Visualizing Mitochondrial Membrane Potential with FRET Probes: Integrating Fluorescence Intensity Ratio and Lifetime Imaging.

Mitochondrial membrane potential (MMP) is crucial for mitochondrial function and serves as a key indicator of cellular health and metabolic activity. Traditional lipophilic cationic fluorescence intensity probes are unavoidably influenced by probe concentration, laser intensity, and photobleaching, limiting their accuracy. To address these issues, we designed and synthesized a pair of fluorescence molecules, OR-C8 and SiR-BA, based on the Förster Resonance Energy Transfer (FRET) mechanism, for dual-modality visualization of MMP. OR-C8 anchors to the inner mitochondrial membrane through strong hydrophobic interactions, while SiR-BA is expelled from mitochondria when MMP decreases, thereby regulating the FRET process. During MMP reduction, the fluorescence intensity and lifetime of OR-C8 increase, while the fluorescence intensity of SiR-BA decreases. By combining changes in fluorescence intensity ratio and fluorescence lifetime, dual-modality visualization of MMP was achieved. This method not only accurately reflects MMP changes but also provides a novel tool for in-depth studies of mitochondrial function and related disease mechanisms, offering significant potential for advancing mitochondrial research and therapeutic development.