Ratiometric Covalent Fluorescent Probes for Dynamic Super-Resolution Imaging of Mitochondrial HClO

Dynamic monitoring of hypochlorous acid (HClO) in mitochondria is crucial for elucidating the molecular pathogenesis of ferroptosis-related diseases. Super-resolution microscopy, which surpasses the optical diffraction limit, has emerged as a powerful tool for subcellular dynamic imaging. However, the lack of mitochondrial HClO fluorescent probes with high specificity, stable labeling, low environmental interference, and negligible spectral crosstalk presents a significant challenge for achieving dynamic super-resolution imaging. Here, we designed and screened a series of HClO fluorescent probes, ultimately obtaining a novel HClO probe, YM-P. The combination of a triphenylphosphine group and a chloroacetyl chloride group enables YM-P to achieve specific, covalent mitochondrial labeling, thereby overcoming off-target labeling during ferroptosis. The ratiometric fluorescence response of YM-P to HClO and its detection limit of as low as 35 nM allow it to resist interferences from environmental factors and ensure accurate detection of HClO. The ultralarge Stokes shift of 210 nm exhibited by YM-P also minimizes spectral crosstalk. Using YM-P, we achieved dynamic super-resolution imaging of mitochondrial HClO during ferroptosis. Notably, we observed for the first time that changes in mitochondrial cristae numbers precede alterations in HClO concentration, with an initial increase followed by a decrease, suggesting that mitochondrial cristae are more sensitive to the occurrence of ferroptosis than HClO concentration. This study provides a robust tool for dynamic monitoring of mitochondrial HClO during ferroptosis, as well as potential support for other mitochondrial HClO-related processes.