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Showing 1 to 20 of 26 for “"Total internal reflection fluorescence (TIRF)"”.
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Development of single molecule total internal reflection fluorescence microscope
… hidden in ensemble experiments. I have built a total internal reflection fluorescence (TIRF) microscope with single-molecule sensitivity to study fluorescent molecules. The microscope is modular and multifunctional with capabilities including a wide excitation wavelength range (400-2000 nm) both …
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Single-molecule imaging studies of protein dynamics
Single-molecule fluorescence imaging is a powerful method for studying biological events. The work of this thesis primarily focuses on single molecule studies of the dynamics of Green Fluorescent Protein: GFP) and other fluorescent-labeled proteins by utilizing Total Internal Reflection …
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Single molecule fluorescence spectroscopy of ClpXP-mediated substrate degradation
… occurs. Here, we present the single molecule fluorescence assay for probing the interaction between the ClpXP enzyme and its substrates. A covalently crosslinked ClpX hexamer maintain functionally stable form at the low concentration of single molecule level. Surface passivation through …
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Direct Single Molecule Imaging of Enhanced Diffusion for Enzymes and Enzyme-conjugated Dna origami
… using single-particle tracking (SPT) with total internal reflection fluorescence (TIRF) microscopy. We found that the mobility of individual enzymes was enhanced three-fold in the presence of the substrate, and the motion remained Brownian. We showed that the relative increase in diffusion …
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Entwicklung eines fluoreszenz-optischen Evaneszenzfeldmesssystems für die Echtzeitanalyse von DNA-Mikroarrays
In dieser Arbeit ist das Messprinzip der Total Internal Reflection Fluorescence (TIRF) auf die Mikroarraytechnologie übertragen, angepasst und optimiert worden, um Hybridisierungsereignisse auf DNA-Mikroarrays in Echtzeit analysieren zu können. Dazu wurden verschiedene (mikro)-optische Auflicht- …
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Domain Redistribution in Lipid Bilayers in the Presence of the Antimicrobial Lipopeptide Fengycin
… cyclic lipopeptide fengycin on model membranes. Total internal reflection fluorescence (TIRF) microscopy was used to visualize and analyse lipid phase separation within supported lipid bilayers (SLBs) of various compositions. The initial research investigated the influence of protocol parameters …
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Probing cellular protein complexes using single-molecule pull-down
… biomolecules at single-molecule resolution using total internal reflection fluorescence (TIRF) microscopy. We name this technology single-molecule pull-down or SiMPull. In one reification biotinylated antibody against the protein of interest is immobilized on a flow chamber. The flow chambers are …
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Potential-Dependent-Adsorption and Reorientation Responses Associated With a Phospholipid Monolayer at the Water /Dichloroethane Interface
Total internal reflection fluorescence (TIRF) and electrocapillary measurements were employed to interrogate the potential dependent mechanical and physicochemical properties of adsorbed films at the interface between two immiscible electrolyte solutions (ITIES). Mixed films of a phospholipid, …
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Super-Resolution Fluorescence Microscopy using Transparent Waveguide Chips
Super-resolution fluorescence microscopy (optical nanoscopy) allows us to surpass the optical diffraction limit and observe biological phenomena at the nanoscale using visible light. Different modalities come with their own trade-offs, such as imaging speed vs. achieved resolution or live cell …
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Investigating Spatiotemporal Kinetics, Dynamics, and Mechanism of Exosome Release
… cells (A549) using CD63 fluorescent probes and total internal reflection fluorescence (TIRF) microscopy. The kinetics of release, or loss of fluorescence post-fusion, can relay information about the fate of exosomes. Using kinetic analysis we determined that a portion of exosomes are free to …
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Microfluidic chips for combinatorial screening applications
… of detecting complimentary DNA fragments. Total internal reflection fluorescence (TIRF) microscopy is used for read out of the chip. Four virus-like oligomer targets with sequences corresponding to key fragments of the viruses HIV, HPV, Hepatitis A and Hepatitis B were tested against four …
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Development of tools for single molecule peptide fluorosequencing
… fluorophore labeling of amino acid residues, total-internal reflection fluorescence (TIRF) microscopy, and Edman degradation, is a novel technology that has been developed to sequence peptides. Unique fluorophores are covalently attached to amino acid residues and through TIRF microscopy are …
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Stoichiometry of the interactions between endogenous Orai1 and STIM1 during store-operated Ca2+ entry
… of STIM1 mixed freely and interacted with Orai1. Total internal reflection fluorescence (TIRF) imaging analyses indicated that there was only a modest increase in the average size of STIM1 puncta after store depletion. Stepwise photobleaching analyses revealed that there was an average of 14.5 …
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Exploring the Structural Dynamics of Bacterial Chemotaxis
… the construction and characterization of a total internal reflection fluorescence (TIRF) microscope for use in single-molecule FRET measurements, which permits the observation of hundreds of single molecules in parallel. This enables the high-throughput screening of chemoreceptors with …
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Quantifying the Release of Protein Substrates from AAA+ ATPase ClpX by Single Molecule Total Internal Reflection Fluorescence Microscopy
… ATPase ClpXP. We present a new assay based on Total-Internal-Reflection Fluorescence (TIRF) microscopy that measures the lifetime of the ClpX-substrate complex. We demonstrate that the technique has the potential to identify factors that affect the mean lifetime of the ClpX-substrate complex. …
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USING SINGLE VIRION FUSION ASSAY TO STUDY INFLUENZA VIRUS ENTRY
… each of these processes in vitro. SPT combines total internal reflection fluorescence (TIRF) microscopy with microfluidics and supported lipid bilayers, making it a very powerful tool for host-pathogen membrane fusion studies. One concern about influenza is that the virus can mutate quickly via …
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MEMS-based temperature-dependent characterization of biomolecular interactions
… handling, on-chip temperature control, and total internal reflection fluorescence (TIRF) microscopy, we have studied the temperature dependence of the structural dynamics of transfer RNA (tRNA) translocation through ribosome in protein synthesis. For solution-based biomolecular interactions, …
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High-resolution optical analyses of IP3-evoked Ca2+ signals
… 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 …
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Investigating the molecular pharmacology of class A and B G protein-coupled receptors
… receptor activity-modifying proteins (RAMPs), or internalisation, whereby distinct responses may be propagated by intracellular GPCR populations. Hence, the signalling repertoire subsequent to receptor activation is extremely complex, varying between individual GPCRs and the biological systems in …
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Development of the single-molecule tracking and fluorescence lifetime techniques for biophysical measurements and biomedical applications
… 3). In Chapter 4, I further employed 3D-SMT and fluorescence correlation spectroscopy (FCS) to characterize the diffusivity of an individual JNK2 oligomers in live HeLa cells and demonstrated that JNK2 tetramers can be stable inside mammalian cells. Although total internal reflection fluorescence …
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