title: Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra.
abstract: Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has the inherent ability to resolve donor emission, fluorescence resonance energy transfer (FRET)-sensitized acceptor emission and directly excited acceptor emission. We here develop an ExEm unmixing-based quantitative FRET measurement method (EES-FRET) independent of excitation intensity and detector parameter setting. The ratio factor (rK), predetermined using a donor-acceptor tandem construct, of total acceptor absorption to total donor absorption in excitation wavelengths used is introduced for determining the concentration ratio of acceptor to donor. We implemented EES-FRET method on a wide-field microscope to image living cells expressing tandem FRET constructs with different donor-acceptor stoichiometry.
authors.forename: Mengyan
authors.initials: M
authors.lastname: Du
authors.forename: Lili
authors.initials: L
authors.lastname: Zhang
authors.forename: Shusen
authors.initials: S
authors.lastname: Xie
authors.forename: Tongsheng
authors.initials: T
authors.lastname: Chen