Tethered Bichromophoric Fluorophore Quencher Voltage Sensitive Dyes

Voltage sensitive dyes (VSDs) are used for in vitro drug screening and for imaging of patterns of electrical activity in tissue. Wide application of this technology depends on the availability of sensors with high sensitivity (percent change of fluorescence per 100 mV), high fluorescence quantum yield, and fast response kinetics. A promising approach uses a two-component system consisting of anionic membrane permeable quenchers with fluorophores labeling one side of the membrane; this produces voltage-dependent fluorescence quenching. However, the quencher must be kept at low concentrations to minimize pharmacological effects, thus limiting sensitivity. By developing tethered bichromophoric fluorophore quencher (TBFQ) dyes, where the fluorophore and quencher are covalently connected by a long hydrophobic chain, the sensitivity is maximized and is independent of VSD concentration. A series of 13 TBFQ dyes based on the aminonaphthylethenylpyridinium (ANEP) fluorophore and the dipicrylamine anion (DPA) quencher have been synthesized and tested in an artificial lipid bilayer apparatus. The best of these, TBFQ1, shows a 2.5-fold change in fluorescence per 100 mV change in membrane potential, and the response kinetics is in the 10-20 ms range. This sensitivity is an order of magnitude better than that of commonly used VSDs. However, the fluorescence quantum yield is only 1.6%, which may make this first generation of TBFQ VSDs impractical for in vivo electrical imaging. Nevertheless, the design principles established here can serve as foundation for improved TBFQ VSDs. We believe this approach promises to greatly enhance our ability to monitor electrical activity in cells and tissues.