Near-infrared (NIR) genetically encoded calcium ion (Ca²⁺) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca²⁺ dynamics.

近红外（NIR）基因编码的钙离子（Ca ²⁺）指示剂（GECI）在降低光毒性，与可见光可激发光遗传学工具和荧光探针的光谱串扰最小方面以及在减少散射方面可提供优于可见波长荧光GECI的优势和在哺乳动物组织中的吸收。我们的先前报道的NIR GECI NIR-GECO1具有这些优点，但在用于神经元活动成像时，与最新的可见波长GECIs相比，还具有一些缺点，包括较低的亮度和有限的荧光响应。在这里，我们报告了2个改进的NIR GECI变体，分别命名为NIR-GECO2和NIR-GECO2G，它们衍生自NIR-GECO1。我们表征了新型NIR GECI在培养细胞，急性小鼠脑切片和秀丽隐杆线虫和非洲爪蟾体内。我们的结果表明，NIR-GECO2和NIR-GECO2G在神经元Ca ²⁺动力学成像方面比NIR-GECO1有了实质性的改进。