A Synthetic Fluorescent Mitochondria-Targeted Sensor for Ratiometric Imaging of Calcium in Live Cells

Ca²⁺ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca²⁺ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria-targeted synthetic Ca²⁺ indicators are plagued by several drawbacks, for example, non-specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca²⁺ sensor, named mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca²⁺ indicator fura-2. Mt-fura-2 binds Ca²⁺ with a dissociation constant of ≈1.5 μm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca²⁺ ] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome.