A sensitive orange fluorescent calcium ion indicator for imaging neural activity

Aggarwal, A.; Baker, H. A.; Durst, C. D.; Chen, I.-W.; de Chambrier, P.; Gonzales, J. M.; Marvin, J. S.; Vandal, M.; Lundberg, T.; Sakoi, K.; Patel, R. H.; Wang, C.-Y.; Visser, F.; Fouad, Y.; Sunil, S.; Wiens, M.; Terai, T.; Takahashi-Yamashiro, K.; Thompson, R. J.; Brown, T. A.; Nasu, Y.; Nguyen, M. D.; Gordon, G. R. J.; McFarlane, S.; Podgorski, K.; Holtmaat, A.; Campbell, R. E.; Lohman, A. W.

doi:10.1101/2025.07.28.667269

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bioRxiv · 10.1101/2025.07.28.667269

A sensitive orange fluorescent calcium ion indicator for imaging neural activity

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Abstract

Genetically encoded calcium indicators (GECIs) are vital tools for fluorescence-based visualization of neuronal activity with high spatial and temporal resolution. However, current highest-performance GECIs are predominantly green or red fluorescent, limiting multiplexing options and efficient excitation with fixed-wavelength femtosecond lasers operating at 1030 nm. Here, we introduce OCaMP (also known as O-GECO2), an orange fluorescent GECI engineered from O-GECO1 through targeted substitutions to improve calcium affinity while retaining the favorable photophysical properties of mOrange2. OCaMP exhibits improved two-photon cross-section, responsiveness, photostability, and calcium affinity relative to O-GECO1. In cultured neurons, zebrafish, and mouse cortex, OCaMP outperforms the red GECIs jRCaMP1a and jRGECO1a in sensitivity, kinetics, and signal-to-noise ratio. These properties establish OCaMP as a robust tool for high-fidelity neural imaging optimized for 1030 nm excitation and a compromise-free option within the spectral gap between existing green and red GECIs.

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Aggarwal, A., Baker, H. A., Durst, C. D., Chen, I.-W., de Chambrier, P., Gonzales, J. M., Marvin, J. S., Vandal, M., Lundberg, T., Sakoi, K., Patel, R. H., Wang, C.-Y., Visser, F., Fouad, Y., Sunil, S., Wiens, M., Terai, T., Takahashi-Yamashiro, K., Thompson, R. J., Brown, T. A., Nasu, Y., Nguyen, M. D., Gordon, G. R. J., McFarlane, S., Podgorski, K., Holtmaat, A., Campbell, R. E., Lohman, A. W.. 2025-07-31. A sensitive orange fluorescent calcium ion indicator for imaging neural activity. https://doi.org/10.1101/2025.07.28.667269

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