A Mitochondria-Targeted Single Fluorescent Probe for Orthogonal Dual-Channel Imaging of Polarity and Viscosity during Ferroptosis

Ferroptosis is a regulated cell death process accompanied by drastic fluctuations in the mitochondrial microenvironment, specifically viscosity and polarity. Simultaneous monitoring of these parameters is crucial for unraveling the pathological mechanisms of ferroptosis, yet achieving this with high precision remains a challenge due to the spectral crosstalk often associated with multiprobe cocktails. Herein, we present a mitochondria-targeted single fluorescent probe, Mito-VPL, designed for the low-crosstalk dual-channel imaging of intracellular viscosity and polarity. Engineered with a donor–π–acceptor (D–π–A) architecture containing a twistable pyridinium rotor, Mito-VPL exhibits two channels with differentiated environmental responses: the green channel responds to polarity via the intramolecular charge transfer (ICT) mechanism, while the red channel responds to viscosity via the restriction of intramolecular rotation (RIR) mechanism. This feature enables differentiated dual-channel readout of viscosity- and polarity-related changes with reduced spectral interference. Leveraging this capability, Mito-VPL was successfully applied to visualize the synchronous elevation of mitochondrial viscosity and polarity during Erastin-induced ferroptosis. Furthermore, Mito-VPL was used to monitor the partial reversal of Erastin-induced fluorescence changes under GSH-treatment conditions, suggesting its potential for visualizing rescue-associated mitochondrial microenvironmental changes. Moreover, an in vivo application in 4T1 tumor-bearing mice demonstrated the feasibility of Mito-VPL for polarity- and viscosity-related fluorescence imaging in a cancer model. This study provides an orthogonal dual-channel molecular tool for monitoring ferroptosis-associated mitochondrial microenvironmental changes.