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

Abstract

Quantum yield ratio (QA /QD ) and absorption ratio (KA /KD ) 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 QA /QD and KA /KD values by linearly unmixing the excitation-emission spectra of at least two different donor-acceptor tandem constructs with unknown FRET efficiency. To measure the QA /QD and KA /KD 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 (SDA ). Every SDA was linearly unmixed into the contributions (weights) of three excitation-emission spectra of donor (WD ) and acceptor (WA ) as well as donor-acceptor sensitisation (WS ). Plot of WS /WD versus WA /WD for the four C-V plasmids from at least 40 cells indicated a linear relationship with 1.865 of absolute intercept (QA /QD ) and 0.273 of the reciprocal of slope (KA /KD ), which was validated by quantitative FRET measurements adopting 1.865 of QA /QD and 0.273 of KA /KD for C32V, C5V, CVC and VCV constructs respectively in living HepG2 cells.