Voltage-sensitive rhodol with enhanced two-photon brightness
We have designed, synthesized, and applied a rhodol-based chromophore to a molecular
wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage
sensing using two-photon (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a
voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain
samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the
nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5
demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability
relative to first-generation voltage sensors. This photostability enables multisite
optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse
model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased
activity relative to wild-type neurons and additionally show that the proportion of
active neurons in the network increases with the loss of Tsc1. The high photostability
and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the
ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds
affords the unique opportunity to image neuronal voltage changes in acutely prepared
mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked
spiking activity that was readily discerned with bath-applied RVF5, demonstrating the
utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores
for imaging voltage in intact brain tissue.