Quantitative Dual-Readout Mapping of Mitochondrial Viscosity Dynamics via Excited-State Conformational Restriction

Accurate mapping of mitochondrial viscosity is critical for understanding cellular metabolism, yet it remains challenging due to the scarcity of probes that combine stable mitochondrial retention with robust, self-calibrated quantification. To address this gap, we developed VNB, an excited-state conformation-sensitive fluorescent probe engineered on a vibration-induced emission (VIE) scaffold for dual-modal imaging of mitochondrial viscosity via simultaneous ratiometric fluorescence and fluorescence lifetime dual-readout. In viscous media, restricted vibrational relaxation impedes excited-state planarization of VNB, leading to a synchronous and linear increase of both the green-to-red emission ratio and the fluorescence lifetime. This dual-response mechanism enables mutual calibration between the two independent quantification modes, effectively eliminating artifacts arising from probe- or system-related errors. With its superior mitochondrial anchoring capability, VNB allows for high-fidelity mapping of viscosity dynamics during starvation-induced mitophagy and drug-induced mitochondrial dysfunction. Notably, real-time imaging reveals that viscosity surges coincide with mitochondrial network disintegration and subsequent cytoskeletal collapse. By integrating ratiometric and lifetime-based quantification within a single molecular framework, VNB establishes a reliable platform for dissecting the role of microviscosity in mitochondrial pathophysiology.