The study of the in vitro circadian oscillator of the cyanobacterium Synechococcus elongatus has uncovered a complex interplay of its three protein components. Synchronization of the clock's central oscillatory component, KaiC, has been thought to be achieved through subunit shuffling at specific intervals during the clock’s period. By utilizing an established fluorescence-based analysis on completely phosphorylated and dephosphorylated mutants as well as wild-type KaiC, this study has shown that shuffling rates are largely unaffected by phosphorylation state. These findings conflict with previous reports and hence revise our understanding of this oscillator.

Non-invasive dynamic optical imaging of small animals requires the development of a novel fluorescence imaging modality. Herein, fluorescence imaging is demonstrated with sub-second camera integration times using agents specifically targeted to disease markers, enabling rapid detection of cancerous regions. The continuous-wave fluorescence imaging acquires data with an intensified or an electronmultiplying charge-coupled device. The work presented in this dissertation (i) assessed dose-dependent uptake using dynamic fluorescence imaging and pharmacokinetic (PK) models, (ii) evaluated disease marker availability in two different xenograft tumors, (iii) compared the impact of autofluorescence in fluorescence imaging of near-infrared (NIR) vs. red light excitable fluorescent contrast agents, (iv) demonstrated dual-wavelength fluorescence imaging of angiogenic vessels and lymphatics associated with a xenograft tumor model, and (v) examined dynamic multi-wavelength, whole-body fluorescence imaging with two different fluorescent contrast agents. PK analysis showed that the uptake of Cy5.5-c(KRGDf) in xenograft tumor regions linearly increased with doses of Cy5.5-c(KRGDf) up to 1.5 nmol/mouse. Above 1.5 nmol/mouse, the uptake did not increase with doses, suggesting receptor saturation. Target to background ratio (TBR) and PK analysis for two different tumor cell lines showed that while Kaposi’s sarcoma (KS1767) exhibited early and rapid uptake of Cy5.5-c(KRGDf), human melanoma tumors (M21) had non-significant TBR differences and early uptake rates similar to the contralateral normal tissue regions. The differences may be due to different compartment location of the target. A comparison of fluorescence imaging with NIR vs. red light excitable fluorescent dyes demonstrates that NIR dyes are associated with less background signal, enabling rapid tumor detection. In contrast, animals injected with red light excitable fluorescent dyes showed high autofluorescence. Dual-wavelength fluorescence images were acquired using a targeted 111In- DTPA-K(IRDye800)-c(KRGDf) to selectively detect tumor angiogenesis and an untargeted Cy5.5 to image lymphatics. After acquiring the experimental data, fluorescence image-guided surgery was performed. Dynamic, multi-wavelength fluorescence imaging was accomplished using a liquid crystal tunable filter (LCTF). Excitation light was used for reflectance images with a LCTF transmitting a shorter wavelength than the peak in the excitation light spectrum. Therefore, images can be dynamically acquired alternating frame by frame between emission and excitation light, which should enable image-guided surgery.

The vulnerability of the Arctic to climate change has been realized due to disproportionately large increases in surface air temperatures which are not uniformly distributed over the seasonal cycle. Effects of this temperature shift are widespread in the Arctic but likely include changes to the hydrological cycle and permafrost thaw, which have implications for the mobilization of organic carbon into rivers. The focus of this research was to describe the seasonal variability of the chemical composition of dissolved organic matter (DOM) in the six largest Arctic rivers (Yukon, Mackenzie, Ob, Yenisei, Lena and Kolyma) using optical properties (UV-Vis Absorbance and Fluorescence) and lignin phenol analysis. We also investigated differences between rivers and how watershed characteristics influence DOM composition. Dissolved organic carbon (DOC) concentrations followed the hydrograph with highest concentrations measured during peak river flow. The chemical composition of peak-flow DOM indicates a dominance of freshly leached material with elevated aromaticity, larger molecular weight, and elevated lignin yields relative to base-flow DOM. During peak flow, soils in the watershed are still frozen and snowmelt water follows a lateral flow path to the river channels. As the soils thaw, surface water penetrates deeper into the soil horizons leading to lower DOC concentrations and likely altered composition of DOM due to sorption and microbial degradation processes. The six rivers studied here shared a similar seasonal pattern and chemical composition. There were, however, large differences between rivers in terms of total carbon discharge reflecting the differences in watershed characteristics such as climate, catchment size, river discharge, soil types, and permafrost distribution. The large rivers (Lena, Yenisei), with a greater proportion of permafrost, exported the greatest amount of carbon. The Kolyma and Mackenzie exported the smallest amount of carbon annually, however, the discharge weighted mean DOC concentration was almost 2-fold higher in the Kolyma, again, indicating the importance of continuous permafrost. The quality and quantity of DOM mobilized into Arctic rivers appears to depend on the relative importance of surface run-off and extent of soil percolation. The relative importance of these is ultimately determined by watershed characteristics.

The objective of this work is to develop a micro/nanofluidic-based single molecule detection (SMD) scheme, which would allow us to inspect individual protein or protein complex study protein-protein interactions and their dynamics. This is a collaboration work with MD Anderson Cancer Center and we applied this scheme to study functions of various proteins related to cancer progression in hope to shed new light on cancer research. State-of-the-art micro/nano-fabrication technology is used to provide fused silica micro/nano-fluidic channel devices as our detection platform. Standard contact photolithography, projection photolithography and advanced electron-beam lithography are used to fabricate micro/nano-fluidic channel with width ranging from 100nm to 2?m. The dimensions of these miniaturized biochips are designed to ensure single molecule resolution during detection and shrinking the detection volume leads to increase in signal-to-noise ratio, which is very critical for SMD. To minimize surface adsorption of protein, a fused silica channel surface coating procedure is also developed and significantly improved the detection efficiency. A fluorescent-labeled protein sample solution is filled in the fluidic channel by capillary force, and proteins are electro-kinetically driven through the fluidic channel with external voltage source. Commercial functionalized Quantum Dots (Qdots) are used as fluorescent labels due to its various advantages over conventional organic dyes for single molecule multi-color detection application. A fluorescence correlation spectrometer system, equipped with a 375nm diode laser, 60x water immersion objective with N.A. of 1.2 and two avalanche photodiodes (APD) is implemented to excite single molecules as well as collect emitted fluorescence signals. A two-dimensional photon burst analysis technique (photon counts vs. burst width) is developed to analyze individual single molecule events. We are able to identify target protein or protein complex directly from cell lysate based on fluorescence photon counts, as well as study the dynamics of protein-protein interactions. More importantly, with this technique we are also able to assess interactions between three proteins, which cannot be done with current ensemble measurement techniques. In summary, the technique described in this work has the advantages of high sensitivity, short processing time (2-3 minutes), very small sample consumption and high resolution quantitative analysis. It could potentially revolutionize the area of protein interaction research and provides us with more clues for the future of cancer diagnostics and treatments.

This thesis presents a lithic attribute and geochemical analysis of the lithic material recovered from coastal site of Fatumafuti, on Tutuila Island, in the Samoan archipelago during 1050-520 BP. The goal of this thesis is to clarify the nature of stone tool production and to add to our current understanding of the cultural transformations from Lapita to a Polynesian identity. To complete this goal four research questions are addressed. What is the stage of reduction (cha ne operatoire) at Fatumafuti? Does the assemblage vary over space and time? Where did the source material come from? And, what was the organization of lithic craft production? Specifically, is there evidence for specialization? The lithics at Fatumafuti contain multiple segments in the technical sequence of tool manufacture (cha ne operatoire). The two major segments are middle stage and late stage reduction, and two minor segments are early stage reduction and tool rejuvenation. Expedient tools found on site indicate that prehistoric groups did not rely on a completely curated technology. Tool manufacture was geared toward producing a variety of tools, as opposed to a specific product. Production was most intense towards the coastal portion of the site during the earlier cultural component and then shifted towards the talus base during the later cultural component. Using non-destructive Energy Dispersive X-Ray Fluorescence (EDXRF), elemental concentrations were analyzed and compared to those of Tataga-matau, Lau?agae, Asiapa and Alega. One, possibly two, sources were utilized at this site; however, they are not chemically similar to Tatagamatau, Lau'agae, Asiapa and Alega. I conclude that people of Fatumafuti practiced independent household production at the end of the Aceramic and beginning of the Recent period. Either the intensification of lithic craft production that is seen during the height of complex chiefdoms is not seen at Fatumafuti, or these social transformations had not yet taken hold. With more cases that date to this time, we may find that Samoan chiefdoms had not attained full complexity at this point.

Coherent control of laser induced processes is based on the quantum interference among multiple excitation pathways. Progress in the field has been fueled by advances in pulse shaping techniques, allowing modulation of phase and amplitude across the bandwidth of ultra short pulses. This dissertation makes use of coherent control technique for the optimization of two-photon fluorescence (TPF) and its applications in selective excitation for biomedical imaging. Different physical processes, e.g. TPF, second harmonic generation (SHG) and their ratios (e.g. TPF/SHG) were optimized by using feedback control pulse shaping technique with an evolutionary algorithm. Various nonlinear effects, e.g. filamentation, intensity clamping and white light generation were studied using two-photon fluorescence and Z-scan technique with different dyes and biomarkers. Simultaneous measurements of different nonlinear effects were performed. Novel methods were proposed and implemented to obtain two-photon excitation characteristics in intensity-resolved manner. Understanding of these nonlinear effects can give new solution to the issues of spatial resolution and molecular contrast for cellular and tissue imaging.

Vulnerable plaque is a clinically silent condition of atherosclerotic plaque that leaves a large number of patients at risk of a coronary event. A method to detect vulnerable atherosclerotic plaque would greatly enhance the ability of clinicians to diagnose and treat patients at risk. Fluorescence lifetime imaging microscopy (FLIM) offers a way to extract both spatial and biochemical information from plaque by taking several wide-field images over time. The goal of this study was to determine the potential of a FLIM angioscopy system to detect and differentiate coronary atherosclerotic plaques ex-vivo into several groups including thin, fibrotic, lipid-laden, thick-cap fibroatheroma (FA), and fibrocalcified. Samples were extracted post-mortem weekly and sliced open to have their lumens imaged. For each sample, 51 time resolved wide-field images were taken over 10 nanoseconds at 390 (±40) nm, 450 (±40) nm, and 550 (±88) nm wavelengths. To analyze the samples, the intensity map and lifetime map were created at each wavelength. The intensity map was simply the wide-field images summed in time and normalized. In order to calculate lifetime at each point, a fast, model-free Laguerre deconvolution algorithm was recently developed for FLIM data analysis and was used. This allowed for fast, efficient estimations of the fluorescence decay curves at each pixel of the FLIM images and facilitated the computation of quantitative parameters describing the fluorescence emission of the tissue, specifically, the relative fluorescence intensity and lifetime at defined emission bands. Statistical analysis on these FLIM derived parameters indicated that the autofluorescence emission of the plaques allows for distinguishing relative plaque thickness: thin plaque, whose signal is dominated by elastin fluorophores, shows a marked difference between thicker plaques, such as fibrotic, fibrocalcified and thick-cap FA (who are dominated primarily by collagen). However, the ability of the current FLIM system to differentiate vulnerable plaque remains in question due to the absence of thin-cap FA samples. Further work has also been proposed; of primary concern is gathering thin-cap FA plaque samples needed to validate the system’s ability to differentiate vulnerable plaques from other common groupings.

Many Americans are afflicted with inflammation of the colon. They are also at a higher risk of developing colon cancer. Confocal microscopy of bulk epithelial tissue has the potential to provide information on tissue structural properties that may be lost in the fixation and slicing procedures required for histopathology. Optical sectioning provides images in three dimensions capturing the organizational structure of cells and colon crypts throughout the entire colon. I have constructed a custom built fluorescence and reflectance confocal microscope for imaging molecular and morphological changes associated with development of inflammation in a mouse model. A confocal microscope is a point scanning system that removes out of focus light by placing a pinhole aperture in the conjugate image plane located in front of the detector. We have two sources, 488 nm and 811 nm, for fluorescence and reflectance imaging, respectively. A polygon scanning mirror and a galvanometer scanning mirror allow for a variable scan rate between 8 and 15 fps. The lateral resolution of the system is approximately 3 μm with an axial resolution of 6 μm and 4 μm for reflectance and fluorescence mode, respectively. As colon tissue becomes inflamed, there is a distinct change in the structure and architecture of the tissue. The colon crypts are no longer uniform in size or distribution throughout the tissue. Having a large field of view of 1mm2 allows for many colon crypts to be visualized within a single frame. Histology was performed on the same tissue imaged for the inflammatory study confirming the constructed confocal microscope’s ability to characterize inflamed tissue and the potential use for guided biopsy. Mosaicing, or image tiling, is an imaging technique that stitches single frames together to produce a much larger field of view. An extended frame with 1 mm x 2 cm field of view is achieved within seconds. This extended frame would allow mosaicing of the entire mouse colon much faster than conventional methods without loss of resolution. The acquired confocal images of colon tissue demonstrate the microscope’s ability to resolve cell nuclei lining the colon crypts within a relatively large field of view.

The maximum quantum yield (Fv/Fm), zooxanthellae density, chlorophyll a concentration and protein concentration of non-bleached and bleached colonies of the reef coral Acropora intermedia were measured in inlet of The Third Nuclear Power Plant of Nanwan Bay in southern Taiwan. A significant positive correlation was found between Fv/Fm and chlorophyll a concentration per zooxanthellae of non-bleached colonies. The chlorophyll a concentration per zooxanthellae and zooxanthellae density of non-bleached colonies were lowest in summer, and were significantly negative correlated with total radiant heat and seawater temperature, respectively. This suggests that the seasonal variation exist, and they maybe regulated by seasonal fluctuation of radiation and temperature. The Fv/Fm, chlorophyll a concentration per cm-2, chlorophyll a concentration per zooxanthellae, zooxanthellae density and protein were significantly lower than those of the non-bleached colonies in the bleaching events. However, compared to the non-bleached colonies, zooxanthellae density, chlorophyll a concentration per cm-2 and protein of bleached colonies were increased and significantly higher than those of non-bleached colonies, then decreased to similar level. However, Fv/Fm increased to similar level, but chlorophyll a concentration per zooxanthellae remained lower. It suggests that number of zooxanthellae rapidly increased while remained stable chlorophyll a concentration during recovery.

In this study, we have successfully demonstrated the use of laser scanning confocal microscopy in studying the fluorescence spectroscopy of Cr3+ and Cr4+ ions in Cr:YAG crystal fibers, double-clad crystal fibers, and glass fibers.