Open AccessWide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra
Author(s) -
Mingde Du,
Lili Zhang,
Shusen Xie,
Tongsheng Chen
Publication year - 2016
Publication title -
optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/oe.24.016037
Subject(s) - förster resonance energy transfer , acceptor , materials science , fluorescence , absorption (acoustics) , excitation , excited state , emission spectrum , absorption spectroscopy , spectroscopy , analytical chemistry (journal) , optics , spectral line , chemistry , atomic physics , physics , quantum mechanics , astronomy , chromatography , composite material , condensed matter physics
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.