Far-red fluorescent genetically encoded calcium ion indicators
Rochelin Dalangin, Bill Jia, Yitong Qi, Abhi Aggarwal, Kenryo Sakoi, Mikhail Drobizhev, Rosana S. Molina, Ronak Patel, Ahmed S. Abdelfattah, Jihong Zheng, Daniel Reep, J. Hasseman, The GENIE Project Team, Getahun Tsegaye, Arthur Tsang, Glenn C. Turner, Y Zhao, Jiahui Wu, Kaspar Podgorski, Alison G. Tebo, Eric R. Schreiter, Thomas E. Hughes, Takuya Terai, Marie-Ève Paquet, Sean G. Megason, Adam E. Cohen, Yi Shen, Robert E. Campbell
Abstract
Genetically encoded calcium ion (Ca2+) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca2+ dynamics and neuronal activities with single-cell resolution. Here we report the design and development of two far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent proteins mKelly1 and mKelly2. FR-GECOs have excitation and emission maxima at ~596 nm and ~644 nm, respectively, display large responses to Ca2+ in vitro (ΔF/F0 = 6 for FR-GECO1a, 18 for FR-GECO1c), are bright under both one-photon and two-photon illumination, and have high affinities (apparent Kd = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca2+. FR-GECOs offer sensitive and fast detection of single action potentials in neurons, and enable in vivo all-optical manipulation and measurement of cellular activities in combination with optogenetic actuators. Genetically-encoded indicators with more red-shifted excitation and emission wavelengths are advantageous for in vivo imaging. Here, Dalangin et al. report the engineering of far-red fluorescent Ca2+ indicators and demonstrate their utility for monitoring of all-optical cardiac pacing in embryonic zebrafish.