Abhi Aggarwal, Heather A. Baker, Céline D. Dürst, I-Wen Chen, Pablo de Chambrier, Carrie Stine, Julia Marie Gonzales, Jonathan S. Marvin, Milène Vandal, Torgny Lundberg, Cherie A. Brown-Panton, Kenryo Sakoi, Ronak Patel, C. Wang, Frank Visser, Yannick Fouad, Smrithi Sunil, Matthew Wiens, Takuya Terai, Kei Takahashi, Roger Thompson, Timothy A. Brown, Yusuke Nasu, Minh Dang Nguyen, Grant R. Gordon, Sarah McFarlane, Kaspar Podgorski, Anthony Holtmaat, Robert E. Campbell, Alexander W. Lohman
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. In an effort to overcome these limitations, we developed OCaMP, 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, photostability, and signal-to-noise ratio. Here we show that OCaMP, an orange fluorescent GECI, is a robust tool for high-fidelity neural imaging optimized for wavelengths above 1000 nm and a practical option within the spectral gap between existing green and red GECIs.