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Multi-channel wide-field microscopic FRET imaging based on simultaneous spectral unmixing of excitation and emission spectra
1  South China Normal University


Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has inherent ability to resolve 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 recently developed a spectral wide-field microscope by integrating a liquid crystal tunable filter (LCTF) into a wide-field microscope for microscopic spectral imaging for implementing ExEm unmixing-based quantitative FRET measurement (ExEm-spFRET) in single living cells. However, the very low transmittance of LCTF, about 20% in wavelengths range from 450 to 500 nm, made this system inapplicable to single living cells with low expression levels of FPs, and the measured E values by using ExEm-spFRET method on this system were slightly larger than those measured by other’s methods. Considering the high transmittance up to 95% of optical filters, we here set up a filter-based multi-channel wide-field microscope by integrating an emission-wheel containing six different emission filters with a wide-field microscope for quantitative ExEm-spFRET measurement. Furthermore, a system correction factor (fsc), a constant for a stable instrument, is introduced for modified ExEm-spFRET (m-ExEm-spFRET). We performed m-ExEm-spFRET with four and two excitation wavelengths respectively on our multi-channel wide-field microscope to quantitatively image single living cells expressing FRET constructs, and obtained accurate FRET efficiency (E) and concentration ratio (RC) of acceptor to donor. We also performed m-ExEm-spFRET microscopic imaging for single living HepG2 cells co-expressing CFP-Bax and YFP-Bax, and found that the E values between CFP and YFP were about 0 for control cells and about 28% for staurosporin-treated cells when RC were larger than 1, indicating that staurosporin induced significant oligomerization.