Simultaneous Zn(2+) tracking in multiple organelles using super-resolution morphology-correlated organelle identification in living cells

Zn(2+) plays important roles in metabolism and signaling regulation. Subcellular Zn(2+) compartmentalization is essential for organelle functions and cell biology, but there is currently no method to determine Zn(2+) signaling relationships among more than two different organelles with one probe. Here, we report simultaneous Zn(2+) tracking in multiple organelles (Zn-STIMO), a method that uses structured illumination microscopy (SIM) and a single Zn(2+) fluorescent probe, allowing super-resolution morphology-correlated organelle identification in living cells. To guarantee SIM imaging quality for organelle identification, we develop a new turn-on Zn(2+) fluorescent probe, NapBu-BPEA, by regulating the lipophilicity of naphthalimide-derived Zn(2+) probes to make it accumulate in multiple organelles except the nucleus. Zn-STIMO with this probe shows that CCCP-induced mitophagy in HeLa cells is associated with labile Zn(2+) enhancement. Therefore, direct organelle identification supported by SIM imaging makes Zn-STIMO a reliable method to determine labile Zn(2+) dynamics in various organelles with one probe. Finally, SIM imaging of pluripotent stem cell-derived organoids with NapBu-BPEA demonstrates the potential of super-resolution morphology-correlated organelle identification to track biospecies and events in specific organelles within organoids.