To see the other types of publications on this topic, follow the link: Total internal reflection fluorescence microscopy.
Dissertations / Theses on the topic 'Total internal reflection fluorescence microscopy'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Total internal reflection fluorescence microscopy.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Beck, Markus. "Extended resolution in total internal reflection fluorescence microscopy /." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17974.
Full text
2
Steele, Bridgett L. Thompson Nancy L. "Total internal reflection fluorescence microscopy for characterizing biochemical interactions." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2009. http://dc.lib.unc.edu/u?/etd,2725.
Full textAbstract:
Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.<br>Title from electronic title page (viewed Mar. 10, 2010). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.
3
Chan, Ho Man. "Analysis of biomolecules by total internal reflection fluorescence microscopy." HKBU Institutional Repository, 2011. http://repository.hkbu.edu.hk/etd_ra/1254.
Full text
4
Bethea, Tomika R. C. "Silica Colloidal Crystals as Porous Substrates for Total Internal Reflection Fluorescence Microscopy." Thesis, The University of Arizona, 2006. http://hdl.handle.net/10150/193371.
Full textAbstract:
In cell biology and chemistry, total internal reflection microscopy (TIRFM) has proven to be a useful technique that allows the probing of cellular processes with high-signal-to-noise ratio imaging. However, samples on solid substrates limit the accessibility to probe processes on extracellular membrane surface closest to the microscope objective. Colloidal crystals provide a porous alternative to the traditional solid substrates. Thin crystals exhibit optical properties similar to that of a fused silica coverslip allowing for TIRFM in the same manner as with a typical coverslip as demonstrated by the observance of Chinese hamster ovary cells with fluorescently labeled receptors on both types of substrates. Accessibility of the cell membrane closest to the substrate and the ability to probe fluorophore orientation information was observed by the binding of TIPP-cy5 to the human delta opioid receptor.
5
Chan, Hei Nga. "Analysis of biomarkers of age-related diseases by total internal reflection fluorescence microscopy." HKBU Institutional Repository, 2018. https://repository.hkbu.edu.hk/etd_oa/527.
Full textAbstract:
Total internal reflection fluorescence microscopy (TIRFM) has been widely applied for the study of biomolecules because of their ability to quantify biomolecules in a sample pretreatment and enrichment free manner, when compared with those costly, sample consuming and labor intensive conventional detection assay. Here, we have applied the TIRFM imaging system for the direct quantification and analysis of the biomarkers for the age-related diseases. Three research works on the quantification and study of biomarkers with the aid of TIRFM were herein described.
6
Ogden, Melinda Anne. "Two-photon total internal reflection microscopy for imaging live cells with high background fluorescence." Thesis, Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/34786.
Full textAbstract:
Fluorescence microscopy allows for spatial and temporal resolution of systems which are inherently fluorescent or which can be selectively labeled with fluorescent molecules. Temporal resolution is crucial for imaging real time processes in living samples. A common problem in fluorescence microscopy of biological samples is autofluorescence, fluorescence inherent to the system, which interferes with detection of fluorescence of interest by decreasing the signal to noise ratio.
Two current methods for improved imaging against autofluorescence are two-photon excitation and total internal reflection microscopy. Two-photon excitation occurs when two longer wavelength photons are absorbed quasi-simultaneously by a single fluorophore. For this to take place there must be a photon density on the order of 1030 photons/(cm2)(s), which is achieved through use of a femtosecond pulsed laser and a high magnification microscope objective. Two-photon excitation then only occurs at the focal spot, significantly reducing the focal volume and therefore background autofluorescence.
The second method, total internal reflection, is based on evanescent wave excitation, which decreases exponentially in intensity away from the imaging surface. This allows for excitation of a thin (~200 nm) slice of a sample. Since only a narrow region of interest is excited, an optical slice can be imaged, decreasing excitation of out-of-focus autofluorescence, and increasing the signal to noise ratio.
By coupling total internal reflection with two-photon excitation, an entire cell can be imaged while still maintaining the use of lower energy photons to irradiate the sample and achieve two-photon excitation along the length traveled by the evanescent wave. This system allows for more sensitive detection of fluorescence of interest from biological systems as a result of a significant decrease in excitation volume and therefore a decrease in autofluorescence signal. In the two-photon total internal reflection microscopy setup detailed in this work, an excitation area of 20 μm by 30 μm is achieved, and used to image FITC-stained actin filaments in BS-C-1 cells
7
Suda-Cederquist, Keith David. "Near-Wall Thermometry via Total Internal Reflection Fluorescence Micro-Thermometry (TIR-FMT)." Thesis, Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/14530.
Full textAbstract:
To effectively design systems of microchannels it is necessary for scientists and engineers to understand thermal transport characteristics of microchannels. To experimentally determine the convective heat transfer coefficient of microchannels it is necessary to measure both the bulk and surface temperature fields. This investigation aims to develop a technique, named Total Internal Reflection Fluorescent Micro-Thermometry (TIR-FMT), to measure the temperature of water within several hundred nanometers of a wall--effectively, the surface temperature of the wall. In TIR-FMT, an evanescent-wave is generated in the water near the wall. The intensity of this evanescent-wave decays exponentially with distance from the wall. A fluorophore if illuminated by the evanescent-wave can absorb a photon. Excited fluorophores subsequently emit red-shifted photons, which are called fluorescence. The probability of a fluorescent emission is temperature-dependent. Therefore, by monitoring the intensity of the fluorescence a correlation can be made to the temperature of the region of illumination. Using the TIR-FMT technique the temperature dependence of the fluorescence intensity from buffered fluorescein (pH=9.2) was determined to be 1.35%/C. TIR-FMT can be used to measure the temperature of a fluorophore solution within 600 nm of a wall across a temperature range of 12.5-55C. The average uncertainties (95% confidence) of the temperature measured was determined to be 2.3C and 1.5C for a single 1.5x1.5 and #956;m pixel and the entire 715x950 and #956;m viewfield. By spatial averaging, average uncertainties of 2.0C and 1.8C were attained with spatial resolutions of 16x16 and 100x100 and #956;m, respectively.
8
Kwok, Ka Cheung. "Measuring binding kinetics of ligands with tethered receptors by fluorescence polarization complemented with total internal reflection fluorescence microscopy." HKBU Institutional Repository, 2010. https://repository.hkbu.edu.hk/etd_oa/18.
Full textAbstract:
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.
9
Kaldaras, Leonora. "Single Molecule Studies of Enzymes Horseradish Peroxidase and Alkaline Phosphatase Using Total Internal Reflection Fluorescence Microscopy and Confocal Microscopy." Bowling Green State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1374686174.
Full text
10
Yiu, Kwok Wing. "Measuring the binding between estrogen receptor alpha and potential endocrine disruptors by fluorescence polarization and total internal reflection fluorescence." HKBU Institutional Repository, 2013. http://repository.hkbu.edu.hk/etd_ra/1503.
Full text
11
Phimphivong, Samrane. "Applications of Total Internal Reflection Fluorescence Microscopy for Studies of Chemical Phenomena at the Substrate-Liquid Interface." Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/194335.
Full textAbstract:
Applications of TIRFM for quantitative measurements of cells are limited due to high background fluorescence which can result in a low S/N ratio and therefore contribute to inaccuracy of measurements. Time-resolved total internal reflection fluorescence microscopy (TR-TIRFM) was developed by temporally gating a CCD camera using a liquid crystal shutter to optically filtering the short-live fluorescence and recording only the long-live emission. This technique was then applied to examine the extent of cell-substrate contacts. Tb+3 chelates such as DTPA-PhenylAS-Tb+3 was synthesized and applied as a membrane staining agent but was observed to internalized into the cell nucleus. A modified chelate molecule was therefore synthesized using DOPE as a carrier molecule. DOPE-DTPA-pAS-Tb+3 has a similar emission lifetime (1.5 msec) and appeared to stain only the cell membrane. TR-TIRF was applied to examine adherent cells on polystyrene-coated substrate. TR-TIRF images showed cellular autofluorescence and polystyrene emissions were optically filtered out, while the long-lived emission intensity of Tb+3 chelate was recorded. These results conclude that TR-TIRFM, with the use of long-live emission label (Tb+3 and Eu+3 chelates), is suitable as an analytical tool for probing a large number of cellular and molecular events occurring in the cell membrane and on the cell surface where background fluorescence would usually be problematic. Detection of K+ transported across a cell membrane is a prerequisite in the development of devices for screening drugs targeting K+ ion channels. K+ sensing film was fabricated by encapsulating a squaraine dye (aza-crown-SQR) in a sol-gel matrix for detection of K+. Sol-gel films were prepared by the hydrolysis and condensation reactions of TEOS or TMOS, APTS and GOPS mixtures. Formation of a DPhPC bilayer on sol-gel films was achieved by the vesicle fusion method and had diffusion coefficients of 2.3 and 2.1x10-8 cm2s-1 as measured by FRAP on TEOS-APTS-GOPS and TMOS-APTS-GOPS film, respectively. The time-based fluorescence intensity data from the H+ blocking experiments showed the sol-gel-supported DPhPC bilayers are impermeable to H+, and the K+ blocking experiments showed K+ was passively transported across a DPhPC bilayer by valinomycin.
12
Hibbel, Anneke [Verfasser], Stefan [Gutachter] Diez, and Jonathon [Gutachter] Howard. "Characterization of Saccharomyces cerevisiae kinesin Kip2 by total internal reflection fluorescence microscopy / Anneke Hibbel ; Gutachter: Stefan Diez, Jonathon Howard." Dresden : Technische Universität Dresden, 2021. http://d-nb.info/1231917253/34.
Full text
13
Schmoranzer, Jan [Verfasser]. "Imaging Single Fusion Events at the Plasma Membrane using Total Internal Reflection Fluorescence Microscopy - Applications in Membrane Traffic / Jan Schmoranzer." Berlin : Freie Universität Berlin, 2002. http://d-nb.info/1021526401/34.
Full text
14
Fournier, Charlotte. "The spatial organization of the epidermal growth factor receptor on the surface of colorectal carcinoma cells." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:350ade6e-514c-4b1d-98b9-7d440620c9a7.
Full textAbstract:
The discovery of the existence of the cell membrane has led to a search for its organization on a molecular scale. The advent of artificial lipid bilayers and the development of electron microscopy in the 1930's provided direct visual evidence for the existence of the cell membrane and drove forward models of membrane structure based its known composition of proteins, lipids and carbohydrates. The fluid mosaic model of membrane structure, based on thermo- dynamics and newly developed protein structural studies of the time, placed integral globular membrane proteins within a fluid phospholipid bilayer. This model allowed for the association of proteins into groups and the possible mobility of proteins within the lipid bilayer. At the the same time fluorescence microscopy demonstrated movement of proteins in the plane of the lipid bilayer. Since then experimental techniques have been developed that show protein complexes of varying sizes do exist and so this gives us the opportunity to ask how receptor proteins fit into the molecular organization of the cell membrane. This thesis presents an investigation into how the epidermal growth factor receptor (EGFR) organizes in the cell membrane of colorectal carcinoma cells. First a new cell line for studying the receptor by stably expressing the epidermal growth factor receptor conjugated to enhanced green fluorescent protein (EGFR-eGFP) in SW620 cells was developed. This is an interest- ing cell line because it originates from a colonic adenocarcinoma that during the process of metastasis has lost the ability to express the EGFR. It therefore provided an environment for the expression of the fluorescent form of the receptor more in keeping with its natural environment. The technique of total internal reflection fluorescence (TIRF) microscopy was used to visualize the fluorescently tagged receptor in the cell membrane. This technique uses the principles of total internal reflection to excite fluorescence in molecules located only 100 nm into the cell. Because sources of fluorescence from outside the illuminated area are minimized individual fluorescent molecules can be imaged. The spots in the images, produced by the fluorophores, were detected using a single molecule detection and tracking algorithm. The intensities of these detected spots were analysed and compared with that from a single molecule of enhanced green fluorescent protein (eGFP). This gave an estimate of the number of receptors contained within each receptor complex. Before ligand binding most of the receptors were found to be located in complexes containing up to eight molecules and most frequently they were found in complexes of two molecules. Larger complexes of receptors were found to have formed after activation of the receptor by its ligand.
15
Myklatun, Ahne. "Production and Application of Micronsized Polysaccharide Particles - Studying Perturbation of a Model Mucus Barrier with Total Internal Reflection Fluorescence (TIRF) Microscopy and Atomic Force Microscopy (AFM) Indentation." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for fysikk, 2011. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-13742.
Full textAbstract:
The overall aim of this project was to produce homogeneously sized polysaccharide microparticles and apply these and similar sized particles as probes for investigation of mucin layers as a model for a biological barrier. Small polysaccharide particles have many applications, e.g. within the medical field of drug delivery. In this study a microfluidic system was developed to produce alginate beads, which can be used in drug delivery systems. Different designs, continuous phases and concentrations were tested in order to find an optimal system. Beads in the size range of 10 µm were produced using a device with T-shaped design and three inlets. An electrostatic bead generator was also used to make alginate beads, however the beads produced were too large to be used in the experiments with the mucin layers.One of the many challenges when working with drug delivery systems is the mucus barrier protecting the epithelial cells. In this study a model mucus barrier was made by immobilizing mucins, the glycoprotein responsible for the physical properties of the barrier. A procedure for fluorescence labelling of polystyrene beads with quantum dots was developed, and penetration of these beads into the model barrier was measured with total internal reflection fluorescence (TIRF) microscopy. In TIRF the excitation field intensity decays exponentially, and the emitted fluorescence intensity from the beads gives an indication of the distance between the beads and the surface. Measurements performed on mucin layers of different concentrations indicate that mucin concentrations above 0,5 mg/ml will result in a layer too thick or too dense to give a intensity signal. At mucin concentration 0,05 mg/ml fluorescence was observable in TIRF, and it was clearly weaker than for the control with a bead directly on a glass surface. This indicates that the beads hover over the surface due to the mucin layers, and show that it is in principle possible to measure the penetration depth of beads into mucin layer using TIRF. To simulate the condition in the lungs of cystic fibrosis (CF) patients, the mucin layers were incubated with alginate. Measurements were performed to see how this affected the penetration of the beads into the layer. A weaker fluorescent signal was obtained for these samples in TIRF, which suggests that there has been interaction between mucin and alginate. It was in addition investigated how different concentrations of G-blocks in the solution affected the penetration into the mucin-alginate layer. These testes were carried out using both TIRF and atomic force microscopy (AFM) nanoindentation experiments. The TIRF measurements were inconclusive, while the nanoindentation experiments showed decreased interaction between mucin-alginate layer and a bead.
16
Pu, Mingming. "Investigations of the Mechanism for Activation of Bacillus Thuringiensis Phosphatidylinositol-specific Phospholipase C." Thesis, Boston College, 2009. http://hdl.handle.net/2345/1179.
Full textAbstract:
Thesis advisor: Mary F. Roberts<br>Thesis advisor: Steven D. Bruner<br>The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is specifically activated by low concentrations of a non-substrate lipid, phosphatidylcholine (PC), presented as an interface. However, if the PC concentration in the interface is too high relative to substrate, the enzyme exhibits surface dilution inhibition. Understanding this bacterial enzyme, which shares many kinetic features with the larger and more complex mammalian PI-PLC enzymes, requires elucidating the mechanism for PC activation and inhibition. Various techniques were applied to study the interaction of the protein with vesicles composed of both the activator lipid PC and the substrate lipid (or a nonhydrolyzable analogue). Fluorescence correlation spectroscopy (FCS), used to monitor bulk partitioning of the enzyme on vesicles, revealed that both the PC and the substrate analogue are required for the tightest binding of the PI-PLC to vesicles. Furthermore, the tightest binding occurred at low mole fractions of substrate-like phospholipids. Field cycling 31P NMR (fc-P-NMR) spin-lattice relaxation studies provided information on how bound protein affects the lipid dynamics in mixed substrate analogue/PC vesicles. The combination of the two techniques could explain the enzyme kinetic profile for the PC activation and surface dilution inhibition: small amounts of PC in an interface enhanced PI-PLC binding to substrate-rich vesicles while high fractions of PC tended to sequester the enzyme from the bulk of its substrate leading to reduced specific activity. FCS binding profiles of mutant proteins were particularly useful in determining if a specific mutation affected a single or both phospholipid binding modes. In addition, an allosteric PC binding site was identified by fc-P-NMR and site directed spin labeling. A proposed model for PC activation suggested surface-induced dimerization of the protein. Experiments in support of the model used cysteine mutations to create covalent dimers of this PI-PLC. Two of these disulfide linked dimers, formed from W242C or S250C, exhibited higher specific activities and tighter binding to PC surfaces. In addition, single molecule total internal reflection fluorescence microscopy was used to monitor the off-rate of PI-PLC from surface tethered vesicles, providing us with a direct measure of off-rates of the protein from different composition vesicles<br>Thesis (PhD) — Boston College, 2009<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Chemistry
17
Decan, Matthew. "The Copper(I)-catalyzed Azide–Alkyne Cycloaddition: A Modular Approach to Synthesis and Single-Molecule Spectroscopy Investigation into Heterogeneous Catalysis." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31882.
Full textAbstract:
Click chemistry is a molecular synthesis strategy based on reliable, highly selective reactions with thermodynamic driving forces typically in excess of 20 kcal mol-1. The 1,3-dipolar cycloaddition of azides and alkynes developed by Rolf Huisgen saw dramatic rate acceleration using Cu(I) as a catalyst in 2002 reports by Barry Sharpless and Morten Meldal enabling its click chemistry eligibility. Since these seminal reports, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has become the quintessential click reaction finding diverse utility. The popularity of the CuAAC has naturally led to interest in new catalyst systems with improved efficiency, robustness, and reusability with particular focus on nanomaterial catalysts, a common trend across the field of catalysis. The high surface area of nanomaterials lends to their efficacy as colloidal and heterogeneous nanocatalysts, but the latter boasts the added benefit of easy separation and recyclability. With any heterogeneous catalyst, a common question arises as to whether the active catalyst species is truly heterogeneous or rather homogeneous through metal ion leaching. Differentiating these processes is critical, as the latter would result in reduced efficiency, higher cost, and inevitable environmental and heath side effects.
This thesis explores the CuAAC from an interdisciplary approach. First as a synthetic tool, applying CuAAC-formed triazoles as functional, modular building blocks in the synthesis of optical cation sensors by combining azide and alkyne modified components to create a series of sensors selective for different metal cations. Next, single-molecule spectroscopy techniques are employed to observe the CuNP-catalyzed CuAAC in real time. Combining bench-top techniques with single-molecule microscopy to monitor single-catalytically generated products proves to be an effective method to establish catalysis occurs directly at the surface of copper nanoparticles, ruling out catalysis by ions leached into solution. This methodology is extended to mapping the catalytic activity of a commercial heterogeneous catalyst by applying super-localization analysis of single-catalytic events. The approach detailed herein is a general one that can be applied to any catalytic system through the development of appropriate probes. This thesis demonstrates single-molecule microscopy as an accessible, effective, and unparalleled tool for exploring the catalytic activity of nanomaterials by monitoring single-catalytic events as they occur.
18
Klajner, Piotr. "Experimental study of the kinetics of two systems : DNA complexation by the NCp7 protein and probe dynamics in a glassy colloidal suspension." Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00858886.
Full textAbstract:
In the first part of this thesis, we study the kinetics of the complexation of a double-stranded DNA byNCp7 protein. To do this, we study the evolution of mechanical properties of DNA and its complexation by stretching the DNA/NCp7 complex with a optical trap. We observed that the persistence length of the complex decreases progressively during the complexation. Using astatistical model we describe the evolution of the flexibility of DNA complexed with NCp7. Our main result is that the fraction phi of base pairs that have reacted is not a linear function of time at low phi.We interpret our results assuming that the adsorption of NCp7 on DNA is highly cooperative. In the second chapter, we describe the dynamics of probe particles in a colloidal glassy suspension of Laponite. Laponite is a colloidal discoidal particle of 25 nm in diameter and 0.92 nm thick. We take advantage of evanescent wave microscopy, and follow the movement of fluorescent latex particles.Then we image these particles. We show that for a movement that has a single characteristic time scale, it is simply a linear function of time. We find that, what ever their size, the motion of probe particles can be described by a succession of two dynamic modes, where the fastest mode corresponds to the diffusion of particles in a viscoelastic fluid.
19
Zhao, Gengjing. "Single-molecule studies of bacterial DNA replication and translesion synthesis." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/276234.
Full textAbstract:
Faithful replication of genomic DNA is crucial for the survival of a cell. In order to achieve high-level accuracy in copying its genome, all cells employ replicative DNA polymerases that have intrinsic high fidelity. When an error occurs on the template DNA strand, in the form of lesions caused by diverse chemicals, reactive oxygen species, or UV light, the high-fidelity replicative DNA polymerases are stalled. To bypass these replication blocks, cells harbor multiple specialized translesion DNA polymerases that are error-prone and therefore able to accommodate the lesions and continue DNA synthesis. As a result of their low fidelity, the translesion polymerases are associated with increased mutagenesis, drug resistance, and cancer. Therefore, the access of the translesion polymerases to DNA needs to be tightly controlled, but how this is achieved has been the subject of debate. This Thesis presents the development of a co-localization single-molecule spectroscopy (CoSMoS) method to directly visualize the loading of the Escherichia coli replicative polymerase on DNA, as well as the exchange between the replicative polymerase and the translesion polymerases Pol II and Pol IV. In contrast to the toolbelt model for the exchange between the polymerases, this work shows that the translesion polymerases Pol II and Pol IV do not form a stable complex with the replicative polymerase Pol IIIα on the β-clamp. Furthermore, we find that the sequential activities of the replication proteins: clamp loader, clamp, and Pol IIIα, are highly organized while the exchange with the translesion polymerases is disordered. This exchange is not determined by lesion-recognition but instead a concentration-dependent competition between the replicative and translesion polymerases for the hydrophobic groove on the surface of the β-clamp. Hence, our results provide a unique insight into the temporal organization of events in DNA replication and translesion synthesis.
20
Mataragka, Stefania. "High-resolution optical analyses of IP3-evoked Ca2+ signals." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/289124.
Full textAbstract:
Ca2+ is a universal intracellular messenger that regulates many cellular responses. Most cells express inositol 1,4,5-trisphosphate receptors (IP3R) that mediate Ca2+ release from the endoplasmic reticulum (ER) when they bind IP3 produced after activation of cell-surface receptors. Vertebrate genomes encode three closely related subtypes of IP3R (IP3R1-3). High-resolution optical analyses have revealed a hierarchy of IP3-evoked Ca2+ signals that are thought to arise from the co-regulation of IP3Rs by IP3 and Ca2+. The smallest events ('blips') report the opening of single IP3Rs, Ca2+ 'puffs' report the almost simultaneous opening of a few clustered IP3Rs, and as stimulus intensities increase further Ca2+ signals propagate regeneratively as Ca2+ waves. The aim of this study was to establish whether all three IP3R subtypes can generate Ca2+ puffs. I first used a haploid cell line (HAP1 cells) to generate, using CRISPR/Cas9, a line lacking all endogenous IP3Rs. However, for analyses of Ca2+ puffs, I used HEK cells that had been engineered, using CRISPR/Cas9 to disrupt endogenous genes, to express single IP3R subtypes. Local Ca2+ signals evoked by flash-photolysis of caged- IP3 were recorded using Cal520 and total internal reflection fluorescence (TIRF) microscopy in human embryonic kidney (HEK) cells. The Flika algorithm was used, and validated, for automated detection of Ca2+ puffs and to measure their properties. IP3 evoked Ca2+ puffs in wild-type HEK cells and in cells expressing single IP3R subtypes. In wild-type cells, the Ca2+ signals invariably propagated regeneratively to give global increases in cytosolic [Ca2+]. This occurred less frequently in cells expressing single IP3R subtypes, commensurate with their lower overall levels of IP3R expression. The properties of the Ca2+ puffs, including their rise and decay times, durations, the size of the unitary fluorescence steps as channels closed channel during the falling phase, and the estimated number of active IP3Rs in each Ca2+ puff, were broadly similar in each of the four cell lines. The latter observation suggests that despite lower overall levels of IP3R expression (~30%) in cells with single subtypes relative to WT cells, there is a mechanism that ensures formation of similarly sized IP3R clusters. The only significant differences between cell lines were the slower kinetics of the Ca2+ puffs evoked by IP3R2, which may suggest dissociation of IP3 from its receptor contributes to the termination of Ca2+ puffs. My results demonstrate, for the first time, that all three IP3R subtypes can generate Ca2+ puffs. I conclude that Ca2+ puffs are fundamental building blocks of all IP3-evoked Ca2+ signals.
21
Malcolm, Dominic W. "An Investigation of a G-Quadruplex and Its Interactions with Human Replication Protein A at the Single Molecule Level." Kent State University Honors College / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1335812645.
Full text
22
Do, Le Duy. "Relation entre l’annexine A6 et la phospholipase D1 pendant le processus d’exocytose dans les cellules PC12." Thesis, Lyon 1, 2014. http://www.theses.fr/2014LYO10160/document.
Full textAbstract:
L'exocytose régulée, est un processus qui permet la communication entre les cellules à travers la sécrétion des hormones et des neurotransmetteurs. Dans les neurones et les cellules neuroendocrines, l'exocytose est strictement contrôlée par des signaux extracellulaires tels que le potentiel trans-membranaire et la fixation des ligands sur des récepteurs. Des progrès substantiels ont été effectués afin de comprendre le mécanisme moléculaire de l'exocytose. Les composants majeurs de la machinerie de sécrétion ont été dévoilés. Maintenant, la question qui émerge concerne le rôle de la plateforme de protéines qui semble avoir une action coordonnée entre chaque protéine. Dans le cas de la famille des annexines, qui est bien connue pour son action dans l'exocytose, leurs modes d'interactions séquentielles ou concertées avec d'autres protéines ainsi que leurs effets régulateurs sur l'exocytose ne sont pas encore bien établis. Des résultats précédents indiquent que l'Annexine A6 (AnxA6) affecte l'homéostasie du calcium et la sécrétion de la dopamine à partir des cellules PC12, utilisées comme un modèle cellulaire de neurosécrétion (Podszywalow Bartnicka et al., 2010). Afin de déterminer l'effet inhibiteur de l'AnxA6 sur l'exocytose de la dopamine, nous cherchons des partenaires moléculaires de l'AnxA6 dans les cellules PC12. Nous faisons l'hypothèse que l'AnxA6 interagit avec la PLD1, une enzyme active dans l'étape de la fusion des vésicules avec la membrane plasmique. En utilisant la microscopie confocale et la microscopie à onde évanescente, nous avons trouvé que l'isoforme 1 de l'AnxA6 et la PLD1 sont tous les deux recrutés sur la surface des vésicules au cours de la stimulation des cellules PC12. AnxA6 inhibait l'activité de la PLD comme indiqué par notre méthode d'analyse enzymatique au moyen de la spectroscopie infrarouge. En conclusion, nous proposons que l'AnxA6 n'est pas seulement impliquée dans la réorganisation des membranes par ses capacités à se lier avec des phospholipides négativement chargés et avec le cholestérol, mais elle influence également l'activité de la PLD1, changeant la composition lipidique des membranes<br>The regulated exocytosis is a key process allowing cell-cell communication through the release of hormone and neurotransmitters. In neurons and neuroendocrine cells, it is strictly controlled by extracellular signal such as transmembrane potential and ligand bindings to receptors. Substantial progress has been made to understand the molecular mechanism of exocytosis. Major components of secretory machinery have been brought to light. Now the emergent question concerns the role of scaffolding proteins that are thought to coordinate the action of each other. In the case of annexin family well known to be involved in exocytosis, their modes of –sequential or concerted- interactions with other proteins, and their regulatory effects on exocytosis are not very well established. Previous findings indicated that Annexin A6 (AnxA6) affected calcium homeostasis and dopamine secretion from PC12 cells, used as cellular model of neurosecretion (Podszywalow-Bartnicka et al., 2010). To determine the inhibitory effect of AnxA6 on exocytosis of dopamine, we were looking for molecular partners of AnxA6 in PC12 cells. We hypothesized that AnxA6 interacts with phospholipase D1 (PLD1), an enzyme involved in the fusion step. By using confocal microscopy and total internal reflection fluorescence microscopy, we found that isoform 1 of AnxA6 and Phospholipase D1 are both recruited on the surface of vesicles upon stimulation of PC12 cells. AnxA6 inhibited phospholipase D activity as revealed by our enzymatic assay based on infrared spectroscopy. To conclude, we propose that AnxA6 is not only implicated in membrane organization by its capacity to bind to negative charged phospholipids and to cholesterol, but AnxA6 is also affecting PLD1 activity, changing membrane lipids composition
23
Robertson, Rebecca. "The development of electroactive total internal reflection integrated optical waveguide and total internal reflection fluorescence devices for the characterization of metalloprotein films." Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/289791.
Full textAbstract:
Orientation distributions of redox active films that correlate with an electron transfer behavior were reported for the first time using electroactive total internal reflection fluorescence spectroscopy (EA-TIRF) and electroactive integrated optical waveguide (EA-IOW) absorbance technology developed here. The mean tilt angle and the angular distribution about the mean were recovered using a Gaussian model. Previous anisotropy calculations were only possible using a theoretical approach (Hansen's model). In the work presented here, a novel method was developed that was based on experimental measurements. This method used Fresnel's equations and Snell's law to determine the relative polarized electric field intensity at the interface of the EA-TIRF substrate. Optically transparent semiconductor surfaces of indium tin oxide (ITO) were used as the adlayer for EA-TIRF and EA-IOW devices. The ITO surface morphology, optical and conduction properties were characterized. The ITO was found to have adequate conduction, optical and surface morphology properties for molecular orientation distribution measurements. The results indicated the ITO morphology contributes a minimal degree of error to the calculated distribution. Surface-bound films of model methylene blue were used to characterize the EA-TIRF and EA-IOW techniques prior to the investigation of horse heart cytochrome c. The studies demonstrated potential control of redox active adsorbed films. The mean tilt angle and the angular distribution about that mean were successfully determined. In addition, the studies of the methylene blue films indicated the possibility of orientation-dependent quenching (the loss of an electron from the excited state LUMO to the ITO semiconductor conduction band). Studies of the cytochrome c films indicated anisotropic submonolayers electrostatically bound to the negative ITO surface. Cyclic voltammetry measurements showed the films to be electroactive, exhibiting quasi-reversible electrochemistry. An average tilt angle and the orientation distribution about the angle, as a function of potential, were reported for horse heart cytochrome c. This potential-dependent orientation distribution of submonolayer films is reported for the first time.
24
Burgess, Helen Jane. "A bioelectrochemical FRET switch : combined total internal reflection (TIRF) microscopy and electrochemistry." Thesis, University of Oxford, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437177.
Full text
25
Byrne, Gerard. "Total internal reflection microscopy studies on colloidal particle endocytosis by living cells." Thesis, University of Nottingham, 2009. http://eprints.nottingham.ac.uk/10979/.
Full textAbstract:
The purpose of this study was to develop novel optical microscopy techniques in order to investigate colloidal drug particle endocytosis by mammalian cells. A total internal reflection microscope (TIRM) was initially developed for high resolution cellular imaging. TIRM is a non-fluorescent imaging technique based on the principle of ‘scattering’ of the evanescent field created when a light beam undergoes total internal reflection at an interface between two media with different refractive indices, such as glass and air. The key design considerations with respect to development of a TIRM instrument are discussed. The technique is also compared and contrasted to the more commonly known non-fluorescent RICM (Reflection Interference Contrast Microscopy) technique using computer simulations. Time-lapse video TIRM is applied to imaging the interaction between A549 and 3T3 cells, and a polylysine coated substrate. Real-time label-free visualisation of 0.5 and 1 m polystyrene particle endocytosis by living cells is then demonstrated. Modifications to the TIRM system to include a dual-colour fluorescent TIRF (Total Internal Reflection Fluorescence) microscope are described in detail. Results are shown which demonstrate the ability of a combined TIRM/TIRF instrument to selectively image the basal cell membrane both label-free and fluorescently. 3T3 fibroblast cells were genetically modified using standard molecular biology protocols to express the fluorescent fusion protein EGFP-Clathrin LCa (enhanced green fluorescent protein clathrin light chain a). Finally, colloidal particle endocytosis by the genetically modified cell was imaged using the TIRM/TIRF microscope. Direct visualisation of the internalisation of 500 nm particles via clathrin coated pits in 3T3 cells was shown for the first time.
26
Haughey, Daniel A. "Studies of colloidal interactions." Thesis, Queen's University Belfast, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295218.
Full text
27
Parsons, David. "The use of total internal reflection fluorescence spectroscopy to study polymer and particle adsorption." Thesis, University of Salford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304598.
Full text
28
Ash, William Mason III. "Total Internal Reflection Holographic Microscopy (TIRHM) for Quantitative Phase Characterization of Cell-Substrate Adhesion." Scholar Commons, 2010. https://scholarcommons.usf.edu/etd/1564.
Full textAbstract:
Total Internal Reflection Holographic Microscopy (TIRHM) combines near-field microscopy with digital holography to produce a new form of near-field phase microscopy. Using a prism in TIR as a near-field imager, the presence of microscopic organisms, cell-substrate interfaces, and adhesions, causes relative refractive index (RRI) and frustrated TIR (f-TIR) to modulate the object beam's evanescent wave phase front. Quantitative phase images of test specimens such as Amoeba proteus, Dictyostelium Discoideum and cells such as SKOV-3 ovarian cancer and 3T3 fibroblasts are produced without the need to introduce stains or fluorophores. The angular spectrum method of digital holography to compensate for tilt anamorphism due to the inclined TIR plane is also discussed.
The results of this work conclusively demonstrate, for the first time, the integration of near-field microscopy with digital holography. The cellular images presented show a correlation between the physical extent of the Amoeba proteus plasma membrane and the adhesions that are quantitatively profiled by phase cross-sectioning of the holographic images obtained by digital holography. With its ability to quantitatively characterise cellular adhesion and motility, it is anticipated that TIRHM can be a tool for characterizing and combating cancer metastasis, as well as improving our understanding of morphogenesis and embryogenesis itself.
29
Salavessa, Laura. "Single-molecule analysis of IL-2 receptor reveals the importance of its clustering for endocytosis and signaling in lymphocytes Shigella promotes major alteration of gut epithelial physiology and tissue invasion by shutting off host intracellular transport Stoichiometry of receptors at the plasma membrane during their endocytosis using Total Internal Reflection Fluorescent (TIRF) microscopy live imaging and single molecule tracking." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL031.
Full textAbstract:
La signalisation par le récepteur de l'interleukine-2 (IL-2R), est régulée par son endocytose indépendante de la clathrine (EIC) et sa dégradation ultérieure, tout en jouant un rôle essentiel dans l'immunité. L'endocytose indépendante de la clathrine étant dépourvue de manteau protéique induisant la formation des puits, la question qui se pose est de savoir comment la vésicule d’IL-2R est-elle initiée. Le regroupement local des protéines peut induire des changements de conformation de la membrane et créer une invagination. Sachant que l'IL-2R s'accumule à la base des protubérances membranaires avant d’initier sa vésicule d’endocytose, nous avons analysé si le regroupement des récepteurs pourrait favoriser son internalisation. Pour étudier l’importance du regroupement de l'IL-2R, nous avons généré une lignée lymphocytaire humaine éditée (CRISPR) pour exprimer GFP-IL-2Rᵧ. Nous avons ensuite analysé la stoechiométrie du récepteur au niveau de la membrane plasmique, par microscopie TIRF couplée à une méthode de suivi de GFP-IL-2Rᵧ en molécule unique et identifié des populations distinctes d’oligomérisation. L'IL-2Rᵧ semble atteindre la surface cellulaire sous forme d'oligomères pré-assemblés auxquels d'autres molécules sont ajoutées, atteignant ainsi une taille d'oligomérisation optimale qui est essentielle à son internalisation. La liaison de l'IL-2 favorise la formation de ces oligomères, compétents pour l’endocytose, ce qui souligne l'importance du regroupement des récepteurs dans l'internalisation EIC.De plus, nous avons constaté que la déplétion du cholestérol augmentait la proportion de gros oligomères, non compétents pour l’endocytose mais induisant une forte signalisation. La perturbation du réseau d'actine favorise également la formation de gros oligomères, mais cette fois en diminuant la signalisation de l’IL-2R. Ainsi, les deux facteurs régulent l'endocytose et la signalisation de l'IL-2R de manière distincte. Nos résultats apportent de nouvelles connaissances sur les mécanismes régulant la signalisation des récepteurs et l’EIC<br>Signaling by the interleukin-2 receptor (IL-2R) is regulated by its clathrin-independent endocytosis (CIE) and subsequent degradation, while playing a critical role in immunity. Interestingly, CIE lacks a coat protein that drives pit formation, raising the question of how the CIE vesicle is initiated. Protein clustering generates forces that can induce membrane conformational changes. Notably, IL-2R has been shown to accumulate at the base of membrane protrusions, where receptors might cluster and thereby initiate the pit.To study the relevance of IL-2R clustering in its endocytosis, we generated a CRISPR-edited T cell line expressing GFP-IL-2Rᵧ and analyzed its stoichiometry at the plasma membrane, by TIRF microscopy coupled to a single-molecule endocytic tracking method. We identified distinct IL-2Rᵧ cluster populations. IL-2Rᵧ seems to reach the cell surface as a preassembled cluster to which further molecules are added, reaching an optimal cluster size that is key for its internalization. Binding of IL-2 promotes the formation of endocytic clusters and receptor uptake, highlighting the importance of clustering for CIE internalization.Moreover, we found that cholesterol depletion increases the proportion of large, non-endocytic clusters as well as IL-2R signaling. Disruption of the actin meshwork also promotes the formation of large clusters, yet it decreases IL-2R signaling. Thus, both factors regulate IL-2R endocytosis and signaling in a distinct manner. Our results provide new insights into the mechanisms regulating receptor signaling and CIE
30
Yordanov, Stoyan [Verfasser]. "Total internal reflection fluorescence cross-correlation spectroscopy: theory and application for studying boundary slip phenomenon / Stoyan Yordanov." Mainz : Universitätsbibliothek Mainz, 2011. http://d-nb.info/1030030928/34.
Full text
31
Pero, Jamie Kay Thompson Nancy L. "Advancements in methodologies and theories regarding model membrane environments by total internal reflection with fluorescence correlation spectroscopy." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2006. http://dc.lib.unc.edu/u?/etd,1279.
Full textAbstract:
Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.<br>Title from electronic title page (viewed Mar. 27, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.
32
Shah, Suhani Kiran. "Modeling scattered intensity from microspheres in evanescent field." Thesis, [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-2021.
Full text
33
Buckley, Charlotte. "Investigation of juxtaglomerular structure and function." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/16881.
Full textAbstract:
Renin is the initiator and rate-limiting factor of the renin-angiotensin system, a major mechanism of blood pressure regulation. The synthesis and secretion of this active circulatory enzyme is confined exclusively to the dense core granules of kidney juxtaglomerular (JG) cells where its precursor prorenin is packaged, cleaved to the active form and stored for release on a regulated pathway. Given its importance, surprisingly little is known about this process, in part due to the difficulty in culturing primary JG cells in vitro and the lack of reliable cell lines. The initial aim of the current work was to investigate renin-containing granule dynamics in live JG cells. To achieve this, I attempted to derive novel cell lines from triple transgenic mouse models comprising immortalised granulated or non-granulated JG cells. Due to the nature of JG cells in culture, the use of these cell lines to investigate granulation was not feasible; therefore the culture of primary JG cell culture was modified and enhanced to visualise granule dynamics in live, primary JG cells for the first time. By isolating cells using a Percoll gradient and plating them on fibronectin-coated dishes, rapid and full adhesion of JG cells was achieved, as well as prolonged expression of renin from 3 days to up to 8 days post-isolation. Using this protocol to isolate JG cells from RenGFP renin reporter mice and identifying granules using the acidotropic fluorophore Lysotracker, granule dynamics were investigated in primary JG cells. High resolution, rapid image acquisition was performed using widefield and total internal reflection microscopy, showing that dense core granules respond dynamically to the β-adrenergic agonist isoproterenol, a known renin secretory stimulus. Two different pools of granules of varying granule diameters and dynamic parameters were identified optically, suggesting that separate granule pools were being identified. Mice null for the Ren-1d gene lack renin storage granules in their JG cells, however granulation was restored in Ren1d-null mice carrying a transgene encompassing the human renin (hRen) locus. Therefore in order to investigate the relationship between renin expression and the amount of granulation in JG cells, mice expressing human renin were used. To dissect the granulation phenotype in detail, 2D electron micrographs were taken of JG cells, which were immunogold stained to confirm renin content, and reconstructed in 3D. Female hRen mice showed a significantly higher volume of granulation and an increased granule number compared to males, a finding consistent with the sexually dimorphic expression of the transgene, supporting the hypothesis that granulation in JG cells is dependent on the level of renin expression. The macula densa (MD) is a critical sensor of flow and salt content in the blood; through extensive tubulo-vascular crosstalk known as tubuloglomerular feedback (TGF), it releases key signalling factors stimulating and inhibiting renin synthesis and secretion from JG cells. Ren-1d-/- mice showed a hypercellular and columnar MD plaque, which was not restored by the introduction of the hRen transgene, indicating that TGF may be impaired in these mice. Using an isolated, perfused juxtaglomerular apparatus model it was shown that high salt- and increased flow-induced TGF functioned effectively in Ren1d-/- and huRen+/-Ren1d-/- mice, although animals on a Ren1d-/- background showed decreased sensitivity of glomerular tuft contraction and abnormal calcium signalling within macula densa cells. In conclusion, an appropriate in vitro model was developed for investigating granule dynamics in JG cells, using which granule motion was visualised and quantified for the first time in these cells. Although JG cell granulation is required for normal MD morphology, it was shown to not affect JGA function.
34
Everett, William Neil. "Evanescent wave and video microscopy methods for directly measuring interactions between surface-immobilized biomolecules." Thesis, [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1585.
Full text
35
Rashidi, Aidin. "DYNAMICS AND SURFACE FORCES EXPERIENCED BY AN ANISOTROPIC COLLOIDAL PARTICLE NEAR A BOUNDARY." Cleveland State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=csu1588861411603402.
Full text
36
Wang, Huan, and Huan Wang. "Flow Field Penetration in Thin Nanoporous Polymer Films under Laminar Flow by Förster Resonance Energy Transfer Coupled with Total Internal Reflectance Fluorescence Microscopy." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/565916.
Full textAbstract:
Tethered polymer layers at solid-fluid interfaces are used widely in a variety of surface science applications. Although many of these applications require exposure to dynamic flow conditions, flow field penetration into densely grafted polymer brushes, is still a question open to debate despite the fact that it is a fundamental process crucial to mass transport through these polymer films. Although most theoretical work has indicated flow field penetration into polymer films, with varying predicted penetration depths predicted, the limited experimental attempts to investigate this phenomenon have resulted in inconsistent conclusions due to lack of a proper analytical method. To help resolve this controversy, in this Dissertation, a new spectroscopic method, FRET-TIRFM, based on a combination of Förster resonance energy transfer (FRET) and total internal reflectance fluorescence microscopy (TIRFM), is developed to provide the first direct, quantitative measurements on flow field penetration by measuring linear diffusion coefficients of small molecules through densely grafted, thin poly(N-isopropylacryl-amide) (pNIPAM) films. Decay curves from FRET of the acceptor with a donor covalently attached at the substrate surface were fit to a combined Taylor-Aris-Fickian diffusion model to obtain apparent linear diffusion coefficients of the acceptor molecules for different flow rates. These values can then be used to obtain quantitative estimates of flow field penetration depths. For a pNIPAM surface of 110 nm dry thickness, with a 0.6 chain/nm² grafting density, apparent diffusion coefficients ranging from 1.9-9.1 × 10-12 cm²/s were observed for flow rates ranging from 100 to 3000 μL/min. This increase in apparent diffusion coefficient with applied fluid flow rate is indicative of flow field penetration of the polymer film. The depth of penetration of the flow field is estimated to range from ~6% of the polymer film thickness to ~57% of the film thickness in going from 100 to 3000 μL/min flow rate of a good solvent. Factors other than flow rate that may impact flow field penetration were also investigated using this new FRET-TIRFM method. Solvent quality and polymer brush grafting density are the two most important parameters due to the fact that they influence changes in tethered polymer chain conformation. This work demonstrates that polymer films are most penetrable in a good solvent and least penetrable in a poor solvent under identical flow conditions. These findings are consistent with polymer chain conformational changes going from extended brushes to compact globules. For flow rates ranging from 100 to 3000 μL/min, flow field penetration depth ranges from ~6% of the film thickness to ~57% of film thickness for a good solvent compared to ~4% to ~19% for a poor solvent. Thus, by simply changing solvent quality from good to poor, flow field penetration decreases by about 38%. Grafting density has a less pronounced effect than solvent quality on penetration depth, probably due to the small range of grafting densities chosen for study. However, a roughly 10-20% difference in penetration depth was observed between high density (0.60 chain/nm²) and low density (0.27 chain/nm²) pNIPAM surfaces in the same solvent. Changes in grafting density have a less significant impact in a good solvent compared to a poor solvent. This is most likely caused by the fact that grafting density impacts polymer chain conformation mainly through polymer-polymer repulsion, which becomes less significant in a solvent that better solvates the polymer. For the two extreme cases studied here at flow rates ranging from 100 to 3000 μL/min, the penetration depth is estimated to range from ~4-19% of the original solvent-swollen film thickness for high density pNIPAM films in a poor solvent and from ~7-67% for low density films in a good solvent. One important assumption that underlies all of this work is that the dominant mass transport mechanism for small molecules in dense polymer brushes is diffusion. This assumption was further validated through the use of two different small molecule quenchers, RhB and 2-nitrobenzylalcohol. These molecules are significantly different in size, charge, and structure, and operate by different quenching mechanisms. Despite these differences, the results for flow field penetration are statistically the same for both. These observations validate the assumption of diffusive mass transport in these films.
37
Olshausen, Philipp von [Verfasser], and Alexander [Akademischer Betreuer] Rohrbach. "Total internal reflection microscopy: super-resolution imaging of bacterial dynamics and dark field imaging = Totalreflexionsmikroskopie: Superauflösende Mikroskopie von Dynamik in Bakterien und Dunkelfeld Mikroskopie." Freiburg : Universität, 2012. http://d-nb.info/1123475865/34.
Full text
38
Abdalla, Hope Cook. "Investigation of Amyloid β Oligomer Dissociation Mechanisms by Single Molecule Fluorescence Techniques". UKnowledge, 2019. https://uknowledge.uky.edu/chemistry_etds/109.
Full textAbstract:
Alzheimer’s disease (AD) is currently considered the most prevalent neurodegenerative disease and places a large financial burden on society as healthcare resources are limited and the disease does not have a cure. Alzheimer’s disease is characterized by the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles; however current literature suggests Aβ oligomers are the main aggregating species leading to AD symptoms. Therefore, the underlying cause of Alzheimer’s, accumulation of amyloid beta, is currently being studied in hopes of developing treatment options. Our research aims at determining the mechanism and kinetics of Aβ oligomer dissociation into non-toxic monomers in the presence of denaturants or small molecule dissociators. These highly active small molecule dissociators, selected from the Apex Screen 5040 library, were previously identified by ELISA studies by the laboratory of Dr. Harry LeVine. We have used fluorescence correlation spectroscopy (FCS) to characterize the size distribution and mole fraction of synthetically prepared fluorescein labeled Aβ (1-42) oligomers. Our FCS results show that in the presence of denaturants or small molecule dissociators, oligomer dissociation may proceed by at least two different mechanisms; high order cooperative dissociation and linear dissociation. A cooperative mechanism is more desirable for therapeutics as oligomer directly dissociates into monomer rather than through various oligomer intermediates. Our FCS studies show the most efficient dissociators proceed through the cooperative dissociation mechanism. We also observed a large retardation of the oligomer dissociation in the presence of gallic acid. We also started preliminary work to develop a total internal reflection fluorescence (TIRF) spectroscopy method to image Aβ (1-42) oligomers. This technique if successful will help to verify the two distinct mechanisms seen by FCS or determine if there is one mechanism that occurs at different rates as TIRF allows for faster analysis.
39
Jia, Baohua, and n/a. "A study on the complex evanescent focal region of a high numerical aperture objective and its applications." Swinburne University of Technology, 2006. http://adt.lib.swin.edu.au./public/adt-VSWT20070205.150740.
Full textAbstract:
In recent years, optical near-field has received an ever-increasing attention owing to
its ability to localise optical signals beyond the diffraction limit. Optical near-field is
a non-propagating field existing in the close vicinity of a matter within a range less
than the wavelength of the illumination light and it carries the high spatial frequency
information showing the fine details of the matter.
An optical near-field can be generated by a near-field optical microscope with a
nano-aperture or a metal-coated fibre tip. However, common difficulties associated
with this approach, such as a fragile probe, a low throughput and signal-to-noise ratio,
and a slow response of gap controlling between the probe and the sample, make it
less applicable. Alternatively, optical near-field can be produced by total internal
reflection (TIR) occurring at the interface of a prism, which is capable of localising
the electromagnetic (EM) field in the close vicinity of the interface. However, in this
geometry, no confinement of the field can be achieved in the transverse direction,
whereas, in most applications such as optical trapping, micro-fabrication and optical
data storage, a transverse confinement of the light field is essential.
In order to achieve a transverse confinement of the light field, maintaining the
high spatial resolution of the optical near-field, and at the same time eliminating
the drawbacks associated with the conventional near-field optical microscope, a novel
near-field probe based on a high numerical aperture (NA) TIR objective combined
with annular illumination has been developed recently. In this arrangement, an
obstruction disk is inserted at the back aperture of the objective to block the light
with a convergence angle lower than the critical angle determined by the refractive
indices of the two media, resulting in a pure focused evanescent field in the second
medium.
The evanescent field produced by this method provides a useful tool for studying
light-matter interaction at the single molecule level not only because of its high
resolution but also due to its inherent merits such as no distance regulation, no heating
effect and simple experimental setup. But, the most significant advantage that makes
this method unique and superior to the other approaches in terms of producing the
optical near-field is that it allows the dynamic control of the focal field by simply
modulating the phase or amplitude or even the polarisation state of the incident beam
before it enters the objective so that complex illumination beams can be generated,
whereas in other fibre probe based approaches this goal is extremely difficult to achieve.
To make use of such a novel near-field probe, a thorough theoretical and
experimental investigation is required. A complete knowledge of the focused evanescent
field is a prerequisite for a wide range of applications including single molecule
detection, Raman spectroscopy, near-field non-linear imaging and near-field trapping.
Therefore, it is not only necessary but also urgent to exploit the focusing properties
of a focused evanescent field under complex field illumination both experimentally and
theoretically and this is the major aim of this thesis.
The complex fields, which are of particular interest in this thesis, are the radially
polarised beam and the Laguerre-Gaussian (LG) beam, because the former owns a
more compact circularly symmetric field distribution in the focal region when focused
by a high NA objective, while the latter is capable of rotating a trapped particle
by transferring the orbital angular momentum. Combining them with the focused
evanescent field is potentially able to induce novel functions in the near-field region,
which cannot be fulfilled by other near-field approaches. In this thesis, in order to
generate these two types of beams, a single liquid crystal spatial light modulator
(LCSLM) is employed to produce useful phase modulation to the incident beam.
Experimental characterisation of an evanescent focal spot is performed with
scanning near-field optical microscopy (SNOM), which is capable of providing the direct
mapping of the focused evanescent field not only because of its high spatial resolution
and its ability to detect the near-field and far-field signals simultaneously, but also due
to the motion of the piezzo-stage enables a three-dimensional characterisation of the
evanescent focal spot.
In this thesis, a SNOM system with an aluminum coated aperture probe is
implemented. The field distributions at both the interface and parallel planes with
a small distance away from the interface are obtained. To verify the applicability of
SNOM as a characterisation methodology, the field distribution in the focal region
of a high NA objective illuminated by a linearly polarised plane wave is measured
first. A focus splitting along the direction of incident polarisation is observed threedimensionally
near the interface under such a circumstance. It has been demonstrated
that the depolarisation effect plays an important role in determining the coupling
behaviour of the light into the fibre probe of SNOM. The good match between the
experimental results and theoretical predications confirms the validity of SNOM.
Theoretical investigation of a tightly focused radially polarised beam is undertaken
based on the vectorial-Debye diffraction theory because under the tight focusing of a
high NA objective, the vectorial nature of the highly localised field has to be carefully
considered in order to represent the field distribution accurately. The calculations
on the focusing properties of a radially polarised beam suggest that the longitudinal
field component in the focal region plays a dominant role in determining the overall
field distribution. Direct measurement of the focused evanescent radially polarised
beam in a three-dimensional manner near the interface is performed with SNOM. A
highly localised focal spot is achieved in the close vicinity of the coverglass. The
measured intensity distributions from SNOM show that correction of the focal spot
deformation associated with a linearly polarised beam is achieved by taking advantage
of the radially symmetric focal spot of a radially polarised beam. A smaller focal spot is
acquired due to the dominant longitudinal polarisation component in the focal region,
which possesses a more compact focal intensity distribution than that of the overall
field. The experimental results demonstrate a good agreement with the theoretical
expectations.
The fact that a radially polarised beam is capable of eliminating the focus
deformation often presented in the focal region of a high NA objective when a linearly
polarised beam is employed can be very useful in many applications, including microfabrication
using two-photon photopolymerisation technique. The theoretical study
on the two-photon point spread function (PSF) of a radially polarised beam indicates
that the focus elongation and splitting associated with a linearly polarised beam are
eliminated and the achievable lateral size of the focal spot is approximately a quarter
of the illumination wavelength, which is less than half of that under the illumination
of a linearly polarised beam. A further reductiont of the lateral size can be expected
by using annular radial beam illumination.
The investigation on the focusing properties of LG beams has also been one of
the major tasks of this thesis. Theoretical investigations of a focused evanescent LG
beam suggest that the phase shift induced by the boundary effect when a light beam
passes the interface satisfying TIR condition plays a vital role in determining the
overall shape of the total field distribution. A severe focal intensity deformation is
predicted theoretically in the case of focused evanescent LG beam illumination, which
might involve new physical phenomena when applied in the near-field trapping. Such
a focal intensity deformation is evidenced experimentally by the direct mapping result
obtained from the SNOM probe. A quantitative cross-section comparison with the
theoretical predication is conducted, which demonstrates a good agreement.
To achieve a controllable optical trap and rotation in the near-field region, complex
optical fields such as LG beams carrying orbital angular momentum, have been induced
for the manipulation of a polystyrene particle. The influence of the focal intensity
deformation on a near-field trapping has been thoroughly investigated. Rotation
motion of the particle is examined by mapping the two-dimensional (2D) transverse
trapping efficiency of the particle. Theoretical investigation reveals that a significant
tangential force component is generated on the particle when it is illuminated by a
focused evanescent LG beam. Such findings may prove useful in introducing a rotation
mechanism in near-field trapping.
The research investigations and methodologies described in this thesis provide a new
approach to characterise the near-field focal spot under complex field illumination.
It enhances the understanding of the novel near-field probe, thus opening the
pathway for numerous near-field applications including optical trapping, two-photon
excitation (photopolymerisation) and spectroscopy. The focal field rotation phenomena
demonstrated in this thesis may prove particularly beneficial in introducing a rotation
mechanism in near-field trapping using a focused evanescent field.
40
Chmyrov, Andriy. "Photo-induced dark states influorescence spectroscopy – investigations & applications." Doctoral thesis, KTH, Experimentell biomolekylär fysik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-12372.
Full textAbstract:
This thesis focuses on investigations of transient dark states of fluorescentmolecules using spectroscopic techniques. The main purpose is to show andconvince the reader that transient dark states are not always a nuisance, butalso represent an additional source of information. Several studies with fluorescencecorrelation spectroscopy were performed, all related to non-fluorescentstates such as triplet state or isomerized states.Photobleaching is one of the main problems in virtually all of the fluorescencetechniques. In this thesis, mechanisms that retard photobleaching arecharacterized. Several compounds, antioxidants and triplet state quenchers,which decrease photobleaching, are studied, and guidelines for achieving optimalfluorescence brightness using these compounds are presented.Triplet state quenching by several compounds was studied. Detailed investigationsof the fluorescence quencher potassium iodide demonstratedthat for some of fluorophores, except of quenching, there is fluorescence enhancementmechanism present. In agreement with the first publication inthis thesis, antioxidative properties were found to play an important role inthe fluorescence enhancement. Quenching of the triplet state is proposedas a tool for monitoring diffusion mediated reactions over a wide range offrequencies.Specially designed fluorophores combining high triplet yields with reasonablefluorescence brightness and photostability were characterized forpossible applications in novel super-resolution imaging techniques based onfluorescence photoswitching. Except of benefits for imaging techniques, photoinducedswitching to non-fluorescent states could be used for monitoringmolecular diffusion, which was also demonstrated in this thesis.Studies of the triplet state kinetics of fluorophores close to dielectric interfaceswere performed using fluorescence spectroscopy. The analysis of thetriplet state kinetic can provide information about the local microenvironmentand electrostatic interactions near dielectric interfaces.<br>QC 20100414
41
Wu, Hung-Jen. "Direct measurements of ensemble particle and surface interactions on homogeneous and patterned substrates." Texas A&M University, 2005. http://hdl.handle.net/1969.1/3747.
Full textAbstract:
In this dissertation, we describe a novel method that we call Diffusing Colloidal
Probe Microscopy (DCPM), which integrates Total Internal Reflection Microscopy
(TIRM) and Video Microscopy (VM) methods to monitor three dimensional trajectories
in colloidal ensembles levitated above macroscopic surfaces. TIRM and VM are well
established optical microscopy techniques for measuring normal and lateral colloidal
excursions near macroscopic planar surfaces. The interactions between particle-particle
and particle-substrate in colloidal interfacial systems are interpreted by statistical
analyses from distributions of colloidal particles; dynamic properties of colloidal
assembly are also determined from particle trajectories.
Our studies show that DCPM is able to detect many particle-surface interactions
simultaneously and provides an ensemble average measurement of particle-surface
interactions on a homogeneous surface to allow direct comparison of distributed and
average properties. A benefit of ensemble averaging of many particles is the diminished
need for time averaging, which can produce orders of magnitude faster measurement
times at higher interfacial particle concentrations. The statistical analyses (Ornstein-
Zernike and three dimensional Monte Carlo analyses) are used to obtain particle-particle
interactions from lateral distribution functions and to understand the role of nonuniformities
in interfacial colloidal systems. An inconsistent finding is the observation of
an anomalous long range particle-particle attraction and recovery of the expected DLVO
particle-wall interactions for all concentrations examined. The possible influence of
charge heterogeneity and particle size polydispersity on measured distribution functions
is discussed in regard to inconsistent particle-wall and particle-particle potentials. In the final part of this research, the ability of DCPM is demonstrated to map potential energy
landscapes on patterned surfaces by monitoring interactions between diffusing colloidal
probes with Au pattern features. Absolute separation is obtained from theoretical fits to
measured potential energy profiles and direct measurement by sticking silica colloids to
Au surfaces via electrophoretic deposition. Initial results indicate that, as colloidal probe
and pattern feature dimensions become comparable, measured potential energy profiles
suffer some distortion due to the increased probability of probes interacting with
surfaces at the edges of adjacent pattern features. Measurements of lateral diffusion via
analysis of mean square displacements also indicated lateral diffusion coefficients in
excellent agreement with rigorous theoretical predictions.
42
Mitsuru, Hirano. "Elucidation of subcellular regulation of voltage-dependent calcium channel functions via β subunit interacting molecules". Kyoto University, 2017. http://hdl.handle.net/2433/226787.
Full text
43
Morten, Michael J. "Developing novel single molecule analyses of the single-stranded DNA binding protein from Sulfolobus solfataricus." Thesis, University of St Andrews, 2015. http://hdl.handle.net/10023/7568.
Full textAbstract:
Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.
44
Huet, Sébastien. "Analyse des mouvements des granules de sécrétion à proximité de la membrane plasmique par microscopie de fluorescence à excitation par onde évanescente." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2006. http://tel.archives-ouvertes.fr/tel-00090014.
Full textAbstract:
La sécrétion régulée d'hormones est un processus décomposable en plusieurs étapes. Les granules de sécrétion (GS) contenant ces hormones sont formés au niveau du réseau trans-golgien puis migrent jusqu'à la périphérie de la cellule. Ces hormones sont libérées dans le milieu extérieur par exocytose en cas de stimulation de la cellule. Grâce à l'observation des GS situés dans la région juxta-membranaire par microscopie de fluorescence à excitation par onde évanescente, les mouvements de ces organites ont été étudiés à l'échelle du granule unique. Une méthode d'analyse permettant la mise en évidence de comportements transitoires au sein des trajectoires tridimensionnelles des GS a été mise au point. Grâce à son application à l'étude des effets de drogues du cytosquelette et à des observations en double marquage, nous avons pu associer chaque type de mouvements décrit par les GS à un environnement particulier (GS lié à la membrane plasmique, au cytosquelette d'actine ou de microtubules). Nous avons de plus étudié le rôle du complexe protéique Rab27A/MyRIP/Myosine Va lors de la capture des GS en périphérie de la cellule et leur accrochage aux filaments d'actine.
45
Le, Cunuder Anne. "Étude expérimentale des forces de Casimir." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSEN007/document.
Full textAbstract:
L'étude des fluctuations dans les milieux confinés constitue un domaine de recherche très récent, que ce soit du point de vue théorique ou expérimental. Afin d'analyser le rôle du confinement sur les propriétés des fluctuations de densité dans un mélange binaire, nous avons développé un système de mesure d'une grande sensibilité, où l'intensité des fluctuations et leur longueur de corrélation peuvent être amplifiées. L'idée consiste à travailler proche du point critique d'une transition de phase de démixion d'un mélange binaire. En effet, la longueur de corrélation augmente exponentiellement lorsqu'on s'approche de la température Tc du point critique de démixion.Nous avons développé un montage permettant de confiner le mélange entre un échantillon plan et une sphère colloïdale attachée à l'extrémité d'un levier de Microscope à Force Atomique (AFM). D'après les prédictions de Fisher et De Gennes, un effet intéressant émerge lorsque la longueur de corrélation est comparable avec la taille du confinement: les deux surfaces vont soit s'attirer, soit se repousser suivant les préférences d'adsorption des composants du mélange pour chacune des surfaces. On nomme cet effet l'effet Casimir critique, en référence à la force de Casimir électrodynamique qui résulte du confinement des fluctuations quantiques du champ électromagnétique.Durant cette thèse, nous avons mesuré la force de Casimir électrodynamique avec le système de mesure que nous avons développé, d'abord dans une atmosphère d'azote puis dans l'éthanol. Ces mesures prouvent que notre appareil de mesure est assez sensible pour mesurer des forces très faibles de l'ordre de la dizaine de pN. Les forces mesurées sont comparées à la théorie de Lifshitz, où les effets de conductivité finie des surfaces sont considérées<br>The study of density fluctuations inside confined liquid systems has received the attention of recent theoretical and experimental papers. In order to analyze the role of confinement on the statistical properties of fluctuations, we developed a highly sensitive system where the intensity of fluctuations, as well as their spatial correlation length can be simply tuned. The idea will be to enhance the role of fluctuations working close to the critical temperature Tc of a second order phase transition in a binary mixture. Indeed, the correlation length dramatically increases when one approaches the critical demixion point.The confinement is obtained by using a sphere-plane geometry with a colloidal particle attached to the cantilever of an Atomic Force Microscope (AFM). When the correlation length is comparable with the distance of confinement, Fisher and De Gennes predicted the existence of an interesting effect: the two surfaces will be submitted to either an attracting or a repelling force, depending on boundary conditions. This effect is called the critical Casimir force in reference to the quantum Casimir force resulting from the confinement of quantum fluctuations of the electromagnetic field.During this thesis, we measured the quantum Casimir force between the sphere and the plate, first in a nitrogen atmosphere and then in ethanol, showing that the developed instrument is sufficiently sensible to measure very weak force, of the same order of magnitude or even weaker than the critical Casimir force. Measurements are compared to Lifshitz theory, taking into account the finite conductivity of surfaces
46
Cheng, Shu-Ya, and 鄭淑雅. "Single Molecule Detection by Total Internal Reflection Fluorescence Microscopy." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/00415403072887196824.
Full textAbstract:
碩士<br>國立臺灣大學<br>化學研究所<br>92<br>Single molecule detection is a powerful and practical approach to explore bio-molecules, because it provides information about actual fluctuation and distribution of dynamic and kinetic parameters, enables relationship between inputs and outputs of events of proteins reaction to be qualified, and removes the need for synchronization. We use a laser of suitable wavelength to excite the selected fluorescent-labeled molecules, and then address the dynamic behaviors of target molecules by analysis and observation of the labeled molecules. This is namely single molecule fluorescence microscopy.
This work is mainly about the construction of a Prism-type Total Internal Reflection Fluorescence Microscopy system for the purpose of single molecule studies. The key of TIRFM is to reduce the background fluorescence effectively. When the light is totally reflected in the interface, the evanescent field is generated. The evanescent field is several times the intensity of the incident light, and diminishes exponentially with the distance form the interface. The penetration depth is quite narrow, around 100nm. Fluorescent-labeled molecules, which happen to be in this region, are excited selectively and emit fluorescence. The signal is collected by a high numerical aperture objective, and detected by CCD and APD. CCD is able to provide spatial information and extract statistics on numbers of individual molecules, while APD offers adequate temporal resolution. The signal pulses from one or double channels were counted and analyzed with time series.
The reporters that are chosen for ATP synthase are CyDye, including Cy3 and Cy5. We use a green laser (wavelength = 532nm) to pump the Cy3. The substrates are spin-coated with Cy3 in agarose solution of 99% water as a test specimen, with concentrations in the range of 10~100pM. Both the CCD visualization and the APD photon counting measurements demonstrate that the TIRFM does work and is capable of probing single molecules. The background fluorescence value in our experiments goes around 20 photo-electrons/sec and the SBR averages 13, 20 at most, and still could be enhanced. Before photo-bleaching, about 100,000∼1,000,000 photo-electrons were detected from each Cy3 molecule by APD.
Since the single molecule detection system has been built up successfully, we will carry on the investigation into the dynamic behaviors of bio-molecules and the study of the enzyme kinetic, such as fluorescence resonance energy transfer between Cy3 and Cy5, so as to elucidate the underlying mechanism of bio-motors from a chemist’s point of view.
47
Chen, Hsiu-Yen, and 陳修彥. "Single fluorescent molecule photobleaching detection using objective type Total Internal Reflection Fluorescence Microscopy." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/89792786749984353758.
Full textAbstract:
碩士<br>國立清華大學<br>生醫工程與環境科學系<br>94<br>In order to detect single molecule fluorescence signal, so I used total internal reflection fluorescence microscopy (TIRFM) to excite the fluorescent molecule. This setup can decrease much noise, and increase the chance of detection single fluorescent signal. Our laboratory used commercial TIRFM to combine with Dual-View and EMCCD, reached to single fluorescent molecule detection.
The sample of my experiment is single strand DNA labeled Cy3. The single strand DNA are fixed to the cover slip surface by antibody-antigen binding force. Then I used the evanescent wave by total internal reflection to excite the fluorescence, and to observe the phenomenon of single fluorescent molecule photobleaching.
In my TIRFM system, we already detected single fluorescent molecule photobleaching. In order to obtain better signal to noise ratio (SNR), I used three methods to analyze my experimental data. The method of mean value of 25 pixels in the fluorescence spot can obtain better SNR. By reducing the laser power, I detected the time of fluorescence photobleaching is increased, and the intensity of single fluorescent molecule is lowered. To enhance the time of fluorescent lifetime is important in the single molecule experiments.
I hope to observe the phenomenon of fluorescence resonance energy transfer (FRET) by using the double strands DNA labeled Cy3 and Cy5. The TIRFM system will combine with flow chamber to reach single molecule dynamical researches, and be integrated to high resolution laser tweezers system to study more biological programs in the future.
48
Hung-HsiuChen and 陳宏修. "The Study of Surface Plasmons Enhanced Total Internal Reflection Fluorescence Microscopy." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/37755946975856426505.
Full textAbstract:
碩士<br>國立成功大學<br>光電科學與工程研究所<br>98<br>In this thesis, we use the method of total internal reflection to excite surface plasmons on metal nanostructures, strengthening the regional electric field of metal nanoparticles to significantly enhance the fluorescent molecule signal. We also use nano-imprint process to fabricate various metal nanostructures as the large-area substrate to extensively enhance the fluorescent signal. Gold nanodisk and gold nanoparticle arrays are used for changing the sizes and interparticle spaces to study the optical enhancement properties of these structures, and the enhancement level of detected fluorescence signals.
The traditional total internal reflection fluorescence microscopy (TIRFM) applies more than the critical angle of light incidence to generate evanescent wave (about several hundred nanometers) on the dielectric film where the fluorescent molecules are significantly stimulated. By this scheme, the TIRFM can achieve the resolution of nanometer level, which offers the image with very low fluorescence background noises and advancing the signal to noise ratio. However, in the real-time live cell imaging, the fluorescence signals from the cell membrane containing the dynamic information of specific molecular interactions are still needed to be enhanced. In order to get stronger fluorescence signals and to reduce the microscopic detection limit, we combine TIRFM and fabricated periodic metal nanostructure arrays to respectively measure fluorescent signals on the nano-imprint lithography-generated large-area polydimethylsiloxane (PDMS) substrates via localized surface plasmon (SP) excitations by means of the evanescent wave generation.
49
Yi-Kuang, Yen, and 顏毅廣. "Real-Time Protien Monitoring and Manipulation Using Total Internal Reflection Fluorescence Microscopy." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/73213440827097249279.
Full textAbstract:
碩士<br>國立臺灣大學<br>應用力學研究所<br>92<br>The study of biomolecular recognition has been becoming crucial to provide insights into molecular genetics, design of biosensor devices, drug design, and development of targeted drug delivery systems. The in-depth understanding of biomolecular recogntion involves adsorption, interaction and desorption as well as associated manipulation between biomolecules. In this present study, we have successfully demonstrated a single biomolecular detection and real-time tracking of anti-IgG in a microchannel using total internal reflection flurorescence (TIRF) microscopy for illustration of protein adsorption and recognition.
TIRF microscopy is a well-suited technique for real-time imaging and monitoring of a single protein molecule in nano-layer fluidics due to its unique evanencent wave at the optically index-mismatch interaface that may excites fluorescences at the transparent near-wall region. Recent advances in charge coupled device (CCD) camera detection efficiency and speed have enabled the microscopy of temporal and spatial resolutions to be far-reaching 0.033 ms and 0.3 μm, respectively. A modified inverted TIRF microscopy was newly established, which allows a directed laser beam underneath through the inverted microscopy to be incident in a critical angle into the surface inside the microchannel. In this microchannel, the conductive ITO film was deposited to be electrically feedthrough for electrical manipulation. Based on the fluorescent beads analysis, which are 1.1 μm in size, the capability of TIRFM for single molecules monitoring and tracking, even the measurement in lateral size of molecules was demonstrated.
In this study, the TIRFM incorporates a MEMS-based electrical control biochip to monitor and track the motion of a single antigen molecule in which the real-time position and velocity of each frames were tracked and measured. The motion of an antigen molecule was founded to be dominated in a hydrodynamic boundary layer. A further comparison of experimental results with theoretics appears to be deviated unexpectedly, which provokes a further thought that a nano-layer fluidics exhibits a novel non-classical fluidic characteristics.
At last, the motion of fluorescence nanobeads manipulated by the application of DC voltage was real-time monitored. The velocities of beads were shown to be in a linear accordance with the applied voltages. As a result of the accordance, the manipulation of nonobeads with electrical control was demonstrated and verified.
50
Hibbel, Anneke. "Characterization of Saccharomyces cerevisiae kinesin Kip2 by total internal reflection fluorescence microscopy." 2015. https://tud.qucosa.de/id/qucosa%3A73767.
Full textAbstract:
Microtubule length control is indispensable for cytoskeletal functions such as mitotic spindle assembly and positioning. In vivo studies have shown that kinesin motor proteins can regulate microtubule length positively and negatively. The mechanisms by which kinesins act as depolymerases and catastrophe factors are well studied. By contrast, how kinesins promote microtubule growth is unknown. The aim of this work was to elucidate the mechanism by which budding yeast kinesin Kip2 regulates microtubule dynamics, using in vitro reconstitution assays combined with total internal reflection fluorescence (TIRF) and differential interference contrast (DIC) microscopy. Kip2 was shown to increase the mean length of microtubules through length-dependent polymerase and anti-catastrophe activities, both with porcine and yeast tubulin, in the absence of accessory proteins. Using single-molecule motility assays, Kip2 was shown to translocate in a highly processive, ATP-dependent manner and to processively target tubulin oligomers to microtubule plus-ends. Mutant studies to probe Kip2 structure-function relationships revealed that the N-terminus of Kip2 is dispensable for promotion of microtubule growth, while the C-terminus is not. An effort to functionally identify a tubulin/microtubule-binding domain in the Cterminus of Kip2 remained unfruitful. Finally, the combinatorial effect of Kip2 with interaction partners Bim1 and Bik1 on microtubule dynamics was reconstituted. This microtubule plus-end tracking complex promoted microtubule growth beyond the effect of Kip2 alone. Together, this work demonstrates that a kinesin motor can act directly as a length-dependent microtubule polymerase and anti-catastrophe factor in the absence of accessory proteins. Thereby, this work provides insight into how kinesins control microtubule length.