UC Berkeley

Fluorescent voltage sensitive dyes (VSDs) provide an attractive, complementary method to traditional electrophysiology techniques and are important tools for studying rapid changes of membrane potential in excitable cells. The VoltageFluor family of VSDs reports this rapid activity with high sensitivity using photoinduced-electron transfer (PeT) as the voltage sensitive trigger. This mechanism provides sub-millisecond response kinetics and our laboratory has shown it can be tuned by exchanging the identity of the electron donor and fluorophore. However, the identity and architecture of the molecular wire remained unexplored. The work presented herein addresses this gap in knowledge, exploring different strategies for tuning PeT through modifications to the molecular wire. Chapter 1 represents an initial attempt, replacing the link between the molecular wire and the fluorophore with an amide. This insulates the fluorophore to slow PeT and results in less sensitive, but brighter, indicators. However, membrane staining was poor with these new indicators and in vitro measurements did not predict brightness in cells. Chapter 2 builds upon the insight of Chapter 1, exchanging the phenylene-vinylene molecular wire for a fluorene monomer. This fluorene-based wire provided indicators with bright membrane stains, maintained voltage sensitivity, and was shown to be less phototoxic. Low voltage sensitivity limited the utility of the indicators. Chapter 3 addresses this limitation by adding conjugation to the scaffold with a vinyl spacer to make indicators with up to a 3.5-fold improvement in sensitivity over fluorene-based indicators. Finally, Chapter 4 describes a collaborative effort to assess the effect of aniline conformation on the voltage sensing properties of VoltageFluor indicators. We found the sensitivity and fluorescence lifetime of these conformationally restricted aniline could be tuned over an order of magnitude. These results show the importance of electronic interaction of the aniline with the molecular wire for a PeT-based voltage sensor.