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Updated: Mar 7, 2026

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Voltage-sensitive rhodol with enhanced two-photon brightness.

Rishikesh U Kulkarni1, Daniel J Kramer2, Narges Pourmandi1

  • 1Department of Chemistry, University of California, Berkeley, CA 94720.

Proceedings of the National Academy of Sciences of the United States of America
|March 1, 2017
PubMed
Summary

Researchers developed Rhodol VoltageFluor-5 (RVF5), a novel voltage sensor for fast and sensitive neural imaging. This bright dye enables clear visualization of neuronal activity in thick tissues and brain slices using two-photon illumination.

Keywords:
fluorescent sensorstwo-photon microscopyvoltage imaging

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Area of Science:

  • Neuroscience
  • Biophotonics
  • Molecular Engineering

Background:

  • Voltage sensing dyes are crucial for monitoring neuronal activity.
  • Existing voltage sensors often lack the sensitivity, brightness, or photostability required for complex biological samples.
  • Two-photon (2P) illumination offers advantages for deep tissue imaging but requires specialized probes.

Purpose of the Study:

  • To design and synthesize a novel rhodol-based chromophore for enhanced voltage sensing.
  • To develop a molecular wire-based platform for fast, sensitive, and bright voltage sensing.
  • To evaluate the performance of the new voltage sensor (RVF5) under one-photon (1P) and two-photon (2P) illumination in biological systems.

Main Methods:

  • Synthesis of Rhodol VoltageFluor-5 (RVF5), a dichlororhodol core with pyrrolidyl substitution.
  • Characterization of RVF5's voltage sensitivity and photostability in mammalian cells under 1P illumination.
  • Application of RVF5 for multisite optical recordings in neurons lacking tuberous sclerosis complex 1 (Tsc1) in a mouse model.
  • Evaluation of RVF5 performance under 2P illumination in brain slices and hippocampus stimulation.

Main Results:

  • RVF5 exhibits high voltage sensitivity (28% ﾎ認/F per 100 mV) and improved photostability compared to first-generation sensors.
  • RVF5 enables multisite recordings in Tsc1 knockout neurons, revealing increased network activity.
  • RVF5 performance is maintained under 2P illumination, with a high 2P cross-section suitable for thick samples.
  • Neuronal voltage changes in acute mouse brain slices were successfully imaged using RVF5 and 2P illumination.

Conclusions:

  • RVF5 is a versatile and effective voltage-sensitive dye for both 1P and 2P imaging.
  • The rhodol scaffold allows for tuning of optical properties, facilitating 2P-optimized voltage sensing.
  • RVF5 and similar molecular wire-based sensors are promising tools for imaging neuronal activity in intact brain tissue.