ANNINE-6plus, a voltage-sensitive dye with good solubility, strong membrane binding and high sensitivity

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 double 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.