Measuring mitochondrial membrane potential

Mitochondrial function determines the balance between health and disease, making understanding their bioenergetics an increasingly important focus across biomedical research. Among the most relevant and accessible measures of mitochondrial function in intact cells is the mitochondrial membrane potential (ΔΨm), typically determined with commercially available fluorescent dyes. However, these probes are prone to misjudgement, as oversimplified interpretation of their fluorescent signal can cause important mistakes during both data acquisition and conclusions. This metabolism methods commentary describes how the interplay between mitochondrial membrane potential, mitochondrial ATP synthesis and oxygen consumption influences ΔΨm as a readout for mitochondrial respiratory function. We then examine how widely used mitochondrial dyes label mitochondria and what their fluorescence really reflects. Finally, we highlight four hallmark principles for the accurate determination of mitochondrial ΔΨm, which we hope will promote the rigorous assessment and interpretation of mitochondrial membrane potential across research fields.