Harnessingan MMP-Independent NIR Probe Unveilingthe Different Mitochondrial Cristae Changes during Mitophagy and Ferroptosisunder STED Microscopy

Mitochondrial cristae remain dynamic structures in order to adapt various physiopathologic processes (e.g., mitophagy and ferroptosis); thus, visualizing and tracking different changes of cristae are crucial for a deeper understanding of these processes. Fluorescent probes that can realize long-term visualization of mitochondrial cristae under stimulated emission depletion (STED) microscopy are powerful tools for their in-depth research. However, there are few reports on such probes, and their constructions remain challenging. Here, we reported a robust squaraine probe (<b>CSN</b>) for visualizing and tracking the changes of mitochondrial cristae in various physiological and pathological processes using STED microscopy. The lipophilic unit of <b>CSN</b> enabled it to firmly immobilize in mitochondria via a hydrophobic interaction, which let the labeling ability of <b>CSN</b> independent of mitochondrial membrane potential (MMP). Using <b>CSN</b>, the mitochondrial cristae were clearly observed at a resolution of 52 nm under STED microscopy. Furthermore, <b>CSN</b> was successfully applied to track the destruction processes of mitochondrial cristae during autophagy and ferroptosis. Interestingly, we found that during mitophagy, mitochondria first underwent swelling and cristae rupture, and then partial vacuolization, and finally complete vacuolization, whereas during ferroptosis, mitochondria first underwent a gradual reduction in the number of cristae, and then partial fracture, and finally vacuolization. This work revealed the difference in mitochondrial cristae changes during mitophagy and ferroptosis, which provided insights into the two physiological and pathological processes. We believed that <b>CSN</b> could serve as a desirable tool to track cristae changes of intracellular activity processes.