The self-assembly of several pyrene-labelled amphiphilic macromolecules in water was characterized by fluorescence. Information on their self-assembly was obtained by monitoring the level of pyrene aggregation in solution. A measure of the level of association was obtained by determining the fraction of aggregated pyrene of the labelled macromolecules from the global analysis of their monomer and excimer fluorescence decays. Global analysis limits the degrees of freedom of the analysis thus reducing the error on the parameters retrieved from the analysis. Extensive developments in the global analysis of the pyrene monomer and excimer decays enabled the first characterization of the molar absorbance coefficient of the pyrene aggregates formed by aqueous solutions of pyrene-labelled poly(N,N-dimethylacrylamide) (PyPDMA) and poly(ethylene oxide) (PyPEO). The molar absorbance coefficients of the pyrene aggregates determined for PyPDMA and PyPEO were both found to be broader and red-shifted compared to that of unaggregated pyrene. These results agree with observations found in the scientific literature made by using absorption and excitation fluorescence measurements. Attempts to determine the molar absorbance coefficient of pyrene-labelled hydrophobically-modified alkali-swellable emulsion (PyHASE) polymers were unsuccessful. The inability to characterize the pyrene aggregates of PyHASE was attributed to the greater complexity of the PyHASE polymer compared to PyPDMA and PyPEO. For these simpler pyrene-labelled polymers, a protocol has been established which uses the global analysis of the pyrene monomer and excimer decays to determine quantitatively the level of association of pyrene-labelled polymers as well as the molar absorbance coefficient of their aggregates. Changes in the level of aggregation of pyrene-labelled lipids (PLLs) having head groups bearing an alcohol (PSOH) or imido diacetic acid (PSIDA) embedded in 1-palmitoyl-2-oleyl-3-sn-phosphatidylcholines (POPC) or distearylphosphatidylcholine (DSPC) liposomes were probed by fluorescence. Distribution of the PLLs in the fluid POPC membrane was found to be homogeneous while the PLLs phase-separated into amorphous channels created in the DSPC membranes. Multivalent cations Cu2+ and La3+ were found to bind to PSIDA, hindering diffusional encounters between unaggregated PSIDA but leaving the PLL aggregates intact. Using the fluorescence quenching ability of Cu2+, the viscosity of the amorphous channels of the DSPC membrane was determined to be about six times greater than that of the more fluid POPC membrane. Simultaneous rheological and fluorescence measurements were achieved by interfacing a rheometer with time-resolved and steady-state fluorometers using fiber-optic cables. This joint set up enabled the simultaneous rheological and fluorescence measurements of PyHASE solutions having concentrations ranging from 0.5 w/w% to 5 w/w%. The level of association of the PyHASE solutions was tracked using fluorescence at shear rates of 0, 0.1 and 100 s–1. Despite the presence of shear thinning leading to viscosity drops of up to four orders of magnitude, no change in the fluorescence and hence the level of association was observed. The lack of change in level of association implied that the mechanism of shear thinning is due to a switching from inter- to intramolecular association rather than a drop in the level of association. This information will prove useful for future models attempting to predict the rheological behaviour of sheared associative polymers.

Color plays a vitally important role in the world we live in. It surrounds us everywhere we go. Achromatic life, restricted to black, white and grey, is extremely dull. Color fascinates artists, for it adds enormously to aesthetic appreciation, directly invoking thoughts, emotions and feelings. Color fascinates scientists. For decades, scientists in color imaging, printing and digital photography have striven to satisfy increasing demands for accuracy in color reproduc- tion. Fluorescence is a very common phenomenon observed in many objects such as gems and corals, writing paper, clothes, and even laundry detergent. Traditional color imaging algo- rithms exclude fluorescence by assuming that all objects have only an ordinary reflective com- ponent. The first part of the thesis shows that the color appearance of an object with both reflective and fluorescent components can be represented as a linear combination of the two components. A linear model allows us to separate the two components using independent component analysis (ICA). We can then apply different algorithms to each component, and combine the results to form images with more accurate color. Displaying color images accurately is as important as reproducing color images accurately. The second part of the thesis presents a new, practical model for displaying color images on self-luminous displays such as LCD monitors. It shows that the model accounts for human visual system’s mixed adaptation condition and produces results comparable to many existing algorithms.

The desert ant Cataglyphis fortis is one of the hyperthermophilic species of Cataglyphis. It lives in the Sahara desert and forages during the hottest hours of the day when it can get up to 70˚C in the sand. The body temperature of the ant during the foraging runs can reach a maximum of 55˚C. Since C.fortis is one of few eukaryotic hyperthermophilic species, its proteins probably have a high thermostability. Investigating the thermostability can give valuable information about the principles of protein folding and stability in hyperthermophiles.Fatty acid binding proteins (FABPs) have an important role in the cell taking up and transporting fatty acids and regulating metabolic and inflammatory pathways. FABPs have been extensively studied and structures from several species have been determined. The determined structures of all FABPs are very similar why thermostability studies of FABP from C.fortis are highly relevant.Fluorescence spectroscopy is an easy and fast method to measure intrinsic protein fluorescence. Tryptophans were genetically introduced into three different positions in FABP to be used as environmental sensitive probes. Complementing the measurement results with a model of the 3D structure of FABP from C.fortis gave additional information about the ligand binding.The (local) thermostability of the mutants can be detected by shift in wavelength maximum during temperature ramping experiments. All mutants are stabilised in the presence of fatty acids. The mutant with tryptophan positioned closest to the supposed ligand binding residues (Y11W) is most affected. The mutant with tryptophan situated farthest from the supposed binding residues (Y52W) shows a stabilisation of Tm less evident than for Y11W. Thus, the structural changes following fatty acid binding are more obvious in the environment close to the binding site.However, the third mutant C87W shows no significant stabilisation although positioned closer to the fatty acid binding site than Y52. This is probably due to the size difference between the original and introduced amino acid in the mutation. Since the high value of the starting λmax for C87W implies that C87W is quite exposed to the aqueous solvent, the residue is likely to not have subsumed in the protein tertiary structure.Further, the myristic acid stabilise the melting temperature of all the mutants while octanoic acid only has a local effect of Y11W increasing the cooperativity. This implies different binding properties and that myristic acid stabilise the entire protein while octanoic acid only has a local stabilisation effect around the ligand binding site.

Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in the ultraviolet to visible wavelength range indicated that the most diagnostic optical signals originate from sub-surface tissue layers. Optical properties extracted from these spectroscopy measurements showed a significant decrease in the hemoglobin saturation, absorption coefficient, reduced scattering coefficient and fluorescence intensity (at 400 nm excitation) in neoplastic compared to normal tissues. The results from these studies indicate that multiphoton microscopy and optical spectroscopy can non-invasively provide information on tissue structure and function in vivo that is related to tissue pathology. / Dissertation

<p>Biosensors incorporating proteins as molecular recognition elements for analytes are used in clinical diagnostics, as biological research tools, and to detect chemical threats and pollutants. This work describes the application of protein engineering techniques to address three aspects in the design of protein-based biosensors; the transduction of binding into an observable, the manipulation of affinities, and the diversification of specificities. The periplasmic glucose-binding protein from the hyperthermophile Thermotoga maritima (tmGBP) was fused with green fluorescent protein variants to construct a fluorescent ratiometric sensor that is sufficiently robust to detect glucose up to 67°C. Ligand-binding affinities of tmGBP were changed by altering a C-terminal helical domain that tunes ligand binding affinity through conformational coupling effects. This method was extended to the Escherichia coli arabinose-binding protein. Computational design techniques were used to diversify the specificity of the E. coli maltose-binding protein (ecMBP) to bind ibuprofen, a non-steroidal antiinflammatory drug. These designs ranged in affinity from 0.24 to 0.8 mM and function as reagentless fluorescent sensors. The ligand affinities of ecMBP are tuned by complex interactions that control conformational coupling. These experiments demonstrate that long-range conformational effects as well as molecular recognition interactions need to be considered in the design of high-affinity receptors.</p> / Dissertation

Fluorescent nano-diamond(FND) is an unique fluorescence bio-labeling materials, which exhibit good fluorescence yield, excellent photostability, and non-toxicity. The emission color of FND is determined by the defect centers in the diamond crystal. When the defect center composed of one vacancy and two nearest-neighborhood nitrogen substitutes, it forms a H3 center. H3 center has a zero-phonon line at 496nm , and a broadband green emission around 530 nm,. When the FND contains lots of H3 centers, the emission color is green, hence it¡¦s called green FND(gFND). Since H3 centers composed of two nitrogen substitutes, it is naturally to fabricate the gFNDs by diamonds with high nitrogen substitutes. However, H3 center is not the only products when the diamond contains many nitrogen substitutes, and high density of vacancies. Other type of defect centers (NV-, NV0, ¡K) exhibit lower energy gap, and quench the emission of H3 centers. In this thesis, it aims to study the spectroscopic homogeneity of the gFNDs. Comparing the intensity of the scattering images and the corresponding fluorescence images, it provides the information of the relation between particle size and the density of color centers. Furthermore, images with different color filters are compared to provide the information of the composition of defect structures. Fluorescence lifetime image is performed for the emission dynamics of the nano-particle. The results indicate that the decay lifetime has an relation to the emission intensity. When the nano-particle contains more color centers, it quenches the emission from H3 centers more.

In recent years, the Surface Plasmon Polariton effect has played an important role for entering the Nano-world. When the metallic materials reach the nanometer level, many special characteristics show up. As the progress of advanced technology development, the equipments which can be operated in nano grade level are more stabilized. Many special surface Plasmonic properties have been discovered through the measurements. This research is to focus on using the Surface Plasmon coupling to excite colloidal quantum dots and observing the emissive behavior of quantum dots. The experiments of changing the distance between the quantum dots and the metal film were performed. The blinking effect disappeared when the quantum dots are very close to the metal film. It showed that some other mechanism is competing with Auger recombination in the quantum dots. The lifetime modification and emission intensity were measured when one quantum dot was placed near a silver cube. The coupling between the surface Plasmon polariton and the quantum dot was discussed.

The photodissociation dynamics of cyanogen iodide (ICN) in the A continuum was studied by the three-dimensional sliced fluorescence imaging (3DSFI) method. The fourth harmonic of a Nd:YAG laser (266 nm) was used to dissociate a low pressure (5 mTorr) sample of ICN, and the resulting CN photofragments were probed by optical-optical double resonance (OODR) detection scheme. A theoretical framework on the emitted fluorescence intensity has been developed to analyze the experimentally obtained image. The vector correlations among the ICN parent molecule transition dipole moment £g, the CN fragment recoil velocity v and the CN rotational angular moment J can be revealed.

Cooperative effect in the radiation process has been studied in for more than half a century. It is important in the sense of both basic physics and applied science. In this work, we study the dynamics of the cooperative spontaneous emission from an ensemble of N atoms which is uniformly excited by absorbing a single photon. We reveal that there are two different regimes in which the system exhibits totally different behaviors. One of them is the superradiance type of behavior: the system decays much quicker than single atom decay, with a decay rate proportional to N(λ/R)2, where N is the atom numbers, R is the size of the atom cloud, and λ is the wavelength. We call it Markovian regime because the sytem does not persist memory effect. The other regime is called non-Markovian regime and the system oscillates with effective Rabi oscillation frequency while slowly decaying with a rate proportional to the photon escaping rate. The effective Rabi oscillation is a new type of dynamics which analogs well known Cavity QED behavior. Particularly in the Markovian regime, we study the system dynamics as a manybody eigenfunction and eigenvalue problem. For a dense cloud, we find analytical solutions for the eigenstates and corresponding eigenvalues, which can help to generally describe the system dynamics for any initial conditions in this regime. One of the applications is in atom microscopy. We propose a scheme to measure the distance between two atoms/molecules beyond diffraction limit. It covers the whole range from half the wavelength to sub-nanometers, utilizing both the atom localization technique and the collective frequency shift effect due to the cooperative effect in the radiation of the two atoms. Another application that we propose is to generate Coherent XUV radiation using Raman-type superradaince. We prove that intense short pulses of XUV radiation can be produced by Raman type superradiance from an ensemble of atoms/ions driven by visible or IR laser pulses.

The quantification of plutonium (Pu) in spent nuclear fuel is an increasingly important safeguards issue. There exists an estimated worldwide 980 metric tons of Pu in the nuclear fuel cycle and the majority is in spent nuclear fuel waiting for long term storage or fuel reprocessing. This study investigates utilizing the measurement of x-ray fluorescence (XRF) from the spent fuel for the quantification of its uranium (U) to Pu ratio. Pu quantification measurements at the front end of the reprocessing plant, the fuel cycle area of interest, would improve input accountability and shipper/receiver differences. XRF measurements were made on individual PWR fuel rods with varying fuel ages and final burn-ups at Oak Ridge National Laboratory (ORNL) in July 2008 and January 2009. These measurements successfully showed that it is possible to measure the Pu x-ray peak at 103.7 keV in PWR spent fuel (~1 percent Pu) using a planar HPGe detector. Prior to these measurement campaigns, the Pu peak has only been measured for fast breeder reactor fuel (~40 percent Pu). To understand the physics of the measurements, several modern physics simulations were conducted to determine the fuel isotopics, the sources of XRF in the spent fuel, and the sources of Compton continuum. Fuel transformation and decay simulations demonstrated the Pu/U measured peak ratio is directly proportional to the Pu/U content and increases linearly as burn-up increases. Spent fuel source simulations showed for 4 to 13 year old PWR fuel with burn-up ranges from 50 to 67 GWd/MTU, initial photon sources and resulting Compton and XRF interactions adequately model the spent fuel measured spectrum and background. The detector simulations also showed the contributions to the Compton continuum from strongest to weakest are as follows: the fuel, the shipping tube, the cladding, the detector can, the detector crystal and the collimator end. The detector simulations showed the relationship between the Pu/U peak ratio and fuel burn-up over predict the measured Pu/U peak but the trend is the same. In conclusion, the spent fuel simulations using modern radiation transport physics codes can model the actual spent fuel measurements but need to be benchmarked.