8 January 2018 Improved spectrometer-microscope for quantitative fluorescence resonance energy transfer measurement based on simultaneous spectral unmixing of excitation and emission spectra
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Abstract
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 ( f sc ) 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 ( R c ) 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 R c for control cells, indicating that Bax did not exist as homooligomer in healthy cells, but positively proportional to R c in the case of R c < 1 and kept constant value (25%) when R c > 1 for staurosporine (STS)-treated cells, demonstrating that all Bax formed homooligomer after STS treatment for 6 h.
© 2018 Society of Photo-Optical Instrumentation Engineers (SPIE)
Fangrui Lin, Fangrui Lin, Mengyan Du, Mengyan Du, Fangfang Yang, Fangfang Yang, Lichun Wei, Lichun Wei, Tongsheng Chen, Tongsheng Chen, } "Improved spectrometer-microscope for quantitative fluorescence resonance energy transfer measurement based on simultaneous spectral unmixing of excitation and emission spectra," Journal of Biomedical Optics 23(1), 016006 (8 January 2018). https://doi.org/10.1117/1.JBO.23.1.016006 . Submission: Received: 18 March 2017; Accepted: 7 December 2017
Received: 18 March 2017; Accepted: 7 December 2017; Published: 8 January 2018
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