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.
Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins
In vivo 2p images of a dendritic segment of a layer 2/3 neuron in the motor cortex transfected with GCaMP6s and CyRFP1. The top panels show a spontaneous calcium elevation in spine 2. The bottom panels show a dendrite calcium transient. Merge, overlay of CyRFP1 and GCaMP6s images.
Max Planck Florida Institute for Neuroscience
