Förster (fluorescence) resonance energy transfer (FRET) is an ideal technique to estimate the distance between interacting protein molecules in live specimens using intensity-based microscopy. The spectral overlap of donor and acceptor— essential for FRET—also generates a contamination of the FRET signal. There are a number of algorithms available to remove this spectral bleedthrough (SBT) contamination and in this paper we compare two popular algorithms to estimate the SBT element and to calculate a more precise level of energy transfer efficiency, and with that a more accurate distance estimate.
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ACKNOWLEDGMENTS
We wish to acknowledge the funding provided by the National Center for Research Resources (NCRR-NIH, RR021202) and the University of Virginia. We also wish to thank Drs. Margarida Barroso, Richard Day and Horst Wallrabe for their valuable suggestions.
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Chen, Y., Periasamy, A. Intensity Range Based Quantitative FRET Data Analysis to Localize Protein Molecules in Live Cell Nuclei. J Fluoresc 16, 95–104 (2006). https://doi.org/10.1007/s10895-005-0024-1
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DOI: https://doi.org/10.1007/s10895-005-0024-1