Time Series Imaging the Mitochondrial Microenvironment and Its Interactions with Lysosomes during Ferroptosis

In the realm of cutting-edge scientific inquiry, the development and application of integrated optical molecular probes for the simultaneous detection and tracing of mitochondrial microenvironments during ferroptosis, as well as the visualization of their interactions with lysosomes, stands as a pivotal advancement. In this work, we developed a probe, IMT, that integrates viscosity sensing with mitochondrial targeting, and used it in conjunction with commercial lysosome green tracers (LGT) to investigate mitochondrial-lysosome interactions (MLIs). This approach avoids the uneven labeling caused by subcellular microenvironment differences when using single-molecule dual-targeting probes. Using the developed IMT, we observed an increase in mitochondrial viscosity during erastin-induced ferroptosis and a decrease during ferrostatin-1-inhibited ferroptosis. Moreover, the time series imaging of the mitochondrial profile lighted by the IMT showed that the mitochondrial area, perimeter, aspect ratio, and mitochondrial form factor changed significantly as ferroptosis progressed. In addition, combined with LGT, we visualized the dynamic process of first contact and then separation between lysosomes and mitochondria during ferroptosis, confirming the complexity and variability of MLIs. This work not only enhances our understanding of the complex biochemical processes underlying ferroptosis but also opens new avenues for therapeutic intervention in diseases characterized by this form of cell death.