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Single molecule imaging of fluorescence anisotropy (Conference Presentation)
Author(s): Thomas Kavanagh; Viviane Devauges; Joseph Tideswell; Camille Perrin; Jin Li; Justin Aluko; Simon P. Poland; Andrew Beavil; Simon M. Ameer-Beg
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Paper Abstract

Given the complexity of biological systems, it is necessary to go beyond ensemble measurements and attain information at the single molecule level to accurately probe molecular properties. Single molecule imaging can examine real-time conformational dynamics [1], which is often the underlying cause of heterogeneity in molecular distributions in terms of dipole orientations, spectra, or intramolecular distances, in both stable and unstable systems. A combination of polarisation-resolved detection and 2-colour alternating laser excitation (ALEX) allows quantification of the anisotropy and stoichiometry of the fluorophores present [2]. As a result, it is possible to accurately quantify energy transfer (e.g. FRET). This technique provides a rapid approach for probing the fluorophore’s environment in terms of viscosity, interactions between molecules, and ligand-substrate binding. Here we present an optimized TIRF microscope in conjunction with ALEX and steady state fluorescence anisotropy detection [3] for single molecule imaging. Validation and determination of the limits of the technique will be by measurement of isolated fluorescent proteins. Preliminary data of single molecules with a fluorescence anisotropy read-out will be presented and future prospects discussed. 1. Santoso Y, et al. (2010) Conformational transitions in DNA polymerase I revealed by single-molecule FRET. PNAS 107: 715–720. 2. Kapanidis A, et al. (2004) Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules PNAS. 101 8936-8941. 3. Devauges V, et al. (2014) Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane. PLoS One 9: e110695.

Paper Details

Date Published: 4 March 2019
Proc. SPIE 10884, Single Molecule Spectroscopy and Superresolution Imaging XII, 108840B (4 March 2019); doi: 10.1117/12.2509832
Show Author Affiliations
Thomas Kavanagh, King's College London (United Kingdom)
Viviane Devauges, King's College London (United Kingdom)
Joseph Tideswell, King's College London (United Kingdom)
Camille Perrin, King's College London (United Kingdom)
Jin Li, King's College London (United Kingdom)
Justin Aluko, King's College London (United Kingdom)
Simon P. Poland, King's College London (United Kingdom)
Andrew Beavil, King's College London (United Kingdom)
Simon M. Ameer-Beg, King's College London (United Kingdom)

Published in SPIE Proceedings Vol. 10884:
Single Molecule Spectroscopy and Superresolution Imaging XII
Zygmunt Karol Gryczynski; Ingo Gregor; Felix Koberling, Editor(s)

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