Ratiometric detection of pH fluctuation in mitochondria with a new fluorescein/cyanine hybrid sensor

The homeostasis of mitochondrial pH (pH(m)) is crucial in cell physiology. Developing small-molecular fluorescent sensors for the ratiometric detection of pH(m) fluctuation is highly demanded yet challenging. A ratiometric pH sensor, Mito-pH, was constructed by integrating a pH-sensitive FITC fluorophore with a pH-insensitive hemicyanine group. The hemicyanine group also acts as the mitochondria targeting group due to its lipophilic cationic nature. Besides its ability to target mitochondria, this sensor provides two ratiometric pH sensing modes, the dual excitation/dual emission mode (D(ex)/D(em)) and dual excitation (D(ex)) mode, and its linear and reversible ratiometric response range from pH 6.15 to 8.38 makes this sensor suitable for the practical tracking of pH(m) fluctuation in live cells. With this sensor, stimulated pH(m) fluctuation has been successfully tracked in a ratiometric manner via both fluorescence imaging and flow cytometry.