How and when to measure mitochondrial inner membrane potentials

The scientific literature on mitochondria has increased significantly over the years due to findings that these organelles have widespread roles in the onset and progression of pathological conditions such as metabolic disorders, neurodegenerative and cardiovascular diseases, inflammation, and cancer. Researchers have extensively explored how mitochondrial properties and functions are modified in different models, often using fluorescent inner mitochondrial membrane potential (ΔΨm) probes to assess functional mitochondrial aspects such as protonmotive force and oxidative phosphorylation. This review provides an overview of existing techniques to measure ΔpH and ΔΨm, highlighting their advantages, limitations, and applications. It discusses drawbacks of ΔΨm probes, especially when used without calibration, and conditions where alternative methods should replace ΔΨm measurements for the benefit of the specific scientific objectives entailed. Studies investigating mitochondria and their vast biological roles would be significantly advanced by the understanding of the correct applications as well as limitations of protonmotive force measurements and use of fluorescent ΔΨm probes, adopting more precise, artifact-free, sensitive, and quantitative measurements of mitochondrial functionality.