Real-time monitoring viscosity during ferroptosis with a novel mitochondria-specific fluorescent probe

Ferroptosis is a form of regulated cell death that depends on iron and is associated with various diseases. Recent studies have shown that mitochondria play a critical role in ferroptosis. Monitoring viscosity changes in the microenvironment of mitochondria is essential for a deeper understanding of ferroptosis, as mitochondrial activity is closely related to viscosity. To address this, we have developed a novel fluorescence probe (HFB) that is both viscosity-sensitive and mitochondria-specific. HFB has a large Stokes shift, minimal background interference, high selectivity, and significant response capability, making it an ideal tool for real-time investigation of mitochondrial events in ferroptosis. Moreover, the physiochemical properties of HFB make it suitable for use with standard fluorescent inverted microscopes. Importantly, we have successfully used HFB to visualize the increase in mitochondrial viscosity during erastin-induced and RSL3-induced ferroptosis. This study presents a robust tool for investigating the intricacies of ferroptosis, which has great potential for use in diverse biological contexts. •HFB possessed remarkable large Stokes shift, small background interference, and excellent mitochondria-targetable ability.•The specific wavelength coverage of HFB enabled it to fit with the red channel of most fluorescent inverted microscopes.•HFB was performed to reveal the alteration details of the mitochondrial viscosity during ferroptosis.•HFB was successfully applied in real-time monitoring the changes of endogenous viscosity induced by ferroptosis inducers.