Development of an iminocoumarin-based zinc sensor suitable for ratiometric fluorescence imaging of neuronal zinc.
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 Zn2+, using iminocoumarin as a fluorophore and (ethylamino)dipicolylamine as a Zn2+ chelator. ZnIC exhibited almost the same fluorescence properties as 4, and the emission spectrum of this probe was red-shifted on addition of Zn2+ under physiological conditions. ZnIC is selective for Zn2+ over other biologically important metal ions, such as Ca2+ and Mg2+, and has high affinity for Zn2+. To confirm the suitability of ZnIC for biological applications, we employed it for the ratiometric detection of changes in intracellular Zn2+ 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 Zn2+.
Komatsu
Kensuke
K
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
Urano
Yasuteru
Y
Kojima
Hirotatsu
H
Nagano
Tetsuo
T
D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D002460:Cell Line; D003374:Coumarins / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence / Q000379:methods; D015394:Molecular Structure; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity; D015032:Zinc / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism
D003374:Coumarins; D005456:Fluorescent Dyes; D015032:Zinc