Dual Ratio and Ultraprecision Quantification of Mitochondrial Viscosity in Ferroptosis Enabled by a Vibration-Based Triple-Emission Fluorescent Probe

Ferroptosis is a new mode of cell death with major morphological changes in mitochondria, including structural shrinkage and increased membrane density, indicating the mitochondrial abnormality during this process. Viscosity, as one of the crucial microenvironmental parameters for characterizing the mitochondrial state, is thought to be highly involved in the ferroptosis. Herein, we present a single fluorescent probe (PPAC-C4) for the dual ratio and ultrahigh-accuracy quantification of mitochondrial viscosity. This probe is constructed by linking a mitochondria-targeting cation fragment on a vibration-based fluorescent scaffold whose fluorescence exhibits the rare triple emission (480, 533, and 628 nm) depending on the viscosity. The intensity ratios of 480 nm/628 nm and 533 nm/628 nm can be used to monitor the viscosity changes in a double self-calibration manner and finally afford an average viscosity value with improved precision. By virtue of this pattern, we reveal that the mitochondrial viscosity will increase from 43.58 to 152.05 cP in A549 cells during the ferroptosis. This dual-ratio probe with triemission not only shows great potential in the study of ferroptosis and ferroptosis-related diseases but also proposes a new concept for ultraprecision quantitative analysis.