Quantitative dual-channel FRET microscopy

• DOI: 10.1364/oe.25.026089
• Publication Date: 2017-10-12T14:57:51Z
• Authors: Lichun Wei; Jiang Zhang; Zihao Mai; Fangfang Yang; Mengyan Du; Fangrui Lin; Junle Qu; Tongsheng Chen
• Keywords: Acceptor; Crosstalk

Acceptor-sensitized quantitative Förster resonance energy transfer (FRET) measurement (E-FRET) is mainly impeded by donor emission crosstalk and acceptor direct excitation crosstalk. In this paper, we develop a novel E-FRET approach (Lux-E-FRET) based on linear unmixing (Lux) of the fluorescence intensity ratio between two detection channels with each different wavelengths. The need not to selectively collect or acceptor, wavelengths excite acceptor. For tandem FRET sensor, Lux-E-FRET only needs single wavelength. We performed measurements our dual-channel wide-field microscope for constructs in living cells, obtained consistent efficiencies those measured other methods. Collectively, completely overcomes all spectral crosstalks thus applicable donor-acceptor pair larger overlapping.