Environment-insensitive two-photon ratiometric probe for in cellulo quantitative measurement of hydrogen peroxide

Abstract The quantitative analysis of a biological analyte directly through fluorescence imaging is essential in biomedical sciences but remains as a challenge owing to the environment-sensitive nature of fluorescence intensity. We show that a fluorescent hydrogen peroxide probe based on an in cellulo super-bright benzothiazolyl-benzocoumarin dye of which emission properties are insensitive to environmental changes offers a reliable and practical means for the quantitative analysis of hydrogen peroxide in cells and tissues directly through ratiometric imaging both under one-photon and two-photon excitation conditions. Different cellular hydrogen peroxide concentrations were determined for several cell lines and also for tumour and normal tissues.