Fluorescence Lifetime Super-Resolution Imaging Unveil the Dynamic Relationship between Mitochondrial Membrane Potential and Cristae Structure Using the Förster Resonance Energy Transfer Strategy

Mitochondrial cristae, invaginations of the inner mitochondrial membrane (IMM) into the matrix, are the main site for the generation of ATP via oxidative phosphorylation, and mitochondrial membrane potential (MMP). Synchronous study of the dynamic relationship between cristae and MMP is very important for further understanding of mitochondrial function. Due to the lack of suitable IMM probes and imaging techniques, the dynamic relationship between MMP and cristae structure alterations remains poorly understood. We designed a pair of FRET-based molecular probes, with the donor (OR-LA) being rhodamine modified with mitochondrial coenzyme lipoic acid and the acceptor (SiR-BA) being silicon-rhodamine modified with a butyl chain, for simultaneous dynamic monitoring of mitochondrial cristae structure and MMP. The FRET process of the molecular pair in mitochondria is regulated by MMP, enabling more precise visualization of MMP through fluorescence intensity ratio and fluorescence lifetime. By combining FRET with FLIM super-resolution imaging technology, we achieved simultaneous dynamic monitoring of mitochondrial cristae structure and MMP, revealing that during the decline of MMP, there is a progression involving cristae dilation, fragmentation, mitochondrial vacuolization, and eventual rupture. Significantly, we successfully observed that the rapid decrease in MMP at the site of mitochondrial membrane rupture may be a critical factor in mitochondrial fragmentation. These data collectively reveal the dynamic relationship between cristae structural alterations and MMP decline, laying a foundation for further investigation into cellular energy regulation mechanisms and therapeutic strategies for mitochondria-related diseases.