High-content microscopy quantification of mitochondrial membrane potential

In nearly all pathophysiological processes, mitochondrial membrane potential serves as a crucial indicator of mitochondrial function and activity. However, there remains a need for high-content imaging techniques that incorporate multiparametric measurements for comprehensive mitochondrial assessment. This paper introduces a novel unbiased approach for quantifying mitochondrial membrane potential in vitro, applicable to both two-dimensional and three-dimensional experimental systems. Furthermore, the incorporation of automated image analysis with machine learning algorithms enabled precise identification and segregation of distinct cell types within complex co-culture systems, allowing for targeted evaluation of individual subpopulations. Here, we provide a protocol for large-scale profiling of mitochondrial activity across various experimental contexts.