A ratiometric fluorescent molecular probe with enhanced two-photon response upon Zn2+ binding for in vitro and in vivo bioimaging

A bipyridine centered donor–acceptor–donor (D–π–A–π–D) type ratiometric fluorescent molecular probe exhibited an unprecedented enhancement in the two-photon absorption (2PA) cross section upon Zn2+ binding. Moreover, owing to the excited state charge-transfer of the fluorophore π-backbone, a significant enhancement in the two-photon (2P) excited fluorescence intensity was observed upon Zn2+ binding, resulting in a 13-fold enhancement in the 2PA cross section and a 9-fold enhancement in fluorescence brightness at 620 nm when compared to the cation-free fluorophore. The large 2PA cross section of 1433 GM and 2P action cross section (860 GM), with an excellent 2P excited fluorescence variation from 517 to 620 nm upon Zn2+ binding, facilitated the ratiometric monitoring of free zinc ions in cells. The low cytotoxicity and good photostability of the fluorophore allowed two-photon Zn2+ imaging of HeLa cells. In addition, in vivo two-photon imaging of Zn2+ ions in hepatocytes of live rats illustrated the viability of the probe in tissue imaging and monitoring of free zinc ions in live cells.