Dual-detection of mitochondrial viscosity and SO2 derivatives with two cross-talk-free emissions employing a single two-photon fluorescent probe

A versatile two-photon fluorescent probe MCB for dual-detection of viscosity and SO2 derivatives in mitochondria with two cross-talk-free emissions (wavelength difference of 174 nm) has been rationally designed and synthesized. To our delight, probe MCB can not only recognize SO2 derivatives utilizing a blue fluorescence emission at 392 nm with a low detection limit (2 nM) and a fast response time (˜ 60 s), but also monitor viscosity by a red fluorescence emission at 566 nm in the methanol/glycerol mixed viscosity system. The viscosity value showed a good linearity with the fluorescence lifetime of MCB (R = 0.99, x = 0.43). Probe MCB was successfully applied to monitor viscosity and exogenous/endogenous SO2 utilizing the two cross-talk-free channels (red and blue) in mitochondria via two-photon microscopy (TPM) owe to its good two-photon absorption performance and mitochondria-located capability. Probe MCB can detect the change of mitochondrial viscosity during nystatin-induced apoptosis. Mitochondrial viscosity mapping was studied quantificationally via fluorescence lifetime imaging microscopy (FLIM). Furthermore, TP fluorescence dual-color imaging of MCB in live zerbrafishes demonstrate it can realize the dual-response of viscosity and SO2 derivatives in vivo.