The local Ca²⁺ release from the heterogeneously distributed endoplasmic reticulum (ER) calcium store has a critical role in calcium homeostasis and cellular function. However, single fluorescent protein-based ER calcium probes experience challenges in quantifying the ER calcium store in differing live cells, and intensity-based measurements make it difficult to detect local calcium microdomains in the ER. Here, we developed a genetically encoded ratiometric ER calcium indicator (GCEPIA1-SNAP_ER) that can detect the real-time ER calcium store and local calcium microdomains in live cells. GCEPIA1-SNAP_ER was located in the lumen of the ER and showed a linear, reversible and rapid response to changes in the ER calcium store. The GCEPIA1-SNAP_ER probe effectively monitored the depletion of the ER calcium store by TG or starvation treatment, and through its use we identified heterogeneously distributed calcium microdomains in the ER which were correlated with the distribution of STIM1 clusters upon ER calcium store depletion. Lastly, GCEPIA1-SNAP_ER can be used to detect the ER calcium store by high-throughput flow cytometry and confers the ability to study the function of calcium microdomains of the ER.

Single-molecule localization microscopy (SMLM) has the highest spatial resolution among the existing super-resolution imaging techniques, but its temporal resolution needs further improvement. An sCMOS camera can effectively increase the imaging rate due to its large field of view and fast imaging speed. Using an sCMOS camera for SMLM imaging can significantly improve the imaging time resolution, but the unique single-pixel-dependent readout noise of sCMOS cameras severely limits their application in SMLM imaging. This paper develops a Hessian-based SMLM (Hessian-SMLM) method that can correct the variance, gain, and offset of a single pixel of a camera and effectively eliminate the pixel-dependent readout noise of sCMOS cameras, especially when the signal-to-noise ratio is low. Using Hessian-SMLM to image mEos3.2-labeled actin was able to significantly reduce the artifacts due to camera noise.