Multimodal sensing of biological microenvironment using aggregation-induced emission fluorophores

Fluorescence microscopy is a central technique in molecular imaging, yet many traditional dyes suffer from aggregationcaused quenching, especially in high-concentration or crowded environments. Aggregation-induced emission (AIE) chromophores have been developed as an alternative, exhibiting increased fluorescence upon aggregation due to restriction of intramolecular motion. However, their use has remained largely limited to intensity-based imaging. Here, we present a new class of tetraphenylethylene (TPE)-based chromophores that combine AIE behavior with a strong intramolecular charge transfer character. By introducing an electron-withdrawing group near the TPE core, we tuned the photophysical properties to respond to environmental polarity. Spectroscopic analyses confirmed the shift in emission wavelength in response to polarity changes (solvatochromism) and extended the excited-state lifetimes in more polar or aggregated environments. Time-correlated single photon counting revealed that lifetimes vary by orders of magnitude across solvents. When embedded into giant unilamellar vesicles with distinct microdomains, fluorescence lifetime imaging microscopy showed that the dyes could distinguish polarity differences based on lifetime contrast. Additionally, biotoxicity tests on breast cancer cell lines revealed no impact on cell viability at biologically relevant concentrations. Together, these results highlight a dual-mode imaging platform in which a single chromophore can report on both spatial proximity (via AIE) and the local environment (via lifetime). This integrated approach enables the molecular design of imaging probes capable of tracking dynamic molecular interactions.