We describe a red-shifted fluorescence resonance energy transfer (FRET) pair optimized for dual-color fluorescence lifetime imaging (FLIM). This pair utilizes a newly developed FRET donor, monomeric cyan-excitable red fluorescent protein (mCyRFP1), which has a large Stokes shift and a monoexponential fluorescence lifetime decay. When used together with EGFP-based biosensors, the new pair enables simultaneous imaging of the activities of two signaling molecules in single dendritic spines undergoing structural plasticity.
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