Environmentally sensitive fluorescent probes with long emission lifetimes are essential for fluorescence lifetime imaging microscopy (FLIM) in living cells. Here, we report that substituting fluorine atoms in the BODIPY-based molecular rotors with cyano groups produced viscosity-sensitive probes with significantly increased fluorescence lifetimes.

The synthesized 4,4′-dicyano-BODIPY derivatives exhibited physicochemical and spectroscopic properties comparable to 4,4′-difluoro-BODIPY analogues. Importantly, time-resolved measurements revealed prolonged fluorescence lifetimes across a wide viscosity range, with increases of up to 1.6 ns, reaching 5.18 ns in some cases.

A membrane-targeting cyano-BODIPY probe was successfully applied to live Staphylococcus epidermidis cells, where it localized to the plasma membrane and enabled quantitative FLIM-based viscosity measurements.

Our result demonstrate that CN-BODIPY molecular rotors are promising viscosity-sensitive fluorophores for biological applications.