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Journal of Biomedical Optics / Volume 17 / Issue 1 / Special Section on FRET at 65

Quantifying the influence of yellow fluorescent protein photoconversion on acceptor photobleaching–based fluorescence resonance energy transfer measurements

J. Biomed. Opt. 17, 011010 (Feb 13, 2012); http://dx.doi.org/10.1117/1.JBO.17.1.011010

Arne Seitz, Stefan Terjung, Timo Zimmermann, and Rainer Pepperkok

European Molecular Biology Laboratory, Advanced Light Microscopy Facility, Meyerhofstr 1, 69117 Heidelberg, Germany

Fluorescence resonance energy transfer (FRET) efficiency measurements based on acceptor photobleaching of yellow fluorescent protein (YFP) are affected by the fact that bleaching of YFP produces a fluorescent species that is detectable in cyan fluorescent protein (CFP) image channels. The presented quantitative measurement of this conversion makes it possible to correct the obtained FRET signal to increase the accuracy of intensity based CFP/YFP FRET measurements. The described method can additionally be used to compare samples with very different fluorescence levels.

© 2012 Society of Photo-Optical Instrumentation Engineers

History
Received Jun 01, 2011
Accepted Dec 20, 2011
Revised Dec 19, 2011
Published online Feb 13, 2012
Digital Object Identifier
http://dx.doi.org/10.1117/1.JBO.17.1.011010
Citation
Arne Seitz, Stefan Terjung, Timo Zimmermann and Rainer Pepperkok, "Quantifying the influence of yellow fluorescent protein photoconversion on acceptor photobleaching–based fluorescence resonance energy transfer measurements", J. Biomed. Opt. 17, 011010 (Feb 13, 2012); http://dx.doi.org/10.1117/1.JBO.17.1.011010

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