Chemical Control of Fluorescence Lifetime of Molecular Rotors Provides Insights into Subcellular Viscosities during Ferroptosis

Cellular viscosity plays an essential role in physiology and pathology. Quantitative measurements of viscosity could be acquired from fluorescence lifetime microscopy. However, it remains a challenge to regulate how the excited-state lifetime of a given molecular rotor responds to local viscosity. Herein, we chose BODIPY as a model probe and present a chemical strategy to rationally control how its derivatives could report on viscosity via the fluorescence lifetime. Through experimental and computational analyses, we found that modulating π-electron density at the <i>meso</i>-position can regulate the viscosity sensitivity of BODIPY. Strong π-excessive or π-deficient rotors at the <i>meso</i>-position lower the rotational barrier, thus enhancing BODIPY's viscosity sensitivity. Based on this mechanism, we designed BODIPY derivatives in the green and red fluorescent channels. Using these probes, we quantitatively studied the viscosity changes for subcellular organelles during ferroptosis. We observed that mitochondrial viscosity decreases during induced ferroptosis. Furthermore, we found that lysosomes and mitochondria are influenced in the early stage of ferroptosis, whereas the endoplasmic reticulum is engaged in the later stage. In summary, this work exemplifies how electron density could regulate the viscosity sensitivity of molecular rotors. We envision that this strategy could be applied to develop probes whose fluorescence lifetime could quantify the surrounding viscosity.