Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators

PLoS One. 2013 Jun 19;8(6):e66959. doi: 10.1371/journal.pone.0066959. Print 2013.


A longstanding goal in neuroscience has been to develop techniques for imaging the voltage dynamics of genetically defined subsets of neurons. Optical sensors of transmembrane voltage would enhance studies of neural activity in contexts ranging from individual neurons cultured in vitro to neuronal populations in awake-behaving animals. Recent progress has identified Archaerhodopsin (Arch) based sensors as a promising, genetically encoded class of fluorescent voltage indicators that can report single action potentials. Wild-type Arch exhibits sub-millisecond fluorescence responses to trans-membrane voltage, but its light-activated proton pump also responds to the imaging illumination. An Arch mutant (Arch-D95N) exhibits no photocurrent, but has a slower, ~40 ms response to voltage transients. Here we present Arch-derived voltage sensors with trafficking signals that enhance their localization to the neural membrane. We also describe Arch mutant sensors (Arch-EEN and -EEQ) that exhibit faster kinetics and greater fluorescence dynamic range than Arch-D95N, and no photocurrent at the illumination intensities normally used for imaging. We benchmarked these voltage sensors regarding their spike detection fidelity by using a signal detection theoretic framework that takes into account the experimentally measured photon shot noise and optical waveforms for single action potentials. This analysis revealed that by combining the sequence mutations and enhanced trafficking sequences, the new sensors improved the fidelity of spike detection by nearly three-fold in comparison to Arch-D95N.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials
  • Animals
  • Archaea / genetics
  • Archaea / metabolism*
  • Archaeal Proteins / genetics*
  • Archaeal Proteins / metabolism
  • Cells, Cultured
  • Fluorescence Resonance Energy Transfer / methods
  • Mutation*
  • Neurons / physiology*
  • Rats


  • Archaeal Proteins
  • archaerhodopsin protein, Archaea

Grant support

This work was supported by the Stanford CNC program, the Stanford BioX Interdisciplinary Initiatives Program (IIP) grant, and a National Academies Keck Futures Initiative (NAKFI) research grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.