13 October 2016 IIem-spFRET: improved Iem-spFRET method for robust FRET measurement
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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 ρ factor denoting the ratio of two molar extinction coefficient ratios (γ) of acceptor to donor at two excitations into IIem-spFRET for direct measurement of the γ values using a tandem construct with unknown FRET efficiency (E). We performed IIem-spFRET on our microscope–spectrometer platform to measure the γ 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.
© 2016 Society of Photo-Optical Instrumentation Engineers (SPIE)
Jiang Zhang, Jiang Zhang, Fangrui Lin, Fangrui Lin, Liuying Chai, Liuying Chai, Lichun Wei, Lichun Wei, Tongsheng Chen, Tongsheng Chen, } "IIem-spFRET: improved Iem-spFRET method for robust FRET measurement," Journal of Biomedical Optics 21(10), 105003 (13 October 2016). https://doi.org/10.1117/1.JBO.21.10.105003 . Submission:

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