UC Berkeley

Voltage sensitive fluorescent indicators have been a useful method for monitoring cardiac electrophysiology for nearly 50 years, complementing traditional methods of electrophysiology such as patch clamp techniques. The impacts fluorophore phototoxicity on cell health have long been a concern in fluorescent imaging. VoltageFluors (VF dyes) are a class of voltage sensitive indicators that rely on photoinduced-electron transfer to sense changes in membrane potential. While our lab has made significant strides towards expanding the wavelengths of indicators offered and improving indicator voltage sensitivity, efforts to reduce fluorophore phototoxicity in the context of VF dyes was largely unexplored. The work presented in this dissertation explores a generalizable method for reducing the phototoxicity of our VF dyes, with the tethering of our probes to the triplet state quencher cyclooctatetraene (COT). Chapter 1 presents the development of VF-COT, a green, xanthene-based indicator intramolecularly tethered to COT. VF-COT can report cardiac action potentials for twice as long as its parent dye, without significant impacts on electrophysiology parameters. Chapter 2 presents BeRST-COT, a near-infrared, silicon rhodamine-based VF that demonstrates the generalizability of reducing phototoxicity with an intramolecularly tethered COT molecule. BeRST-COT reports cardiac electrophysiology for 2.7- times as long as its parent dye and reduces the relative production of singlet oxygen during imaging sessions. Chapters 1 and 2 present work utilizing cardiac cells towards efforts of better characterizing ultra-stable VF indicators. Appendix 1 aims to utilize VF dyes and fluorescence lifetime imaging to better quantify cardiac electrophysiology parameters and lays out the work completed and the rationale for future work in this project.