Quantitative FRET measurement using emission‐spectral unmixing with independent excitation crosstalk correction

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 (R_C) 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 R_C 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 R_C 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.     

Simultaneous measurement of quantum yield ratio and absorption ratio between acceptor and donor by linearly unmixing excitation–emission spectra

Quantum yield ratio (Q_A/Q_D) and absorption ratio (K_A/K_D) in all excitation wavelengths used between acceptor and donor are indispensable to quantitative fluorescence resonance energy transfer (FRET) measurement based on linearly unmixing excitation–emission spectra (ExEm‐spFRET). We here describe an approach to simultaneously measure Q_A/Q_D and K_A/K_D values by linearly unmixing the excitation–emission spectra of at least two different donor–acceptor tandem constructs with unknown FRET efficiency. To measure the Q_A/Q_D and K_A/K_D values of Venus (V) to Cerulean (C), we used a wide‐field fluorescence microscope to image living HepG2 cells separately expressing each of four different C–V tandem constructs at different emission wavelengths with 435 nm and 470 nm excitation respectively to obtain the corresponding excitation–emission spectrum (S_DA). Every S_DA was linearly unmixed into the contributions (weights) of three excitation–emission spectra of donor (W_D) and acceptor (W_A) as well as donor–acceptor sensitisation (W_S). Plot of W_S/W_D versus W_A/W_D for the four C–V plasmids from at least 40 cells indicated a linear relationship with 1.865 of absolute intercept (Q_A/Q_D) and 0.273 of the reciprocal of slope (K_A/K_D), which was validated by quantitative FRET measurements adopting 1.865 of Q_A/Q_D and 0.273 of K_A/K_D for C32V, C5V, CVC and VCV constructs respectively in living HepG2 cells.