"Red-edge" excitation of fluorescent proteins

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/colocalization-analysis-tp786850p1075160.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Recently I've been trying to detect homo-FRET between fluorescent proteins using various FRET techniques and then I came across this paper that talks about "red-edge" excitation of fluorescent probes which I had never heard of:

Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells

Author(s): Squire A, Verveer PJ, Rocks O, Bastiaens PIH

Source: JOURNAL OF STRUCTURAL BIOLOGY    Volume: 147    Issue: 1    Pages: 62-69    Published: JUL 2004

As it turns out, I'm using 532 nm in TIRF to image YFP, and 532 nm does sit very far on the "red-edge" of the YFP absorption spectrum. As a consequence, I may be unintentionally missing the homo-FRET I'm trying to detect because of the effect described in this paper.

Can anyone out there explain to me the "red-edge failure of energy transfer" effect originally reported by Weber and Shinitzky? I has always been under the impression that exciting a dye at any wavelength along the absorption spectrum didn't matter (except for the intensity of fluorescence that comes back of course), but I suppose when FRET is involved, things are more complicated. What's happening at the molecular level to prevent homo-FRET when you excite with longer wavelengths?

Thank you.

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022