The fluorescent probes MitoAR and MitoHR (Koide, Urano, Kenmoku, Kojima, Nagano, JACS 2007) enabled the first selective imaging of highly reactive oxygen species (hROS: hydroxyl radical, peroxynitrite, hypochlorous acid) in mitochondria of living cells, but left a precise open agenda that remains substantially unresolved in 2026. First, the probes, like nearly all mitochondria-targeted small-molecule ROS probes, accumulate in mitochondria by Nernstian uptake driven by the negative mitochondrial membrane potential, and their fluorescent oxidation product leaks out when that potential collapses under severe oxidative stress — compromising spatial fidelity exactly in the pathological states of greatest interest. Second, the observed signals confound intra- and extra-mitochondrial hROS sources: myeloperoxidase-positive HL-60 cells generate hypochlorous acid outside mitochondria yet yield a strong mitochondrial probe response, while HeLa cells show none. Third, the precise biological roles and enzymatic sources of mitochondrial hROS across cell types remain unestablished. Solving the problem requires hROS detection whose localization and reaction-product retention survive depolarization, validated source attribution, and deployment of such tools to map the origins and downstream consequences of mitochondrial hROS.

Background

Reactive oxygen species (ROS) — such as superoxide (O2∙−O_2^{\bullet-}), hydrogen peroxide (H2O2H_2O_2), and the hydroxyl radical (∙OH{}^{\bullet}OH) — are oxygen-derived oxidants generated in cells, and mitochondria are a major production site because electrons leak from the respiratory chain to oxygen. The term hROS (highly reactive oxygen species) denotes the most strongly oxidizing subset — the hydroxyl radical ∙OH{}^{\bullet}OH, peroxynitrite ONOO−ONOO^- (formed from superoxide and nitric oxide), and hypochlorous acid HOCl — which directly oxidize DNA, proteins, and lipids and cannot be detected by the H2O2H_2O_2- or superoxide-selective probes. Because the mitochondrial interior is negatively charged relative to the cytosol (a membrane potential, Δψm\Delta\psi_m, of roughly −150 to −180 mV), lipophilic cations such as the triphenylphosphonium (TPP) group or cationic rhodamine scaffolds accumulate inside mitochondria by Nernstian electrostatic uptake; this is the dominant mitochondrial-targeting strategy for small-molecule probes, including genetically unencoded fluorescent ROS probes. In 2007 Koide, Urano, Kenmoku, Kojima and Nagano built the first mitochondria-targeted hROS probes, MitoAR and MitoHR: a tetramethylrhodamine fluorophore (which is itself the cationic targeting group) bears a 4-aminophenyl or 4-hydroxyphenyl aryl-ether appendage that quenches fluorescence by photoinduced electron transfer (PET — intramolecular electron donation that suppresses the excited emitting state); hROS irreversibly cleave the ether, releasing the fluorescent rhodamine product HMTMR. The probes are highly selective for ∙OH{}^{\bullet}OH, ONOO−ONOO^- and HOCl over O2∙−O_2^{\bullet-}, NO and H2O2H_2O_2. Three limitations defined the paper's open agenda. (1) Spatial fidelity: after adding NaOCl to probe-loaded cells, the fluorescent product partly diffused out of mitochondria, which the authors attributed to loss of Δψm\Delta\psi_m under oxidant stress permitting leakage — so the probe's localization is maintained by the very property that oxidative pathology destroys. (2) Source attribution: myeloperoxidase (MPO, the HOCl-generating enzyme of neutrophil-lineage cells, located in azurophilic granules) is absent from HeLa cells, which showed no response to H2O2H_2O_2, while HL-60 leukemia cells (MPO-positive) responded strongly — yet that HOCl is generated outside mitochondria, so a cationic mitochondrial probe can report extramitochondrially generated hROS, and the enzymatic origins and subcellular locations of hROS signals differ across cell types in an unmapped way. (3) Biology: the authors state that the precise roles of mitochondrial hROS in physiology and pathology are still elusive, in part because no method existed for direct, mitochondrion-selective hROS detection. Later reviews (2015–2026) reiterate all three points as standing limitations of lipophilic-cation mitochondrial ROS probes: probe uptake and retention depend on Δψm\Delta\psi_m, generating artifacts precisely under metabolic disruption and depolarization, and irreversible reaction-based dosimeters capture only cumulative oxidant exposure.

Problem Statement

Establish reliable, spatially faithful detection of highly reactive oxygen species (hROS — hydroxyl radical, peroxynitrite, hypochlorous acid) in mitochondria of living cells, and use it to resolve the origins and functional roles of mitochondrial hROS. The problem, as posed by the MitoAR/MitoHR work and still open in 2026, comprises three jointly linked components serving this single objective: (1) an hROS detection method whose mitochondrial localization does not depend on the mitochondrial membrane potential — both probe accumulation and retention of the fluorescent reaction product must survive the depolarization that occurs under severe oxidative stress (the NaOCl — sodium hypochlorite, delivering HOCl — induced leakage scenario of the source paper), so that the measured signal remains attributable to mitochondria in pathological states; (2) validated discrimination between intra- and extramitochondrial hROS generation, resolving the attribution problem illustrated by myeloperoxidase (MPO)-positive HL-60 cells (extramitochondrial HOCl) versus MPO-negative HeLa cells, and establishing the enzymatic sources (e.g., myeloperoxidase, nitric oxide synthase-derived peroxynitrite, Fenton-chemistry hydroxyl radical from respiratory-chain-derived precursors) and subcellular locations of hROS in different cell types; and (3) application of such validated tools to determine the precise functional consequences of mitochondrial hROS in physiological and pathological contexts. Component (1) is intended to support (2) and (3): potential-independent, product-retentive detection is one route by which mitochondrial hROS signals can be separated from extramitochondrial oxidant generation, which otherwise confounds biological conclusions drawn from cationic hROS probes under oxidative-stress conditions. The problem is resolved only when all three components are established: (1) an hROS detection method that keeps both its mitochondrial accumulation and its retention of the fluorescent reaction product when the membrane potential collapses; (2) discrimination of intra- from extramitochondrial hROS generation over a defined set of cell types that includes the MPO-positive versus MPO-negative contrast, together with identification of the enzymatic sources and subcellular locations responsible; and (3) a functional consequence of mitochondrial hROS in at least one defined physiological context and one defined pathological context. Resolving (1) alone therefore does not resolve the problem.

References

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