This study introduces a paradigm-shifting approach to optimize mitochondrial targeting. Employing a new fluorescent probe strategy, we unravel a combined influence of both Nernst potential (Ψ) and partitioning (P) contributions. Through the synthesis of new benz[e]indolinium-derived probes, our findings redefine the landscape of mitochondrial localization by optimizing the efficacy of mitochondrial probe retention in primary cortical neurons undergoing normoxia and oxygen–glucose deprivation. This methodology not only advances our understanding of subcellular dynamics, but also holds promise for transformative applications in biomedical research and therapeutic development.
