A new molecular imaging modality has been developed to detect and locate positive axillary and sentinel lymph nodes non-invasively in breast cancer patients undergoing lymphoscintigraphy. The modality is based on fluorescent photon detection to locate the presence of indocyanine green (ICG) in the lymph subsequent to peritumoral injection of ICG into the breast. The imaging system consists of a gain-modulated intensified charge-coupled device (ICCD) camera, which captures low-intensity, near-infrared, and frequency-modulated photons. A four-fold ‘optical lymphography’ study was conducted to (1) examine fluorescence depth penetration and ICCD system accuracy at clinically relevant depths, (2) compare image quality of the ICCD system vs. conventional gamma imaging, (3) measure ICG pharmacokinetics in vivo, and (4) develop a clinical protocol while examining pre-clinical factors such as the outcome of combining ICG with sulfur colloids used in lymphoscintigraphy. The frequency-domain ICCD system was found to precisely detect modulation amplitude, IAC, and phase, θ, at depths up to 9 cm and with IAC accuracy less than 20% and θ less than 2º using an 80-mW laser incident on phantoms having ranging tissue optical properties. Significant differences in the mean depth of penetration owing to 0.62-ns lifetime and 100-MHz frequency increases were detected. An in vivo optical vs. nuclear image quality comparison demonstrated statistically similar (α=0.05) target-to-background ratios for optical (1.4+/-0.3) and nuclear (1.5+/-0.2). Alternatively, resulting image signal-to-noise ratios (SNR) from the ICCD system were greater than that achieved with a conventional gamma camera (pvalue<<0.01). Analysis of SNR versus contrast showed greater sensitivity of optical over nuclear imaging for subcutaneous tumors. In vivo and rapid detection of ICG in the blood-stream of nude mice was accomplished with a home-built avalanche photodiode dynamic fluorescence measurement system. Intensity data upon i.v. injection were regressed with a pharmacokinetic model describing the partitioning of ICG from the blood to the surrounding tissues. ICG blood-clearance was detected approximately 15 min after injection. Lastly, a human subject protocol was written, practiced, and federally approved for the application of optical lymphography. Furthermore, ICG was unaffected when mixed with sulfur colloids thus supporting the feasibility for combining fluorescence imaging with lymphoscintigraphy in breast cancer patients.

<p>This thesis represents a quantum chemical treatise ofvarious types of interactions between radiation and molecularsystems, with special emphasis on the nonlinear opticalprocesses of Multi-Photon Absorption and Excited StateAbsorption. Excitation energies, transition dipole moments,two-photon and three-photon tensor elements have beencalculated from different approaches; density functional theoryand<i>ab-initio</i>theory, employing different orders ofcorrelation treatment with the purpose to provide accuratevalues as well as evaluate the quality of the lower ordermethods. A combined study of the Multi-Photon Absorption andExcited State Absorption processes is motivated partly becausethey both contribute to the total optical response of a systemsubjected to intense radiation, but also because of theirconnection through so-called sum-over-states expressions. Thelatter feature is exploited in a generalized few-states model,which incorporates the polarization of the light and thedirections of the transition dipole moments constructing anexcitation channel, which thereby enables a more comprehensivecomparison of the attained transition dipole moments withexperimental data. Moreover, by decomposing a complex nonlinearresponse process such as Two-Photon Absorption into moreintuitive quantities, generalized few-states models may alsoenable a more elaborate interpretation of computed orexperimental results from which guidelines can be extracted inorder to control or optimize the property of interest. Ageneral conclusion originating from these models is that thetransition dipole moments in an excitation channel should bealigned in order to maximize the Two-Photon Absorptionprobability. The computational framework employed is responsetheory which through the response functions (linear, quadratic,cubic) offers alternative routes for evaluating the propertiesin focus; either directly and untruncated through the singleresidue of the quadratic or cubic response func- tions orthrough various schemes of truncated sum-over-statesexpressions where the key ingredients, transition dipolemoments, can be identified from the single residue of thelinear response function and double residue of the quadraticresponse function. The range of systems treated in the thesisstretches from diatomics, such as carbon monoxide and lithiumhydride, via small to large fundamental organic molecules, suchas formaldehyde, tetrazine and the trans-polyenes, to largechro- mophores, such as<i>trans</i>-stilbene, cumulenes, dithienothiophene,paracyclophane and organo-metallic systems, such as theplatinum(II)ethynyl compounds.</p>

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Supervisor: Joe C. Campbell. Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (M. S.)--Electrical and Computer Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Shen, Shyh-Chiang; Committee Member: Doolittle, William A.; Committee Member: Dupuis, Russell Dean. Part of the SMARTech Electronic Thesis and Dissertation Collection.

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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 thesis focuses on the ultrafast isomerization reaction of retinal in both rhodopsin and bacteriorhodopsin, examples of sensory and energy transduction proteins that exploit the same photoactive chromophore for two very different functions. In bacteriorhodopsin, retinal isomerizes from an all-trans to 13-cis conformation as the primary event in light- driven proton pumping. In the visual pigment rhodopsin, the retinal chromophore isomerizes from an 11-cis to all-trans geometry as the primary step leading to our sense of vision. This diversity of function for nominally identical systems raises the question as to just how optimized are these proteins to arrive at such drastically different functions? Previous work has employed transient absorption spectroscopy to probe retinal protein photochemistry, but many of the relevant electronic and nuclear dynamics of isomerization are masked by inhomogeneous broadening effects and strong spectral overlap between reactant and photoproduct states. This work exploits the unique properties of two-dimensional photon echo spectroscopy to deconvolve inhomogeneous broadening and spectral overlap effects and fully reveal the dynamics that direct retinal isomerization in proteins. In bacteriorhodopsin, vibrational coupling to the reaction coordinate results in a surface crossing event prior to the conventional conical intersection associated with isomerization to the J intermediate. In rhodopsin, however, a similarly early vibrationally-mediated barrier crossing event is observed, resulting in spectral signals consistent with the known photoproduct state appearing an order of magnitude faster than determined from conventional transient absorption measurements. The competing overlapping spectral signals that obscured the initial dynamics when probed with transient absorption spectroscopy are now clearly resolved with two-dimensional photon echo spectroscopy. These experiments illustrate the critical role of the protein in directing the outcome of retinal photochemistry. The protein controls the reaction pathway through steric interactions between the binding pocket and the retinal chromophore, the result of which directly sets the isomerization coordinate and indirectly controls the vibrational coupling to the reaction coordinate based on the local retinal structure. The new insight from this work is the extraordinary degree of selective vibrational coupling involved in directing the isomerization reaction in retinal proteins.

This thesis focuses on the ultrafast isomerization reaction of retinal in both rhodopsin and bacteriorhodopsin, examples of sensory and energy transduction proteins that exploit the same photoactive chromophore for two very different functions. In bacteriorhodopsin, retinal isomerizes from an all-trans to 13-cis conformation as the primary event in light- driven proton pumping. In the visual pigment rhodopsin, the retinal chromophore isomerizes from an 11-cis to all-trans geometry as the primary step leading to our sense of vision. This diversity of function for nominally identical systems raises the question as to just how optimized are these proteins to arrive at such drastically different functions? Previous work has employed transient absorption spectroscopy to probe retinal protein photochemistry, but many of the relevant electronic and nuclear dynamics of isomerization are masked by inhomogeneous broadening effects and strong spectral overlap between reactant and photoproduct states. This work exploits the unique properties of two-dimensional photon echo spectroscopy to deconvolve inhomogeneous broadening and spectral overlap effects and fully reveal the dynamics that direct retinal isomerization in proteins. In bacteriorhodopsin, vibrational coupling to the reaction coordinate results in a surface crossing event prior to the conventional conical intersection associated with isomerization to the J intermediate. In rhodopsin, however, a similarly early vibrationally-mediated barrier crossing event is observed, resulting in spectral signals consistent with the known photoproduct state appearing an order of magnitude faster than determined from conventional transient absorption measurements. The competing overlapping spectral signals that obscured the initial dynamics when probed with transient absorption spectroscopy are now clearly resolved with two-dimensional photon echo spectroscopy. These experiments illustrate the critical role of the protein in directing the outcome of retinal photochemistry. The protein controls the reaction pathway through steric interactions between the binding pocket and the retinal chromophore, the result of which directly sets the isomerization coordinate and indirectly controls the vibrational coupling to the reaction coordinate based on the local retinal structure. The new insight from this work is the extraordinary degree of selective vibrational coupling involved in directing the isomerization reaction in retinal proteins.

No description available.

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.