Abstract Genetically encoded calcium ion (Ca 2+ ) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca 2+ dynamics and neuronal activities on a single cell level. Here we report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We characterized these far-red GECIs as purified proteins and assessed their performance when expressed in cultured neurons. FR-GECOs have excitation and emission maxima at ~ 596 nm and ~ 644 nm, respectively, display large responses to Ca 2+ (Δ F / F 0 = 6 for FR-GECO1a, 18 for FR-GECO1c), and are bright under both one-photon and two-photon illumination. FR-GECOs also have high affinities (apparent K d = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca 2+ , and they enable sensitive and fast detection of single action potentials in neurons.

Far-red fluorescent genetically encoded calcium ion indicators