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Multichannel wide‐field microscopic FRET imaging based on simultaneous spectral unmixing of excitation and emission spectra
Author(s) -
DU M.,
MAI Z.,
YANG F.,
LIN F.,
WEI L.,
CHEN T.
Publication year - 2018
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/jmi.12609
Subject(s) - förster resonance energy transfer , excitation , fluorescence , spectral line , spectral imaging , acceptor , emission spectrum , chemistry , fluorescence lifetime imaging microscopy , analytical chemistry (journal) , optics , physics , chromatography , quantum mechanics , astronomy , condensed matter physics
Summary Simultaneous spectral unmixing of excitation and emission spectra ( ExEm unmixing) has inherent ability resolving spectral crosstalks, two key issues of quantitative fluorescence resonance energy transfer (FRET) measurement, of both the excitation and emission spectra between donor and acceptor without additional corrections. We here set up a filter‐based multichannel wide‐field microscope for ExEm unmixing‐based FRET imaging ( m ‐ ExEm‐spFRET ) containing a constant system correction factor ( f sc ) for a stable system. We performed m‐ExEm‐spFRET with four‐ and two‐wavelength excitation respectively on our system to quantitatively image single living cells expressing FRET tandem constructs, and obtained accurate FRET efficiency ( E ) and concentration ratio of acceptor to donor ( R C ). We also performed m‐ExEm‐spFRET imaging for single living cells coexpressing CFP‐Bax and YFP‐Bax, and found that the E values were about 0 for control cells and about 28% for staurosporin‐treated cells when R C were larger than 1, indicating that staurosporin induced significant oligomerisation.