Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

Ahmed S. Abdelfattah, Jihong Zheng, Amrita Singh, Yi Chieh Huang, Daniel Reep, Getahun Tsegaye, Arthur Tsang, Benjamin J. Arthur, Monika Rehorova, Carl V.L. Olson, Yichun Shuai, Lixia Zhang, Tian Ming Fu, Daniel E. Milkie, Maria V. Moya, Timothy D. Weber, Andrew L. Lemire, Christopher A. Baker, Natalie Falco, Qinsi ZhengJonathan B. Grimm, Mighten C. Yip, Deepika Walpita, Martin Chase, Luke Campagnola, Gabe J. Murphy, Allan M. Wong, Craig R. Forest, Jerome Mertz, Michael N. Economo, Glenn C. Turner, Minoru Koyama, Bei Jung Lin, Eric Betzig, Ondrej Novak, Luke D. Lavis, Karel Svoboda, Wyatt Korff, Tsai Wen Chen, Eric R. Schreiter, Jeremy P. Hasseman, Ilya Kolb

Research output: Contribution to journalArticlepeer-review

16 Scopus citations


The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Original languageEnglish (US)
Pages (from-to)1547-1563.e9
Issue number10
StatePublished - May 17 2023
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General Neuroscience


  • biosensors
  • fluorescence imaging
  • fluorescent proteins
  • genetically encoded indicators
  • voltage imaging


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