A wash-free fluorescent probe with red-emission for monitoring mitochondrial viscosity changes during cellular metabolism and different death processes.

The abnormal viscosity of mitochondria is closely related to various cell dysfunction. Therefore, the development of effective tools that can visually monitor mitochondrial viscosity is critical for maintaining mitochondrial homeostasis. Here, based on twisted intramolecular charge transfer (TICT) mechanism, we designed and synthesized a series of viscosity sensitive fluorescent probes (CTP1-CTP7) using thiophene-containing carbazole as parent unit, single bond connecting ethylene bond as viscosity sensitive "rotator" site, and pyridine salt as strong electron-withdrawing group. The fluorescence response of these probes exhibited interesting "on" and "off" behaviors in high and low viscosity environments, respectively. Not only that, all these probes were highly sensitive and exclusively selective for viscosity in vitro and have excellent chemical and photostability. Biological experiments demonstrated that the positive charge in the probe molecule enables it to be efficiently localized in mitochondria in cells (except for CTP2). In addition, probe CTP1 displayed a high signal-to-noise ratio in fluorescence imaging and was fully capable of precise targeting of mitochondria in living cells by a wash-free procedure. More importantly, probe CTP1 has been successfully applied to visualize mitochondrial viscosity changes in a variety of drug-induced cell models and during cell death. This has practical applications for studies to elucidate the potential link between mitochondrial viscosity changes and related diseases in vivo.