Development of an Iminocoumarin-Based Zinc Sensor Suitable for Ratiometric Fluorescence Imaging of Neuronal Zinc

摘要

Ratiometric imaging is a technique to reduce artifacts by minimizing the influence of extraneous factors on the fluorescence of a sensor and is particularly useful for cellular imaging studies. Here we characterized the iminocoumarin fluorophore as a new scaffold for sensors for ratiometric imaging. The iminocoumarin 4 showed a high quantum yield in aqueous media on excitation in the visible wavelength region, while its coumarin analogue showed little fluorescence. We therefore developed a novel fluorescence probe, ZnIC, for ratiometric imaging of Zn~(2+), using iminocoumarin as a fluorophore and (ethylamino)-dipicolylamine as a Zn~(2+) chelator. ZnIC exhibited almost the same fluorescence properties as 4, and the emission spectrum of this probe was red-shifted on addition of Zn~(2+) under physiological conditions. ZnIC is selective for Zn~(2+) over other biologically important metal ions, such as Ca~(2+) and Mg~(2+), and has high affinity for Zn~(2+). To confirm the suitability of ZnIC for biological applications, we employed it for the ratiometric detection of changes in intracellular Zn~(2+) in cultured cells and in rat hippocampal slices. The results indicate that iminocoumarin is a useful fluorophore for fluorescence microscopic imaging and that ZnIC should be useful for studies on the biological functions of Zn~(2+).