Rational design of a red-emitting AIE-ESIPT probe for real-time visualization of lipid droplet dynamics in metabolic stress and ferroptosis

Lipid droplets (LDs), central to lipid homeostasis and implicated in metabolic disorders , cancer, and neurodegenerative diseases , urgently require advanced imaging probes to overcome limitations of conventional tools—such as small Stokes shifts , low photostability, and complex washing steps. To address these challenges, we engineered three ESIPT-active fluorescent probes ( HBT-CF 3 , Al-HBT-CF 3 , In-HBT-CF 3 ) through systematic molecular design. By progressively integrating stronger electron-withdrawing groups into the HBT scaffold, we achieved In-HBT-CF 3 , a red-emitting probe (λ em : 620 nm) with a 8038 cm −1 Stokes shift, 41 % quantum yield , and negligible phototoxicity . Its performance stems from synergistic action between restricted intramolecular rotation and hydrophobic shielding, which reinforce intramolecular hydrogen bonds and suppress non-radiative decay. Demonstrating rapid (<2 min), wash-free LD labeling, In-HBT-CF 3 enabled real-time tracking of LD dynamics under metabolic stress (starvation/oleic acid) and during ferroptosis , revealing erastin-induced LD accumulation and Fer-1-mediated suppression. This work not only resolves critical drawbacks of existing probes but also establishes a design framework for next-generation imaging tools, advancing mechanistic studies of LD-related pathologies and oxidative stress-driven diseases. • Three ESIPT-active dyes with progressive red-shifted emission engineered via stepwise electron-withdrawing substitution. • ESIPT-AIE synergy validated by DFT calculations and single-crystal structural analysis. • In-HBT-CF 3 achieves 41 % Φ f in aqueous media, 8038 cm −1 Stokes shift, and robust photostability. • Rapid lipid droplet labeling enabled by AIE-driven emission in live cells. • Real-time visualization of lipid droplet metabolism and ferroptosis .