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Measuring binding kinetics of ligands with tethered receptors by fluorescence polarization complemented with total internal reflection fluorescence microscopyKwok, Ka Cheung 02 July 2010 (has links)
The study of the binding between estrogen receptors (ER) and their ligands in vitro has long been of interest mainly because of its application in anti-estrogen drug discovery for breast cancer treatment as well as in the screening of environmental contaminants for endocrine disruptors. Binding strength was conventionally quantified in terms of equilibrium dissociation constant (KD). Recently, emphasis is shifting towards kinetics rate constants, and off-rate (koff) in particular. This thesis reported a novel method to measure such binding kinetics based on fluorescence polarization complemented with total internal reflection fluorescence (FP-TIRF). It used tethered receptors in a flow cell format. For the first time, the kinetics rate constants of the binding of full-length human recombinant ERα with its standard ligands were measured. koff was found to range from 1.3 10-3 to 2.3 10-3 s-1. kon ranged from 0.3 105 to 11 105 M-1 s-1. The method could also be used to screen potential ligands. Motivated by recent findings that ginsenosides might be functional ligands of nuclear receptors, eleven ginsenosides were scanned for binding with ER and peroxisome proliferator-activated receptor gamma (PPAR). None of the ginsenosides showed significant binding to ER, but Rb1 and 20(S)-Rg3 exhibited significant specific binding with PPAR.
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Dual-View Inverted Selective Plane Illumination Microscopy (diSPIM) Imaging for Accurate 3D Digital PathologyJanuary 2020 (has links)
archives@tulane.edu / For decades, histopathology and cytology have provided the reference standard for cancer diagnosis, prognosis prediction and treatment decisions. However, they are limited to 2D slices, which are created via cutting and/or smearing, thus not faithfully representing the true 3D structures of the cellular or tissue material. Multiple imaging methods have been utilized for non-destructive histologic imaging of tissues, but are usually limited by varying combinations of low resolution, low penetration depth, or a relatively slow imaging speed, and all suffer from anisotropic resolution, which could distort 3D tissue architectural renderings and thus hinder new work to analyze and quantify 3D tissue microarchitecture. Therefore, there is a clear need for a non-destructive imaging tool that can accurately represent the 3D structures of the tissue or cellular architecture, with comparable qualities and features as traditional histopathology.
In this work, dual-view inverted selective plane illumination microscopy (diSPIM) has been customized and optimized for fast, 3D imaging of large biospecimens. Imaging contrast of highly scattering samples has been further improved by adding confocal detection and/or structured illumination (SI) as additional optional imaging modes. A pipeline of dual-view imaging and processing has also been developed to achieve more isotropic 3D resolution, specifically on DRAQ5 and eosin (D&E) stained large (millimeter to centimeter size) biopsies.
To determine the impact of 3D, high-resolution imaging on clinical diagnostic endpoints, multiple prostate cancer (PCa) biopsies have been collected, imaged with diSPIM, and evaluated by pathologists. It has been found that the pathologist is “equally” confident on the PCa diagnosis from viewing 3D volumes and 2D slices, and the diagnostic agreement between 3D volumes is significantly higher than 2D slices.
The high-resolution and large-volume coverage of diSPIM may also help verify results from other lower-resolution modalities by serving as a 3D histology surrogate. Tissue correlations have been found between images acquired by diSPIM and photo-acoustic imaging, or by diSPIM and biodynamic imaging, proving diSPIM as a useful tool to aid in validation of lower-resolution imaging tools. The potential of diSPIM imaging has also been demonstrated in other applications, such as in the study of in-vitro neural models. / 1 / Bihe Hu
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Novel biophysical appliations [sic] of STICSVaillancourt, Benoit. January 2008 (has links)
No description available.
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Direct observation of correlated motions in colloidal gels and glassesGao, Yongxiang. January 2008 (has links)
No description available.
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Dynamic and compressed memory coding in the hippocampusPriestley, James Benjamin January 2022 (has links)
A longstanding goal in neuroscience is to provide a biological understanding of episodic memory, our conscious recollection of prior experience. While the hippocampus is thought to be a critical locus for episodic learning in the mammalian brain, the nature of its involvement is unsettled. This thesis details several investigations that attempt to probe the neural mechanisms that support the encoding and organization of new experiences into memory.
Throughout the included works, we utilize in vivo two-photon fluorescence microscopy and calcium imaging to study the functional dynamics of hippocampal networks in mice during memory-guided behavior. To begin, Chapter 2 examines how neural coding in hippocampal area CA1 is modified during trace fear conditioning, a common model of episodic learning in rodents that requires linking events separated in time. We longitudinally tracked network activity throughout the entire learning process, analyzing how changes in hippocampal representations paralleled behavioral expression of conditioned fear. Our data indicated that, contrary to contemporary theories, the hippocampus does not generate sequences of persistent activity to learn the temporal association. Instead, neural firing rates were reorganized by learning to encode the relevant stimuli in a temporally variable manner, which could reflect a fundamentally different mode of information transmission and learning across longer time intervals.
The remaining chapters concern place cells---neurons identified in the hippocampus that are active only in specific locations of an animals' environment. In Chapter 3, we use mouse virtual reality to explore how the hippocampus constructs representations of novel environments. Through multiple lines of analysis, we identify signatures of place cells that acquire spatial tuning through a particularly rapid form of synaptic plasticity. These motifs were enriched specifically during novel exploration, suggesting that the hippocampus can dynamical tune its learning rate to rapidly encode memories of new experiences. Finally, Chapter 4 expands a model of hippocampal computation that explains the emergence of place cells through a more general mechanism of efficient memory coding. In theory and experiment, we identified properties of place cells that systematically varied with the compressibility of sensory information in the environment. Our preliminary data suggests that hippocampal coding adapts to the statistics of experience to compress correlated patterns, a computation generically useful for memory and which naturally extends to representation of variables beyond physical space.
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Synthesis, Characterizations, And Evaluation Of New Reactive Two-photon Absorbing Dyes For Two-photon Excited Fluorescence Imaging ApplicationsHales, Katherine J. 01 January 2005 (has links)
Recent, cooperative advances in chemistry, biology, computing, photophysics, optics, and microelectronics have resulted in extraordinary developments in the biological sciences, resulting in the emergence of a novel area termed 'biophotonics'. The integrative and interdisciplinary nature of biophotonics cuts across virtually all disciplines, extending the frontiers of basic cellular, molecular, and biology research through the clinical and pharmaceutical industries. This holds true for the development and application of the novel imaging modality utilizing multiphoton absorption and its extraordinary contribution to recent advances in bioimaging. Intimately involved in the revolution of nonlinear bioimaging has been the development of optical probes for probing biological function and activity. The focus of this dissertation is in the area of probe development, particularly conjugated organic probes, optimized for efficient two-photon absorption followed by upconverted fluorescence for nonlinear, multiphoton bioimaging applications. Specifically, [pi]-conjugated fluorene molecules, with enhanced two-photon absorbing (2PA) properties and high photostability, were prepared and characterized. Contemporary synthetic methods were utilized to prepare target fluorene derivatives expected to be highly fluorescent for fluorescence imaging, and, in particular, exhibit high two-photon absorptivity suitable for two-photon excitation (2PE) fluorescence microscopy. The flexibility afforded through synthetic manipulation to integrate hydrophilic moieties into the fluorophore architecture to enhance compatibility with aqueous systems, more native to biological samples, was attempted. Incorporation of functional groups for direct covalent attachment onto target biomolecules was also pursued to prepare fluorene derivatives as efficient 2PA reactive probes. Linear and two-photon spectroscopic characterizations on these novel compounds reveal they exhibit high 2PA cross-sections on the order of ~100 GM units, nearly an order of magnitude greater than typical, commonly used fluorophores utilized in nonlinear, multiphoton microscopy imaging of biological samples. Photostability studies of representative fluorene derivatives investigated and quantified indicate these derivatives are photostable under one- and two-photon excitation conditions, with photodecomposition quantum yields on the order of 10[super-5]. Preliminary cytotoxicity studies indicate these fluorene derivatives exhibit minimal cytotoxic effects on proliferating cells. Finally, their ultimate utility as high-performance, 2PA fluorescent probes in 2PE fluorescence microscopy imaging of biological samples was demonstrated in both fixed and live cells. Due to the low cytotoxicity, high photostability, efficient 2PA, and high fluorescence quantum yield, the probes were found suitable for relatively long-term, two-photon fluorescence imaging of live cells, representing a significant advance in biophotonics.
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EVIDENCE FOR DNA OXIDATION IN SINGLE MOLECULE FLUORESCENCE STUDIESWylie, Douglas 06 October 2006 (has links)
No description available.
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DEVELOPMENT OF A MULTIPLEXED CONFOCAL FLUORESCENCE LIFETIME IMAGING MICROSCOPE FOR SCREENING APPLICATIONSHirmiz, Nehad January 2019 (has links)
Protein-protein interactions are important for biological processes. Therefore, many small molecules target a specific protein or interaction in the cell to have biological consequence. While we can measure some protein-protein interactions in a test tube, many proteins cannot be purified making it difficult to properly test that a drug is “on target”. An alternative is to measure these interactions in live cells. We express the proteins of interest fused to fluorophores allowing the use of fluorescence techniques. Förster Resonance Energy Transfer (FRET) provides a molecular level ruler to measure the distance, within a few nanometers, between two proteins. FRET indicates binding. The gold standard for measuring FRET in live cells is by quantifying changes in fluorescence lifetime using Fluorescence lifetime imaging microscopy (FLIM). The change in fluorescence lifetime is inversely proportional to the ratio of bound to non-bound proteins. Tradition FLIM-FRET microscopy is too slow for screening applications. Our aim was to develop a highly multiplexed confocal system for rapid FLIM-FRET acquisition.
We present the development of multiple prototypes for confocal multiplexing. In this work, our final design includes 32×32 multiplexed excitation points which scan the sample using refractive window scanners. We coupled this excitation scheme to a 64×32 time-gated single-photon avalanche photodiode (SPAD) sparse array detector. This multiplexed setup allows the use of the sparse array with high frame rate and sub-nanosecond time-gating to achieve high throughput FLIM acquisition. Using our multiplexed FLIM prototype we measured Bcl-2 family protein-protein interactions in live cells (310×310 μm FOV) with two-channel confocal FLIM in 1.5 s. Protein binding affinities were estimated by measuring the changes in FRET as a function of acceptor to donor ratio. The resulting speed of this system meets requirements for implementation in screening applications. / Thesis / Candidate in Philosophy / Inside a cell, proteins are the “workers” and they interact with each other, doing that work. Many of these interactions are important for the cell to live. Pharmaceutical companies may design drugs that can interfere with a specific interaction in order to cause an effect in the cell. Scientists are interested in measuring these interactions and we can do this by “taking a picture” of the interaction using a specialized microscope. One of the major issues with these microscopes is that it takes scientists a long time to collect pictures of these interactions. This means only a few drugs can be tested in a day. To speed up the drug discovery and testing we want to design faster microscopes that can test hundreds of drugs in a day. In my thesis I contributed to building a state-of-the-art super fast microscope. We made progress in steps, and by the third attempt we successfully measured interactions in cells in seconds! Our new microscope is ~400x faster than current technologies. We hope that this research will be useful to speed up drug discovery in the future.
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Identifying Novel Contributors to RNA Interference in Aedes aegyptiSaadat, Angela P. 02 September 2015 (has links)
Aedes aegypti is an important vector of human pathogens including the viruses yellow fever, dengue and chikungunya. The small interfering RNA (siRNA) pathway is a critical immune response for controlling viral replication in Aedes aegypti. The goal of this research is to identify components of the Aedes aegypti genome that influence this pathway.
A transgenic mosquito strain that reports the status of the siRNA pathway via enhanced green fluorescent protein (EGFP) intensity was employed to differentiate silencing abilities among individuals. Extreme EGFP expression phenotypes, representing efficient and poor silencing abilities, were enriched over five generations.
Transcriptome sequencing and analyses were performed from pools of individuals from each enriched phenotype, revealing potential RNAi contributors. 1,120 transcripts were significantly different (FDR<0.0001) among the extreme phenotypes.
Four genes were chosen, amplified, sequenced for SNP analysis. These analyses were performed on samples obtained by crossing enriched, extreme phenotype F0 individuals, intercrossing their progeny, then selecting individuals representing the extreme phenotypes from the F2 population. Though further verification is needed, findings from these analyses imply the regions of Aedes aegypti, Liverpool strain (AAEL) gene identifiers AAEL005026, AAEL013438 and AAEL011704 amplified do not contribute to the two extreme, opposite RNAi silencing in the sensor strain used here. SNP analyses of AAEL000817 indicate this gene either influences extreme RNAi phenotypes or is closely linked to a gene(s) that contributes to RNAi in Aedes aegypti.
The 1,120 genes identified can be validated or eliminated as potential targets in the quest to mitigate the impact of Aedes aegypti. / Master of Science in Life Sciences
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Single molecule studies of acidity in heterogeneous catalystsSun, Xiaojiao January 1900 (has links)
Doctor of Philosophy / Department of Chemical Engineering / Keith L. Hohn / Amorphous silica-alumina is widely used as a solid acid catalyst for various reactions in oil refining and the petrochemical industry. The strength and the number of the acid sites in the material are most often believed to arise from the alumina atoms inserted into the silica lattice. The existence of the acidity distribution across the framework is a result of the local composition or the short-range interactions on the silica-alumina surface. Conventional techniques used to characterize silica-alumina provide effective information on the average acidity, but may not reflect the heterogeneity of surface acidity within the material.
Recently, it is possible to study individual catalytic sites on solid catalysts by single molecule fluorescence microscopy with high time and space resolution. Fluorophores can be chosen that emit at different wavelengths depending on the properties of the local environment. By doping these fluorophores into a solid matrix at nanomolar concentrations, individual probe molecules can be imaged. Valuable information can be extracted by analyzing changes in the fluorescence spectrum of the guest molecules within a host matrix. In this research, silica-alumina thin films were studied with single molecule fluorescence microscopy. The samples were prepared by a sol-gel method and a wide-field fluorescence microscope was used to locate and characterize the fluorescent behaviors of pH sensitive probes. In mesoporous thin films, the ratio of the dye emission at two wavelengths provides an effective means to sense the effective pH of the microenvironment in which each molecule resides. The goal of this work was to develop methods to quantify the acidity of individual micro-environments in heterogeneous networks. Pure silica films treated with external phosphate solutions of different pH values were used to provide references of the fluorescence signals from individual dye molecules. SM emission data were obtained from mesoporous Al-Si films as a function of Al content in films ranging from 0% to 20% alumina. Histograms of the emission ratio revealed that films became more acidic with increasing Al content.
The acidity on interior surfaces in zeolite pores was also of interest in this work. A microfluidic device was built to isolate the interior surface from the exterior surface. Some preliminary results showed the potential of using SM fluorescence method to study the acidic properties inside the pores of zeolite crystals.
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