Spectral measurement of acceptor-to-donor extinction coefficient ratio in living cells

This report presents a simple method named as sp-ECR to determine the molar extinction coefficient ratio (γ(λex)) of acceptor-to-donor in living cells at excitation wavelength λex, which is closely associated with the acceptor cross-excitation, the hardest issue of FRET quantification. sp-ECR determines γ(λex) by spectrally unmixing the emission spectrum of a donor-acceptor tandem construct under λex excitation without any additional references, such that this method can be performed under optimal imaging condition. We used sp-ECR to measure the γ(458) of Venus/Cerulean in living HepG2 cells on a confocal microscope, and the measured values were consistent with those obtained by lux-FRET method. We also used sp-ECR to measure the γ(458) values of Venus/Cerulean and YFP/CFP as well as YFP/GFP, the commonly used FRET FPs pairs in other two kinds of cancer cell lines on the confocal microscope, and found that the extinction coefficients of FPs depended on cell lines. After predetermining the γ(458) of Venus to ECFP, we used sp-ECR method to monitor the staurosporine (STS)-induced dynamical caspase-3 activation in single live A549 cells expressing SCAT3 by spectrally resolving the absolute FRET efficiency of SCAT3, and found that STS-induced caspase-3 activation in single cells is a very rapid process within 20 min.