A comprehensive review of mitochondrial fluorescent probes: Design principles, fluorophore engineering, and...

Mitochondria are essential organelles that play pivotal roles in cellular energy metabolism, signaling, and homeostasis, and their dysfunction is closely associated with various human diseases. In recent years, the rapid development of mitochondria-targeted fluorescent probes has greatly expanded our ability to visualize mitochondrial structure and monitor diverse physicochemical and biochemical parameters in situ. While existing reviews have primarily categorized these probes by specific physiological readouts, a unified perspective that integrates probe design strategies, fluorophore engineering, and target physiological parameters is stilling lacking. To address this gap, we provide a systematic overview of mitochondrial fluorescent probes, focusing on molecular design principles, fluorophore selection, targeting strategies, and the sensing of key mitochondrial physiological indices. In addition, we critically discuss current limitations and emerging challenges, and outline future directions in this field. This review aims to offer theoretical insights and practical guidance for the rational design of mitochondrial fluorescent probes, promoting their broader applications in life science and biomedical research.