Rhodamine-derived ratiometric fluorescent molecular rotor for mitochondrial viscosity sensing

Fluorescent molecular rotors that can sense local microviscosity in living systems have become essential tools for understanding biological mechanisms. Existing probes typically rely on intensity or lifetime measurements, but ratiometric molecular rotors are still in short supply. Researchers have now introduced a novel design concept using xanthene scaffold with a freely rotating 9-aryl group.

Three rhodamine derivatives were studied, with the 2-carboxyphenyl group replaced by either 4-methoxyphenyl, 2-thienyl, or 2-benzofuryl. The researchers discovered that the five-membered 9-aryl ring provided two crucial effects. The thienyl derivative, confirmed by theoretical calculations, demonstrated classical molecular rotor behavior with intensity and lifetime-based responses to viscosity.

The benzofuryl derivative exhibited a second emission band in the near-infrared region and a fluorescence ratiometric response to viscosity.

Theoretical calculations suggest that the benzofuryl derivative can assume a planar conformation in its excited state, which accounts for the appearance of the near-infrared emitting band. Both new dyes efficiently target mitochondria, with the 9-benzofuryl derivative enabling quantitative ratiometric measurement of the inner mitochondrial membrane's viscosity. Ratiometric imaging revealed significant mitochondrial heterogeneity, with local viscosity values ranging from 250 to 460 centipoise.

Oxidative stress led to a substantial rise in local viscosity within mitochondria, alongside their morphological changes. Overall, the researchers propose a concept that converts bright rhodamine dyes into rotor molecules with a valuable ratiometric response to viscosity. This development opens up numerous potential biological applications.