Requirements for Effective Fluorescence Imaging of Neuronal Zinc

The criteria required for sensing Zn2+ in biological environments create significant design challenges. First and foremost is a selective fluorescence response to Zn2+ over competing biological analytes, especially abundant cellular metal ions like Ca2+ and Mg2+. Sensors must bind rapidly and reversibly to Zn2+ and have dissociation constants (Kd) close to the median concentration of the ana-lyte. Detection systems that allow modular tuning of Kd values are particularly useful because labile pools of biological Zn2+ can vary over a wide range of concentrations. Biological compatibility is also essential. Zn2+ probes should be air-stable, water-soluble, and relatively insensitive to changes in ionic strength and pH. Both hydrophobic and hydrophilic sensors are useful for intracellular and extracellular applications, respectively. In addition, lipophilic, cell-permeable dyes are useful for probing vesicular and organelle stores of Zn2+. Ideal optical properties include visible excitation and emission profiles to minimize cell and tissue damage and avoid autofluorescence from native cellular species, and high extinction coefficients (e) and quantum yields ($) to afford large brightness (e x $) values. For spatial and temporal resolution, probes that report Zn2+ by an increase in emission intensity or a shift in excitation and/or emission profiles are superior to those that quench their emission upon Zn2+ recognition. In particular, sensors that undergo a wavelength shift in excitation or emission maxima can, in principle, provide accurate and quantitative determinations of [Zn2+] by using ratiometric fluorescence imaging [51]. We now present a selection of tools for the fluorescence detection of Zn2+ in biological environments and their application for interrogating zinc metalloneurochemistry. Pertinent optical and biological properties of the zinc probes are displayed in Table 1.

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