title: Quantitative FRET measurement using emission-spectral unmixing with independent excitation crosstalk correction.
abstract: Quantification of fluorescence resonance energy transfer (FRET) needs at least two external samples, an acceptor-only reference and a linked FRET reference, to calibrate fluorescence signal. Furthermore, all measurements for references and FRET samples must be performed under the same instrumental conditions. Based on a novel notion to predetermine the molar extinction coefficient ratio (RC ) of acceptor-to-donor for the correction of acceptor excitation crosstalk, we present here a robust and independent emission-spectral unmixing FRET methodology, Iem-spFRET, which can simultaneously measure the E and RC of FRET sample without any external references, such that Iem-spFRET circumvents the rigorous restriction of keeping the same imaging conditions for all FRET experiments and thus can be used for the direct measurement of FRET sample. We validate Iem-spFRET by measuring the absolute E and RC values of standard constructs with different acceptor-to-donor stoichiometry expressed in living cells. Our results demonstrate that Iem-spFRET is a simple and powerful tool for real-time monitoring the dynamic intermolecular interaction within single living cells.
authors.affiliation: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou, China.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: H
authors.initials: H
authors.lastname: Li
authors.forename: L
authors.initials: L
authors.lastname: Chai
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Qu
authors.forename: T
authors.initials: T
authors.lastname: Chen
mesh_terms: D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods* / Q000592:standards; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000379:methods* / Q000592:standards
chemical_list: D008164:Luminescent Proteins
keywords: Crosstalk; FRET; emission-spectral unmixing; fluorescent proteins; living cells; microscopy