Far-red fluorescent genetically encoded calcium ion indicators

Rochelin Dalangin, Bill Z. Jia, Yitong Qi, Abhi Aggarwal, Kenryo Sakoi, Mikhail Drobizhev, Rosana S. Molina, Ronak Patel, Ahmed S. Abdelfattah, Jihong Zheng, Daniel Reep, Jeremy P. Hasseman, Yufeng Zhao, Jiahui Wu, Kaspar Podgorski, Alison G. Tebo, Eric R. Schreiter, Thomas E. Hughes, Takuya Terai, Marie-Eve Paquet, Sean G. Megason, Adam E. Cohen, Yi Shen () and Robert E. Campbell ()
Additional contact information
Rochelin Dalangin: University of Alberta
Bill Z. Jia: Harvard University
Yitong Qi: Harvard University
Abhi Aggarwal: Howard Hughes Medical Institute
Kenryo Sakoi: The University of Tokyo
Mikhail Drobizhev: Montana State University
Rosana S. Molina: Montana State University
Ronak Patel: Howard Hughes Medical Institute
Ahmed S. Abdelfattah: Howard Hughes Medical Institute
Jihong Zheng: Howard Hughes Medical Institute
Daniel Reep: Howard Hughes Medical Institute
Jeremy P. Hasseman: Howard Hughes Medical Institute
Yufeng Zhao: University of Alberta
Jiahui Wu: University of Alberta
Kaspar Podgorski: Howard Hughes Medical Institute
Alison G. Tebo: Howard Hughes Medical Institute
Eric R. Schreiter: Howard Hughes Medical Institute
Thomas E. Hughes: Montana State University
Takuya Terai: The University of Tokyo
Marie-Eve Paquet: Centre de recherche CERVO
Sean G. Megason: Harvard Medical School
Adam E. Cohen: Harvard University
Yi Shen: University of Alberta
Robert E. Campbell: University of Alberta

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-58485-z

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