Reversible Dual Fluorescence-Lifetime Imaging of Mitochondrial GSH and Microviscosity: Real-Time Evaluation of Ferroptosis Status

Ferroptosis, as a new form of regulated cell death, is implicated in various physiological and pathological processes. Developing a single probe for an independent analysis of multiple analytes related to ferroptosis can provide more accurate information and simplify the detection procedures, but it faces great challenges. In this work, we develop a fluorescent probe for the simultaneous detection of GSH through ratiometric fluorescence response and microviscosity via a fluorescence lifetime model. Based on the reversible Michael addition reaction between GSH and unsaturated C & boxH;C bond, the probe responds reversibly to GSH with a ratiometric fluorescence variation and a fast response time (t(1/2) = 4.7 s). At the same time, the probe is sensitive to environmental viscosity by changing its fluorescence lifetimes. The probe was applied to monitor the drug-induced ferroptosis process through both the classical X-c(-)/GSH/GPX4- and DHODH-mediated defense mechanisms. We hope that the probe will provide a useful molecular tool for the real-time live-cell imaging of GSH dynamics, which is benefit to unveiling related physiological and pathological processes.