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 AN-NINE-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.

We consider the physicochemical basis for enzyme-induced staining of cell membranes by fluorescent voltage-sensitive dyes, a method that may lead to selective labeling of genetically encoded nerve cells in brain for studies of neuronal signal processing. The approach relies on the induction of membrane binding by enzymatic conversion of a water-soluble precursor dye. We synthesized an amphiphilic hemicyanine dye with and without an additional phosphate appendix at its polar headgroup. The fluorescence of these dyes is negligible in water but high when bound to lipid membranes. By fluorescence titration with lipid vesicles it was shown that the phosphate group lowers the partition coefficient from water to membrane by more than an order of magnitude. By isothermal titration calorimetry, we showed that the dye phosphate was a substrate for a water-soluble alkaline phosphatase following MichaelisMenten kinetics. In a suspension of lipid vesicles, the enzyme reaction led to a fluorescence increase due to enhanced membrane binding of the product dye in accord with the MichaelisMenten kinetics of the reaction and the partition coefficients of substrate and product. We successfully tested the staining method by fluorescence microscopy with individual giant lipid vesicles and with individual red blood cells. In both systems, the membrane fluorescence due to bound hemicyanine was enhanced by an order of magnitude, proving the feasibility of enzyme-induced staining with voltage-sensitive dyes.

A novel class of amphiphilic hemicyanine dyes is described where electron-pushing aniline and electronpulling pyridinium are joined by anellated benzene rings. Enhancing the solvent polarity, the absorption band of these ANNINE dyes is shifted to the blue and the fluorescence band is shifted to the red at an invariant 00 energy. The increasing Stokes shift spans the whole visible spectrum. The divergent symmetrical solvatochromism of the positively charged chromophores is parametrized by a monopole-dipole model using a Born-Onsager-Marcus approach. From the intramolecular charge shift that is induced by electronic excitation, the linear Stark effect that is expected when the ANNINE dyes are used as voltage-sensitive probes in a biomembrane is estimated.

Optical recording of the electrical activity of individual neurons in culture or in a tissue requires cell-selective staining with a fluorescent voltage-sensitive dye. In a proof-of-principle experiment, we implement a novel approach to genetically targeted staining. The method relies on a water-soluble precursor dye and an overexpressed cell-surface enzyme that transforms the precursor into a hydrophobic dye that binds to the targeted cell. We fused an alkaline phosphatase to a specifically designed general-purpose membrane anchor, and the fusion protein was expressed on the surface of HEK293 cells, as was corroborated by immuno- and histochemical staining. We next synthesised an amphiphilic hemicyanine dye containing two enzymatically cleavable phosphate groups at its hydrocarbon tails. When the phosphate groups were removed, the binding to membranes was enhanced by a factor of a thousand, as shown by titration with lipid vesicles. We observed selective staining of enzymatically active cells by fluorescence microscopy in a mixed population of phosphatase-transfected and untransfected HEK293 cells. The critical parameters of enzyme-induced cell-selective staining were elucidated by a simple kinetic model to guide further developments of the method.