Multichannel wide-field microscopic FRET imaging based on simultaneous spectral unmixing of excitation and emission spectra.

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has inherent ability resolving spectral crosstalks, two key issues of quantitative fluorescence resonance energy transfer (FRET) measurement, of both the excitation and emission spectra between donor and acceptor without additional corrections. We here set up a filter-based multichannel wide-field microscope for ExEm unmixing-based FRET imaging (m-ExEm-spFRET) containing a constant system correction factor (fsc ) for a stable system. We performed m-ExEm-spFRET with four- and two-wavelength excitation respectively on our system to quantitatively image single living cells expressing FRET tandem constructs, and obtained accurate FRET efficiency (E) and concentration ratio of acceptor to donor (RC ). We also performed m-ExEm-spFRET imaging for single living cells coexpressing CFP-Bax and YFP-Bax, and found that the E values were about 0 for control cells and about 28% for staurosporin-treated cells when RC were larger than 1, indicating that staurosporin induced significant oligomerisation.