We recently developed a quantitative Förster resonance energy transfer (FRET) measurement method based on emission-spectral unmixing (Iem-spFRET). We here developed an improved Iem-spFRET method (termed as IIem-spFRET) for more robust FRET measurement in living cells. First, two background (BG) spectral fingerprints measured from blank living cells are introduced to remove BG and autofluorescence. Second, we introduce a ? factor denoting the ratio of two molar extinction coefficient ratios (?) of acceptor to donor at two excitations into IIem-spFRET for direct measurement of the ? values using a tandem construct with unknown FRET efficiency (E). We performed IIem-spFRET on our microscope–spectrometer platform to measure the ? values of Venus (V) to Cerulean (C) and the E values of C32V, CVC, VCV, and VCVV constructs, respectively, in living Huh7 cells. For the C32V or CVC cells, the Iem-spFRET and IIem-spFRET methods measured consistent E values. However, for the cells especially with low expressing levels of VCV or VCVV, the E values measured by Iem-spFRET showed large deviations and fluctuations, whereas the IIem-spFRET method greatly improved the measured E values. Collectively, IIem-spFRET is a powerful and robust tool for quantitatively measuring FRET signal in living cells.
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