A General Strategy to Control Lifetime-Resolved Polarity Quantification of Push−Pull Fluorophores

Abstract Cellular polarity is important for physiological and biological conditions. Fluorescence lifetime imaging microscopy (FLIM) provides a quantitative approach for resolving microenvironmental polarity in living cells, and a variety of fluorophores have been developed for lifetime-based sensing. However, achieving tunable and quantitative polarity responses in fluorescence lifetime remains challenging, particularly when the range of polarity changes is wide. Herein, we establish a molecular design strategy that enables push−pull fluorophores to exhibit a linear response of their lifetime to environmental polarity in a wide range of polarity variations (dielectric constants from 5.30 to 32.70 or to 70.85). Theoretical calculations demonstrated that the tetrahydroquinoxaline−piperidine (THQP) unit exhibited an appropriate twisted intramolecular charge transfer (TICT) tendency, enabling effective modulation of the excited-state dynamics associated with environmental polarity. This strategy provides broad applicability across multiple push−pull fluorophores including coumarins, difluoride-boron complexes, rhodols, and boranil-derived fluorophores. Notably, THQP−coumarin probes enable quantitative mapping of polarity variations in proteins and subcellular compartments both in vitro and in living cells via FLIM. Owing to their high brightness and photostability, these probes further uncover nucleolar polarity dynamic change during the cell cycle as well as the correlation between mitochondrial polarity and morphology under stress conditions through time-lapse FLIM. In summary, this work establishes a general molecular design principle for tuning lifetime-resolved polarity quantification and expands the applicability of FLIM for quantitative polarity imaging in complex biological systems.