Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells

Significance Impaired copper regulation has been implicated in a range of human diseases, yet the current mechanistic understanding of intracellular copper homeostasis remains incomplete. While Cu(I)-selective synthetic fluorescent probes could shed light on copper homeostasis within living cells by dynamically reporting intracellular Cu(I) availability, the affinity range of previously employed probes was insufficient to achieve significant metallation in the presence of glutathione (GSH) at normal physiological concentrations. Employing an air-stable phosphine-based ligand design, we devised a ratiometric indicator capable of competing for Cu(I) at attomolar buffered levels. Contrary to prior assumptions, our ratiometric imaging data indicate that intracellular Cu(I) is not buffered directly by GSH but by thiol ligands of still higher affinity.