Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra | Zendy

Mengyan Du | Zendy; Lili Zhang | Zendy; Shusen Xie | Zendy; Tongsheng Chen | Zendy

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Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra

Author(s) -

Mengyan 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.

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