Illuminating mitochondrial metabolism with genetically encoded fluorescent indicators

Mitochondria are recognized as dynamic metabolic hubs that integrate bioenergetic, biosynthetic and signaling functions well beyond ATP production. Their matrix hosts the Krebs cycle, fatty acid β-oxidation, and key branches of amino-acid metabolism, while also supporting heme synthesis, the urea cycle, calcium handling and redox homeostasis. Dissecting these intertwined pathways demands tools that can report metabolite dynamics in situ, where native ion gradients and macromolecular crowding are preserved. Genetically encoded fluorescent indicators are protein-based indicators whose spectroscopic properties shift upon binding the target metabolite. Because they are genetically encoded, these fluorescent indicators can be expressed in specific cell types and targeted to subcellular compartments, allowing real-time, non-invasive tracking of metabolic dynamics in living systems. This systematic review surveys the state-of-the-art in mitochondrial GEFIs, cataloguing fluorescent indicators for monocarboxylates, Krebs-cycle intermediates, amino acids, redox cofactors and energy nucleotides. Additionally, we outline core design principles, summarize strategies that ensure efficient targeting into the mitochondrial matrix, and discuss challenges for their correct application. By charting current capabilities and knowledge gaps, this review aims to guide the next generation of mitochondrial GEFIs and to accelerate quantitative mapping of metabolic networks at subcellular resolution.