Self-ReferencedProbe Enables Quantified MonitoringMitochondrial Membrane Potential by Colocalization Coefficients

Mitochondria are dynamic organelles that have a variety of physiological states. Mitochondrial membrane potential (MMP) maintains mitochondrial physiological activities and reflects cellular health and viability. Thus, it is essential to dynamically monitor changes in MMP for understanding mitochondria-related biological processes. However, <i>in situ</i>, synchronous and quantitative tracking of MMP dynamic processes remains a great challenge. In this work, we designed and synthesized a fluorescent probe <b>CBTH</b> that undergoes spirocyclization in response to pH and emits a two-color fluorescence. Relying on the electrostatic interaction with MMP, <b>CBTH</b> is able to dual-color target mitochondria in cells with normal MMP. During the reduction of MMP, the fluorescence signal of the red channel (λ_ex = 543 nm) gradually migrated to the nucleus, while one from the green channel (λ_ex = 405 nm) stayed in the mitochondria. Thus, taking the green channel as an internal reference, the probe can detect changes in MMP through the colocalization coefficient fluctuations between the two fluorescent channels. In cellular experiments, CLC exhibited reversibly detecting ability and a direct proportional relationship to MMP. Overall, this work provides a novel strategy to quantitatively detect MMP. Moreover, a detailed explanation of the design principle and response mechanism of <b>CBTH</b> has been provided for the creation of this kind of probe.