A water-soluble, cell-permeable Mn( <scp>ii</scp> ) sensor enables visualization of manganese dynamics in live mammalian cells

Central roles of Mn²⁺ ions in immunity, brain function, and photosynthesis necessitate probes for tracking this essential metal ion in living systems. However, developing a cell-permeable, fluorescent sensor for selective imaging of Mn²⁺ ions in the aqueous cellular milieu has remained a challenge. This is because Mn²⁺ is a weak binder to ligand-scaffolds and Mn²⁺ ions quench fluorescent dyes leading to turn-off sensors that are not applicable for <i>in vivo</i> imaging. Sensors with a unique combination of Mn²⁺ selectivity, μM sensitivity, and response in aqueous media are necessary for not only visualizing labile cellular Mn²⁺ ions live, but also for measuring Mn²⁺ concentrations in living cells. No sensor has achieved this combination thus far. Here we report a novel, completely water-soluble, reversible, fluorescent turn-on, Mn²⁺ selective sensor, M4, with a <i>K</i> _d of 1.4 μM for Mn²⁺ ions. M4 entered cells within 15 min of direct incubation and was applied to image Mn²⁺ ions in living mammalian cells in both confocal fluorescence intensity and lifetime-based set-ups. The probe was able to visualize Mn²⁺ dynamics in live cells revealing differential Mn²⁺ localization and uptake dynamics under pathophysiological <i>versus</i> physiological conditions. In a key experiment, we generated an in-cell Mn²⁺ response curve for the sensor which allowed the measurement of the endogenous labile Mn²⁺ concentration in HeLa cells as 1.14 ± 0.15 μM. Thus, our computationally designed, selective, sensitive, and cell-permeable sensor with a 620 nM limit of detection for Mn²⁺ in water provides the first estimate of endogenous labile Mn²⁺ levels in mammalian cells.