Mitochondrial chelatable iron contributes to the severity of several injury processes, including ischemia/reperfusion, oxidative stress, and drug toxicity. However, methods to measure this species in living cells are lacking. To measure mitochondrial chelatable iron in living cells, here we synthesized a new fluorescent indicator, mitoferrofluor (MFF). We designed cationic MFF to accumulate electrophoretically in polarized mitochondria, where a reactive group then forms covalent adducts with mitochondrial proteins to retain MFF even after subsequent depolarization. We also show in cell-free medium that Fe²⁺ (and Cu²⁺), but not Fe³⁺, Ca²⁺, or other biologically relevant divalent cations, strongly quenched MFF fluorescence. Using confocal microscopy, we demonstrate in hepatocytes that red MFF fluorescence colocalized with the green fluorescence of the mitochondrial membrane potential (ΔΨ_m) indicator, rhodamine 123 (Rh123), indicating selective accumulation into the mitochondria. Unlike Rh123, mitochondria retained MFF after ΔΨ_m collapse. Furthermore, intracellular delivery of iron with membrane-permeant Fe³⁺/8-hydroxyquinoline (FeHQ) quenched MFF fluorescence by ∼80% in hepatocytes and other cell lines, which was substantially restored by the membrane-permeant transition metal chelator pyridoxal isonicotinoyl hydrazone. We also show FeHQ quenched the fluorescence of cytosolically coloaded calcein, another Fe²⁺ indicator, confirming that Fe³⁺ in FeHQ undergoes intracellular reduction to Fe²⁺. Finally, MFF fluorescence did not change after addition of the calcium mobilizer thapsigargin, which shows MFF is insensitive to physiologically relevant increases of mitochondrial Ca²⁺. In conclusion, the new sensor reagent MFF fluorescence is an indicator of mitochondrial chelatable Fe²⁺ in normal hepatocytes with polarized mitochondria as well as in cells undergoing loss of ΔΨ_m.

#	Concept	H-Index	Publications	Citations
1	Engineering	5	5	155
2	Medicine	2	2	69
3	Biochemistry	1	1	44
4	Biomedical Engineering	1	1	16
5	Biomolecular Engineering	1	1	16

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