Simultaneous two-photon intravital imaging of viscosity and superoxide radical anion by a styrylpyridinium-based fluorescent probe

Simultaneous sensing of cellular microenvironment parameters and redox signaling molecules by a single fluorescent probe allows an efficient access to numerous applications ranging from convenient diagnostic assays to investigation on their pathophysiological roles and cross-link. Focusing on a lack of efficient molecule tools for simultaneous intravital imaging of mitochondrial viscosity and superoxide radical anion (O2 center dot-), we bridged diethylaminobenzene with diphenylphosphinate-modified pyridinium moiety by an olefinic bond to construct a styrylpyridinium-based two-photon fluorescent probe V-OS. V-OS triggered a turn-on response either at 625 nm to viscosity which blocks intramolecular rotation, or at 530 nm to O2 center dot- via nucleophilic attack of O2 center dot- followed by 1,6-elimination. With the aid of the two-photon probe, we not only identified the cross-talk between viscosity and O2 center dot- but successfully mapped the burst of O2 center dot- and the increased viscosity during either cellular ferroptosis process or cerebral ischemia reperfusion injury of living mice.