Synergistic microscopic platform for probing molecular interaction on mitochondria-lysosome contact membrane

In biomedicine, studies on dynamic interactions between mitochondria and lysosomes are critical for understanding cellular metabolism and disease mechanisms. Yet, the interaction mechanism on the molecular level is still unclear due to a lack of suitable investigation tools. Here, we employ a synergistic microscopic platform that integrates quantitative phase contrast microscopy (QPCM), confocal fluorescent imaging, line-scanning fluorescence resonance energy transfer (ls-FRET), and line-scanning fluorescence correlation spectroscopy (ls-FCS) to reveal the mechanism of biomolecular interaction on mitochondria–lysosome contact membranes. Using ls-FRET, we quantified the FRET efficiency between (Rab7A) donors and (TOM20) acceptors situated in the mitochondrial-lysosomal contact membrane. It is found that FRET efficiency decreases under carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment. The joint use of QPCM and ls-FCS elucidates the underlying mechanism behind the reduction of FRET efficiency. The above multi-modality imaging platform enables simultaneous mapping of molecular interactions and dynamics on subcellular membranes in living cells, offering multidimensional and complementary optical readouts.