Long Fluorescence Lifetime Molecular Rotors Based on the 4,4‐Dicyano‐BODIPY Core

Environmentally sensitive fluorescent probes with extended emission lifetimes are highly valuable for fluorescence lifetime imaging microscopy (FLIM) in live cells, as they enable superior temporal separation of the probe's signal from background autofluorescence. Here, we demonstrate that replacing fluorine atoms with cyano groups in BODIPY-based fluorescent molecular rotors yields viscosity-sensitive probes with significantly prolonged emission lifetimes as a result of reduced non-radiative decay rates. The minimal cyano-BODIPY rotor retained a robust Förster-Hoffmann relationship (viscosity sensitivity factor, x = 0.36) and exhibited fluorescence lifetimes up to 1.6 ns longer than those of the parent F-BODIPY rotor in cuvette-based measurements. Notably, the cyano-BODIPY rotor also displayed modestly reduced aggregation in aqueous environments. To enable biological applications, we synthesized a membrane-targeting CN-BODIPY rotor probe bearing a linear alkyl chain. This probe was successfully applied to estimate the apparent plasma membrane microviscosity in live Staphylococcus epidermidis cells. Our results establish CN-BODIPY molecular rotors as promising viscosity-responsive probes with increased fluorescence lifetimes for studying microenvironments in living cells.