Electric field-dependent excited-state twisting in donor-acceptor thiazolothiazole dyes establishes a new class of cell membrane voltage sensors.

Voltage sensitive dyes (VSDs) are versatile and powerful reporters of the membrane potential across cell membranes. Thiazolothiazole (TTz) dyes are a promising new class of VSDs with high photostability and low cytotoxicity. However, the mechanistic basis of their voltage sensitivity is unknown. To address this, we developed a new generation of asymmetrically substituted TTz dyes (asym-TTz). They exhibit strong solvatofluorochromism with large Stokes shifts and exceptionally large changes in dipole moments (23-29 D), representing an 80% increase over those of previously reported TTz VSDs. An asym-TTz derivative, TwistTz-P, bearing a carboxyphenyl acceptor group, shows sustained cellular membrane localization and a voltage sensitivity of ∼10% ΔF/F per 100 mV. Computational analysis of TwistTz-P under varying electric fields revealed a pronounced twist on both sides of the TTz bridging unit in the excited state. The degree of twisting was voltage-dependent, exhibiting an increase upon depolarization, consistent with the experimentally observed fluorescence decrease at an identical voltage. In another asym-TTz dye (FlatTz-P), where a carboxypyrazinyl acceptor group was incorporated, excited-state twisting under varying electric fields was negligible and demonstrated no voltage sensitivity. These observations establish electric field-dependent excited-state twisting as a new voltage sensing mechanism, and, additionally, provide a new strategy for creating highly sensitive VSDs.