Ratiometric imaging of mitochondrial pH in living cells with a colorimetric fluorescent probe based on fluorescein derivative

•A novel strategy used to design ratiometric fluorescent probe (FDI) based on fluorescein fluorophore.•FDI provided ratiometric detection by CLSM in dual excitation/dual emission mode.•FDI exhibited excellent and fast cell-membrane permeability and mitochondrial-targetability.•It would be a potential tool to assess pH fluctuation in mitochondria of live cells.

Mitochondrial pH plays a pivotal role in the regulation of physiological process. Developing ratiometric fluorescent probes for real-time detection of mitochondrial pH fluctuation is still highly demanded yet challenging. Herein, we present a novel strategy to design a ratiometric probe (FDI) by broadening absorption spectrum (360-700 nm) of fluorescein fluorophore. Unsaturated dialkene/indole quaternary ammonium moiety has broadened absorption and emission spectrum of fluorescein fluorophore to provide ratiometric detection by CLSM in dual excitation/dual emission mode, as well as directionally accumulated in mitochondria in living cells. Superior to commercially mitochondrial tracker, Carboxy-SNARF, probe FDI exhibits excellent and fast cell-membrane permeability and mitochondrial-targetability. It has been demonstrated that FDI could permit real-time monitoring of pH alkalization of mitochondria stimulated by chloroquine. Owing to nondestructive process and reversible ratiometric response to pH, image acquisition can be repeated frequently to trace and monitor the time course of mitochondrial pH responses. It is clearly confirmed that FDI would be a promising probe for real-time tracking of mitochondrial pH changes in the biomedical and biological fields.

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