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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.

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Ca 2+ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca 2+ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures.Onthe other hand, existing mitochondriatargeted synthetic Ca 2+ indicators are plagued by several drawbacks,f or example,n on-specific localization, leakage, toxicity.H ere we report the synthesis and characterization of an ew fluorescent Ca 2+ sensor,n amed mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca 2+ indicator fura-2. Mt-fura-2 binds Ca 2+ with ad issociation constant of % 1.5 mm in vitro. When loaded in different cell types as acetoxymethyl ester,the probe shows proper mitochondrial localization and accurately measures matrix [Ca 2+ ]variations,provingits superiority over available dyes.W edescribe the synthesis,characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome.Calcium (Ca 2+ )s ignalling is the trigger of essential physiological processes. [1,2] Mitochondria, by taking up and releasing Ca 2+ to the cytosol, contribute to the shaping of cytosolic Ca 2+ signals.M atrix Ca 2+ ,t ransported by the mitochondrial Ca 2+ uniporter (MCU), is essential for the regulation of specific mitochondrial functions. [3][4][5][6] Several sensors have been generated to measure Ca 2+ inside mitochondria. [7][8][9] They are mostly genetically-encoded Ca 2+ indicators (GECIs) that can

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