Influence of electron-accepting groups on the development of acridan-based two-photon fluorophores for cellular bioimaging

A series of nine acridan-based fluorophores featuring various electron-accepting moieties was designed and synthesized to investigate structure–property relationships governing their one- and two-photon photophysical properties. The synthetic strategy relied on a modular late-stage introduction of charged or neutral acceptor groups, affording a diverse set of push–pull architectures. Comprehensive spectroscopic studies were carried out in organic and aqueous media, including a viscous environments mimicking the cellular one (PBS + BSA 100 eq.). The fluorophores exhibited tunable absorption and emission covering the green-to-NIR spectral range (λabs = 421–549 nm, λem = 505–777 nm), with significant medium-dependent fluorescence behavior attributed to solubility and aggregation effects. Two-photon excited fluorescence (TPEF) measurements in DMSO revealed high two-photon absorption cross-sections between 440 GM and 750 GM, in agreement with TD-DFT calculations. Confocal microscopy experiments on A549 cells demonstrated efficient mitochondrial localization for the benzimidazolium derivative. Despite moderate cytotoxicity (IC50 = 5.4 μM), these results highlight the potential of acridan-based fluorophores as compact, tunable platforms for two-photon bioimaging and photonic material applications.