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Hello Scott,

To some degree the answer to your question depends on the type of FRET 
detection strategy.

Those GFP variants that are the most efficient donor-acceptor pairs for 
FRET measurements show a high degree of spectral overlap; this tends to 
confound accurate analysis by conventional filter-based imaging systems. 
  Spectral imaging and un-mixing technology provides the ability to 
succesfully use FRET pairs with greater overlap, such as GFP-YFP 
(Zimmermann et al., 2002).  The study demonstrates that linear un-mixing 
of convolved spectral signatures for FRET analysis can be performed 
reliably with relatively few, in this case four, wavelength channels 
contributing to the dataset.

By developing a spectral unmixing algorithm (sRET analysis) that 
accounts for the effects of resonant energy transfer on the intensity 
distribution of spectral components eminating from mixtures of donor and 
acceptor molecules, Thaler et al. showed that spectral imaging and 
subsequent computational post-processing can be used to accurately 
measure donor and acceptor concentrations as well as their FRET 
efficiencies.

Sophisticated quantitative FRET methods reported by Gu et al. and Raicu 
et al. both involve combining spectral imaging and acceptor photobleaching.

Gu, Y, Di, WL, Kelsell, DP, and Zicha, D. (2004). ‘Quantitative 
fluorescence resonance energy transfer (FRET) measurement with acceptor 
photobleaching and spectral unmixing.’ J. Microsc. 215, 2: 162-173.

Raicu, V, Jansma, DB, Dwayne Miller, RJ, and Friesen, JD. (2005). 
‘Protein interaction quantified in vivo by spectrally resolved 
fluorescence resonance energy transfer.’ Biochem. J. 385: 265-277.

Thaler, C, Koushik, SV, Blank, PS, Vogel, SS. (2005). 'Quantitative 
Multiphoton Spectral Imaging and its use for Measuring Resonance Energy 
Transfer.' Biophys. J. 89: 2736-2749.

Zimmerman, T, Rietdorf, J, Girod, A, Georget, V, Pepperkok, R. (2002). 
'Spectral imaging and linear un-mixing enables improved FRET efficiency 
with a novel GFP2-YFP FRET pair.' FEBS Letters 531: 245-249.

Cheers,
Karl


Scott Howell wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> 
> List Members,
> 
> 
> Have a member lab that wants to do some FRET work. They are starting 
> at the start, preparing to build their clones. Therefore the question 
> is: What at the moment seems to be the best fret pair that will give 
> us the best chance for success? Have worked with CFP/YFP in the past 
> but guessing there is something superior these days. Thanks.
> 
> Scott J. Howell, Ph.D.
> Manager, Imaging Module
> Visual Sciences Research Center
> Case Western Reserve University
> 2085 Adelbert Rd. 
> Institute of Pathology Room 106
> Cleveland, Ohio 44106
> 216-368-2300
> http://www.case.edu/med/vsrc/

-- 
Karl Garsha
Research Microscopy Specialist
US-Southwest Region
Leica Microsystems-Life Sciences Research
www.leica-microsystems.com