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

Summary 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. Lay description Quantitative fluorescence resonance energy transfer (FRE...
Source: Journal of Microscopy - Category: Laboratory Medicine Authors: Tags: Original Article Source Type: research