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.
Highlights
Fluorescence resonance energy transfer (FRET) microscopy has become an invaluable tool for monitoring intracellular dynamic spatio-temporal activity of biochemical events during signal transduction
Improvements in the spectral characteristics of genetically encoded fluorescent proteins (FPs) and FPs-biosensors enable fluorescence resonance energy transfer (FRET)-based visualization of dynamic signaling events to measure the protein-protein interaction of Bcl-2 family proteins during cell apoptosis [1,2] and the structural changes of fibroblast growth factors receptor dimerization on different ligands binding [3] as well as the dependence of mitochondrial calcium uptake from the cytosolic Ca2+ signals [4] within living cells
ExEm unmixing is capable of simultaneously resolving the donor spectral bleedthrough and acceptor excitation cross without additional correction
Summary
Fluorescence resonance energy transfer (FRET) microscopy has become an invaluable tool for monitoring intracellular dynamic spatio-temporal activity of biochemical events during signal transduction. Overlapping spectra between donor and acceptor, including donor spectral bleedthrough (donor emission into acceptor channel) and acceptor excitation cross (direct excitation of acceptor fluorophores by donor excitation), is inevitable for FP-FRET pairs [8]. Donor spectral bleedthrough can be overcome by Em unmixing, but the acceptor excitation cross must be corrected using additional manner [13,14,15,16]. Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has the inherent ability to resolve donor emission, FRET-sensitized acceptor emission and direct acceptor emission [5,6,17,18]. ExEm unmixing is capable of simultaneously resolving the donor spectral bleedthrough and acceptor excitation cross without additional correction
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