European Biophysics Journal

, 39:241

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

  • A. J. W. G. Visser
  • S. P. Laptenok
  • N. V. Visser
  • A. van Hoek
  • D. J. S. Birch
  • J.-C. Brochon
  • J. W. Borst
Original Paper

Abstract

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive.

Keywords

Time-resolved fluorescence Maximum entropy Lifetime distribution FRET Visible fluorescent proteins 

Supplementary material

249_2009_528_MOESM1_ESM.doc (3 mb)
Supplementary material 1 (DOC 3123 kb)

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Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  • A. J. W. G. Visser
    • 1
    • 2
    • 4
  • S. P. Laptenok
    • 1
    • 3
  • N. V. Visser
    • 1
    • 3
  • A. van Hoek
    • 1
    • 3
  • D. J. S. Birch
    • 4
  • J.-C. Brochon
    • 5
  • J. W. Borst
    • 1
    • 2
  1. 1.Microspectroscopy CentreWageningen UniversityWageningenThe Netherlands
  2. 2.Laboratory of BiochemistryWageningen UniversityWageningenThe Netherlands
  3. 3.Laboratory of BiophysicsWageningen UniversityWageningenThe Netherlands
  4. 4.Department of PhysicsUniversity of Strathclyde, Scottish Universities Physics Alliance, Photophysics GroupGlasgowUK
  5. 5.Laboratoire de Biotechnologies et de Pharmacologie Génétique AppliquéeEcole Normale Supérieure de CachanCachanFrance

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