Mechanistic Basis of the Voltage-Sensitivity of Thiazolothiazole Dyes

Voltage sensitive dyes (VSDs) are versatile and powerful reporters of the membrane potential across living cell membranes. Thiazolothiazole (TTz) dyes are a relatively new and promising class of VSDs. While TTz dyes have high photostability and low cytotoxicity, the mechanistic basis of their excellent voltage sensitivity remains unknown. To answer this question, we have accessed a new generation of TTz dyes (asym-TTz) that are asymmetrically substituted, push-pull dyes. One of these molecules shows sustained cellular membrane localization, high photostability and voltage sensitivity of ~9% dF/F per 100 mV. Asym-TTz derivatives exhibited strong solvatofluorochromism with large Stokes shifts and exceptionally high transition dipole moments that provided a clue towards their voltage- sensitivity. By computationally analyzing their behavior within cell membranes under an electric field, we found that asym-TTz dyes show a surprising twist on either side of the TTz bridging unit in the excited state at more positive membrane potentials, which decreases dye fluorescence. This novel mechanism can be leveraged to create newer classes of highly sensitive voltage reporter dyes for diverse sensing applications.