Peter Fromherz, Gerd Hübener, Bernd Kuhn, Marlon J. Hinner
We present a novel voltage-sensitive hemicyanine dye ANNINE-6plus and describe its synthesis, its properties and its voltage-sensitivity in neurons. The dye ANNINE-6plus is a salt with a doubly positively charged chromophore and two bromide counterions. It is derived from the zwitterionic dye ANNINE-6. While ANNINE-6 is insoluble in water, ANNINE-6plus exhibits a high solubility of around 1 mM. Nonetheless, it displays a strong binding to lipid membranes. In contrast to ANNINE-6, the novel dye can be used to stain cells from aqueous solution without surfactants or organic solvents. In neuronal membranes, ANNINE-6plus exhibits the same molecular Stark effect as ANNINE-6. As a consequence, a high voltage-sensitivity is achieved with illumination and detection in the red end of the excitation and emission spectra, respectively. ANNINE-6plus will be particularly useful for sensitive optical recording of neuronal excitation when organic solvents and surfactants must be avoided as with intracellular or extracellular staining of brain tissue.
Figure 1. Hemicyanine dyes with anellated benzene rings: dye salt ANNINE-6plus with double positively charged chromophore and two bromide counter ions and zwitterionic dye ANNINE-6.
Figure 2. Solubility of the dye salt ANNINE-6plus and of the zwitterionic dye ANNINE-6 as the logarithm of the saturation concentration in water/dioxane mixtures. At 30% (v/v) dioxane, the solubility of the two dyes differs by five orders of magnitude.
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