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Proceedings Paper

Multicolor single-molecule spectroscopy for the study of complex interactions and dynamics
Author(s): Daniel Fetting; Robert Kasper; Philip Tinnefeld
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Paper Abstract

Most biological processes are governed by assemblies of several dynamically interacting molecules. We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. By comparing energy transfer of the different subpopulations, we can disentangle the reasons that lead to the occurrence of three-way junctions lacking one chromophore. A merit of the method is the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously.

Paper Details

Date Published: 13 July 2007
PDF: 9 pages
Proc. SPIE 6633, Biophotonics 2007: Optics in Life Science, 66330H (13 July 2007); doi: 10.1117/12.727832
Show Author Affiliations
Daniel Fetting, Bielefeld Univ. (Germany)
Robert Kasper, Bielefeld Univ. (Germany)
Philip Tinnefeld, Bielefeld Univ. (Germany)
Ludwig-Maximilians-Univ. (Germany)

Published in SPIE Proceedings Vol. 6633:
Biophotonics 2007: Optics in Life Science
Jürgen Popp; Gert von Bally, Editor(s)

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