The synthesis of a difluorofluorescein monocarboxalclehyde platform and its use for preparing ZP8, a new member of the Zinpyr family of neuronal Zn2+ sensors, are described. By combining an aniline photoinduced electron transfer (PET) switch and an electron-withdrawing fluorescein scaffold, ZP8 displays reduced background fluorescence and improved dynamic range compared to previous ZP probes. The bright sensor undergoes an 11-fold increase in fluorescence intensity upon Zn2+ complexation (Phi = 0.03-0.35) with high selectivity over cellular concentrations of Ca2+ and Mg2+. In addition, sensors in the ZP family have been utilized for optical imaging in biological samples using two-photon microscopy (TPM), The cell-permeable ZP3 probe is capable of identifying natural pools of labile Zn2+ within the mossy fiber synapses of live hippocampal slices using TPM, establishing the application of this technique for monitoring endogenous Zn2+ stores.