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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 24 — Nov. 19, 2012
  • pp: 26806–26827

Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy

Ming Zhao, Run Huang, and Leilei Peng  »View Author Affiliations


Optics Express, Vol. 20, Issue 24, pp. 26806-26827 (2012)
http://dx.doi.org/10.1364/OE.20.026806


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Abstract

Förster resonant energy transfer (FRET) is extensively used to probe macromolecular interactions and conformation changes. The established FRET lifetime analysis method measures the FRET process through its effect on the donor lifetime. In this paper we present a method that directly probes the time-resolved FRET signal with frequency domain Fourier lifetime excitation-emission matrix (FLEEM) measurements. FLEEM separates fluorescent signals by their different phonon energy pathways from excitation to emission. The FRET process generates a unique signal channel that is initiated by donor excitation but ends with acceptor emission. Time-resolved analysis of the FRET EEM channel allows direct measurements on the FRET process, unaffected by free fluorophores that might be present in the sample. Together with time-resolved analysis on non-FRET channels, i.e. donor and acceptor EEM channels, time resolved EEM analysis allows precise quantification of FRET in the presence of free fluorophores. The method is extended to three-color FRET processes, where quantification with traditional methods remains challenging because of the significantly increased complexity in the three-way FRET interactions. We demonstrate the time-resolved EEM analysis method with quantification of three-color FRET in incompletely hybridized triple-labeled DNA oligonucleotides. Quantitative measurements of the three-color FRET process in triple-labeled dsDNA are obtained in the presence of free single-labeled ssDNA and double-labeled dsDNA. The results establish a quantification method for studying multi-color FRET between multiple macromolecules in biochemical equilibrium.

© 2012 OSA

OCIS Codes
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.6280) Medical optics and biotechnology : Spectroscopy, fluorescence and luminescence

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: July 24, 2012
Revised Manuscript: October 14, 2012
Manuscript Accepted: November 6, 2012
Published: November 13, 2012

Virtual Issues
Vol. 7, Iss. 12 Virtual Journal for Biomedical Optics

Citation
Ming Zhao, Run Huang, and Leilei Peng, "Quantitative multi-color FRET measurements by Fourier lifetime excitation-emission matrix spectroscopy," Opt. Express 20, 26806-26827 (2012)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-20-24-26806


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