements provided further support for the bulk measurements, with the fluorescence intensity of coupled AF514 molecules being comparable with that of unconjugated molecules. The photobleaching of the dye-AuMPC conjugates took place as a series of consecutive photobleaching events, without additional blinking dynamics within the time resolution of the experiment. These results suggest that the fluorophores on the AuMPCs are either entirely quenched, or remaining unquenched, as is consistent with the ensemble measurements.

This dissertation focuses on laser-based noninvasive photoacoustic microscopy of subsurface structures in vivo. Photoacoustic microscopy is a hybrid imaging modality that combines the high-resolution advantage of ultrasonic imaging in deep tissue with the high-contrast advantage of optical imaging. It detects the short-pulsed laser-induced photoacoustic waves, whose amplitudes reflect the localized laser energy absorption, to image the internal optical absorption distributions. The spatial resolution is determined by the ultrasonic focal ability and the ultrasonic bandwidth. The imaging depth is primarily limited by the acoustic attenuation within the reach of diffuse photons. The ratio of maximum imaging depth to axial resolution in photoacoustic microscopy is greater than 100, which is comparable to that of modern high-resolution optical imaging modalities, such as confocal microscopy, two-photon microscopy, and optical coherence tomography. However, the maximum imaging depth has been much enlarged by taking advantages of absorbed diffuse photons. Based on the intrinsic optical contrast, we have achieved in vivo volumetric imaging of subcutaneous microvasculature, skin melanoma, and acute thermal injuries in high spatial resolution. We have imaged physiological parameters in subcutaneous microvessels, such as total hemoglobin concentration and hemoglobin oxygen saturation, on a single vessel basis in small animals in vivo. We have also monitored changes of hemoglobin oxygen concentration between different systemic physiological states on a vessel-by-vessel basis. Moreover, we have demonstrated the feasibility of human imaging using photoacoustic microscopy by imaging finger tips and subcutaneous palm vessels.

Lithium niobate(LiNbO3)has been widely used in optical frequency converter, electro-optical modulator¡Asurface acoustic waveguide filter¡Aand photorefractive recording due to its ease of growth, high nonlinear coefficient, and excellent optical quality. We think that we can utilize second harmonic generation (SHG) and laser scanning system to construct a reflective SHG system .From this system we can obtain PPLN poling pattern, then we can check the growth condition correctly or not. Compare to traditional method, this approach has the advantage of fast and non-invasive test. Besides, we can find crystal axis of crystal by changing the angle of incidence light and using additional half plate.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 115-131). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 158-169). Available also in a digital version from Dissertation Abstracts.