IndiFluors: A New Full-Visible Color-Tunable Donor–Acceptor–Donor (D1–A–D2) Fluorophore Family for Ratiometric pH Imaging during Mitophagy

Full-visible color-tunable new fluorophores are essential in bioimaging research. However, it is significantly challenging to design fluorophores with the desired optical and biological properties owing to their structural complexity. We report a unified design of an interesting molecular framework, IndiFluors, based on the principle of a donor-acceptor-donor (D1-A-D2) system. The IndiFluors comprise pyrylium, pyridinium, and pyridine derivatives, which exhibit full-visible emission color (375-700 nm) by varying donor and acceptor strengths of the core scaffolds. With a minimal change of structure, the bright fluorophores (Φ: 0.96) can be tuned to become nonfluorescent (Φ: 0.01), which is well explained by time-dependent density functional theory (TD-DFT/PCM) by oscillator strengths in the S1 state. Within IndiFluors, pyridinium offers several advantages, including a large Stokes shift (∼154 nm) and excellent stability, compared to pentacyclic pyrylium fluorophores. Especially, the designed probe, PM-Mito-OH, demonstrated specific colocalization in mitochondria and a monitored ratiometric pH change during mitochondrial damage, autolysosomes, and the mitophagy process. Hence, IndiFluors and the derived probe show great potential for cellular pH imaging in live cells while exhibiting minimal cytotoxicity.