Rhodamine-derived ratiometric fluorescent molecular rotor for mitochondrial viscosity sensing

Fluorescent molecular rotors for sensing local microviscosity have become indispensable tools for better understanding the mechanisms of living systems. Current probes relay on intensity or lifetime measurements, while ratiometric molecular rotors are lacking. Here, we introduce a new design concept of molecular rotor using xanthene scaffold with freely rotating 9-aryl group. We studied three rhodamine derivatives, where 2-carboxyphenyl group was replaced with 4-methoxyphenyl, 2-thienyl and 2-benzofuryl. We found that five membered 9-aryl ring provides two major effects. First, free rotation of thienyl derivative, confirmed by theoretical calculations, makes it classical molecular rotor with intensity and lifetime-based response to viscosity. Second, benzofuryl derivative exhibits second emission band in near-infrared region and fluorescence ratiometric response to viscosity. Theoretical calculations suggest that the benzofuryl derivative can attain planar conformation in the excited state, which explains appearance of the NIR emitting band. Both new dyes efficiently target mitochondria, while the 9-benzofuryl derivative enable quantitative ratiometric measurement of viscosity of inner mitochondrial membrane. Ratiometric imaging revealed mitochondria heterogeneity with local viscosity values ranging from 250 to 460 cP. Oxidative stress induced significant rise in the local viscosity in mitochondria in addition to their morphological changes. Overall, we propose a concept that converts bright rhodamine dyes into molecules rotors with a valuable ratiometric response to viscosity, which opens a bunch of potential biological applications.