Mitochondrial membranes
and their microenvironments directly influence
and reflect cellular metabolic states but are difficult to probe on
site in live cells. Here, we demonstrate a strategy, showing how the
widely used mitochondrial membrane localization fluorophore 10-nonyl
acridine orange (NAO) can be transformed into a multifunctional probe
of membrane microenvironments by monitoring its blinking kinetics.
By transient state (TRAST) studies of NAO in small unilamellar vesicles
(SUVs), together with computational simulations, we found that NAO
exhibits prominent reversible singlet–triplet state transitions
and can act as a light-induced Lewis acid forming a red-emissive doublet
radical. The resulting blinking kinetics are highly environment-sensitive,
specifically reflecting local membrane oxygen concentrations, redox
conditions, membrane charge, fluidity, and lipid compositions. Here,
not only cardiolipin concentration but also the cardiolipin acyl chain
composition was found to strongly influence the NAO blinking kinetics.
The blinking kinetics also reflect hydroxyl ion-dependent transitions
to and from the fluorophore doublet radical, closely coupled to the
proton-transfer events in the membranes, local pH, and two- and three-dimensional
buffering properties on and above the membranes. Following the SUV
studies, we show by TRAST imaging that the fluorescence blinking properties
of NAO can be imaged in live cells in a spatially resolved manner.
Generally, the demonstrated blinking imaging strategy can transform
existing fluorophore markers into multiparametric sensors reflecting
conditions of large biological relevance, which are difficult to retrieve
by other means. This opens additional possibilities for fundamental
membrane studies in lipid vesicles and live cells.
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