Microscopy and Microanalysis


Optimization of Pairings and Detection Conditions for Measurement of FRET between Cyan and Yellow Fluorescent Proteins

Mark A.  Rizzo  a1 , Gerald  Springer  a2 , Katsuhisa  Segawa  a3 , Warren R.  Zipfel  a4 and David W.  Piston  a2 c1
a1 University of Maryland School of Medicine, Baltimore, MD 21201, USA
a2 Department of Molecular Physiology and Biophysics, 735 Light Hall, Vanderbilt University, Nashville, TN 37232, USA
a3 3-204 Murayamadainijutaku, 1-2-46 Fujimicho Higashimurayama-shi, Tokyo 189-0024, Japan
a4 Applied & Engineering Physics, 212 Clark Hall, Cornell University, Ithaca, NY 14853, USA

Article author query
rizzo ma   [PubMed][Google Scholar] 
springer g   [PubMed][Google Scholar] 
segawa k   [PubMed][Google Scholar] 
zipfel wr   [PubMed][Google Scholar] 
piston dw   [PubMed][Google Scholar] 


Detection of Förster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater Förster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET. a

(Received September 1 2005)
(Accepted October 26 2005)

Key Words: FRET; GFP; two-photon microscopy; confocal microscopy; fluorescence spectral imaging; anisotropy; lifetime imaging.

c1 Corresponding author. E-mail: dave.piston@vanderbilt.edu


a Note: M. Rizzo and K. Segawa performed this research at Vanderbilt University (same address as Piston). W. Zipfel performed research at Cornell University.