Improved orange and red Ca2+ indicators and photophysical considerations for optogenetic applications

Jiahui Wu, Lin Liu, Tomoki Matsuda, Yongxin Zhao, Aleksander Rebane, Mikhail Drobizhev, Yu Fen Chang, Satoko Araki, Yoshiyuki Arai, Kelsey March, Thomas E. Hughes, Ken Sagou, Takaki Miyata, Takeharu Nagai, Wen Hong Li, Robert E. Campbell

Research output: Contribution to journalArticlepeer-review

163 Scopus citations


We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca2+) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca2+ indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca2+ indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca2+ imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.

Original languageEnglish (US)
Pages (from-to)963-972
Number of pages10
JournalACS Chemical Neuroscience
Issue number6
StatePublished - Jun 19 2013


  • Ca imaging
  • Ca indicators
  • channelrhodopsin
  • fluorescent proteins
  • photoactivation

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Cognitive Neuroscience
  • Cell Biology


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