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Two-photon excitation of fluorescence (2PEF) is a versatile tool for high-resolution functional microscopy, but few existing fluorophores combine high two-photon brightness with ability to report on key in vivo- and in vitro environmental factors such as pH, ionic strength, or solvent polarity. We describe four novel pH-sensitive derivatives of coumarin 151 with different attached phosphazene moieties, offering high lipophilicity both in their neutral- and protonated forms at near physiological conditions (neutral to mildly basic). We measure the two-photon absorption (2PA) and 2PEF emission spectra, as well as the fluorescence quantum yields in neutral acetonitrile solution, as well as with the addition of small amount of trifluoromethanesulfonic (triflic) or hydrochloric acid. We show that the neutral form has high two-photon brightness in the basic environments, whereas protonation quenches the fluorescence by a factor of ~ 20, while blueshifting the absorption- and emission peaks by 60 nm. By evaluating the ratio of the measured 2PA and 1PA spectra corresponding to the transition to the lowest excited singlet state S1 we show, for the first time, that the permanent molecular dipole moment change is reduced upon the protonation. This conclusion is supported by our quantum-chemical calculations which indicate that protonation leads to reduction of extent of conjugation in the system. We also measure the time-dependent fluorescence and show that in neutral environment the fluorescence displays a single-exponential decay, while upon protonation the decay is distinctly non-single-exponential in character.
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