JournalNeural Excitability, Synapses, and GliaTechniques

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.

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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.


Laviv, T., Kim, B.B., Chu, J., Lam, A.J., Lin, M.Z., and Yasuda, R. (2016). Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins. Nat Meth advance online publication.
DOI: https://dx.doi.org/10.1038/nmeth.4046

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