Intracellular Calcium Imaging (Part II): Practical Methods and Concepts (I) for intra-organellar Ca<sup>2+</sup> imaging in cultured cells

Intracellular calcium (Ca2+) dynamics reflect coordinated Ca2+ fluxes in the cytosol as well as bidirectional exchanges between the cytosol, extracellular space, and intracellular organelles. Among these, the endoplasmic reticulum (ER) and mitochondria serve as major intracellular Ca2+ stores and shape the spatiotemporal organization of cytosolic Ca2+ signals. Organellar Ca2+ imaging provides insights difficult to obtain from cytosolic recordings alone, revealing heterogeneous responses within single cells and uncovering regulatory mechanisms essential for cellular homeostasis. Direct visualization of Ca2+ dynamics within these compartments has been enabled by modifications of genetically encoded Ca2+ indicators (GECIs), which are engineered by adding organelle-targeting sequences and amino acid substitutions in the EF-hand domain of calmodulin to adjust Ca2+ affinity. We developed a family of organelle-optimized GECIs, termed CEPIA, by genetically modifying G-CaMPs and GECOs. CEPIAs permit high-resolution Ca2+ imaging of the ER lumen and mitochondrial matrix. This article outlines the principles and practical aspects of organellar Ca2+ imaging using CEPIA and is intended to guide researchers in applying CEPIA-based approaches to elucidate how organelle-resolved Ca2+ dynamics complement cytosolic signaling in physiology and disease.