Improved spectrometer-microscope for quantitative fluorescence resonance energy transfer measurement based on simultaneous spectral unmixing of excitation and emission spectra

Based on our recently developed quantitative fluorescence resonance energy transfer (FRET) measurement method using simultaneous spectral unmixing of excitation and emission spectra (ExEm-spFRET), we here set up an improved spectrometer-microscope (SM) for implementing modified ExEm-spFRET (mExEm-spFRET), in which a system correction factor (fsc) is introduced. Our SM system is very stable for at least six months. Implementation of mExEm-spFRET with four or two excitation wavelengths on SM for single living cells expressing different FRET constructs obtained consistent FRET efficiency (E) and acceptor–donor concentration ratio (Rc) values. We also performed mExEm-spFRET measurement for single living cells coexpressing cyan fluorescent protein (CFP)-Bax and yellow fluorescent protein (YFP)-Bax and found that the E values between CFP-Bax and YFP-Bax were very low (2.2%) and independent of Rc for control cells, indicating that Bax did not exist as homooligomer in healthy cells, but positively proportional to Rc in the case of Rc 1 for staurosporine (STS)-treated cells, demonstrating that all Bax formed homooligomer after STS treatment for 6 h.