20 March 2015 Miniature fiber optic spectrometer-based quantitative fluorescence resonance energy transfer measurement in single living cells
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Abstract
Spectral measurement of fluorescence resonance energy transfer (FRET), spFRET, is a widely used FRET quantification method in living cells today. We set up a spectrometer-microscope platform that consists of a miniature fiber optic spectrometer and a widefield fluorescence microscope for the spectral measurement of absolute FRET efficiency (E) and acceptor-to-donor concentration ratio (RC) in single living cells. The microscope was used for guiding cells and the spectra were simultaneously detected by the miniature fiber optic spectrometer. Moreover, our platform has independent excitation and emission controllers, so different excitations can share the same emission channel. In addition, we developed a modified spectral FRET quantification method (mlux-FRET) for the multiple donors and multiple acceptors FRET construct (mDnA) sample, and we also developed a spectra-based 2-channel acceptor-sensitized FRET quantification method (spE-FRET). We implemented these modified FRET quantification methods on our platform to measure the absolute E and RC values of tandem constructs with different acceptor/donor stoichiometries in single living Huh-7 cells.
© 2015 Society of Photo-Optical Instrumentation Engineers (SPIE)
Liuying Chai, Jianwei Zhang, Lili Zhang, Tong-sheng Chen, "Miniature fiber optic spectrometer-based quantitative fluorescence resonance energy transfer measurement in single living cells," Journal of Biomedical Optics 20(3), 037008 (20 March 2015). https://doi.org/10.1117/1.JBO.20.3.037008 . Submission:
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