Highly dynamic and sensitive NEMOer calcium indicators for imaging ER calcium signals in excitable cells

The Endoplasmic/sarcoplasmic reticulum (ER/SR) is central to calcium (Ca²⁺) signaling, yet current genetically encoded Ca²⁺ indicators (GECIs) cannot detect elementary Ca²⁺ release events from ER/SR, particularly in muscle cells. Here, we report NEMOer, a set of organellar GECIs, to efficiently capture ER Ca²⁺ dynamics with increased sensitivity and responsiveness. NEMOer indicators exhibit dynamic ranges an order of magnitude larger than G-CEPIA1er, enabling 2.7-fold more sensitive detection of Ca²⁺ transients in both non-excitable and excitable cells. The ratiometric version further allows super-resolution monitoring of local ER Ca²⁺ homeostasis and dynamics. Notably, NEMOer-f enabled the inaugural detection of Ca²⁺ blinks, elementary Ca²⁺ releasing signals from the SR of cardiomyocytes, as well as in vivo spontaneous SR Ca²⁺ releases in zebrafish. In summary, the highly dynamic NEMOer sensors expand the repertoire of organellar Ca²⁺ sensors that allow real-time monitoring of intricate Ca²⁺ dynamics and homeostasis in live cells with high spatiotemporal resolution.