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

ACS Chem Neurosci. 2013 Jun 19;4(6):963-72. doi: 10.1021/cn400012b. Epub 2013 Mar 19.


We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca(2+)) 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 Ca(2+) indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca(2+) indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca(2+) 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Cerebral Cortex / chemistry
  • Cerebral Cortex / metabolism
  • Channelrhodopsins
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Indicators and Reagents / metabolism
  • Mice
  • Optogenetics / methods*
  • Organ Culture Techniques
  • Photochemical Processes*
  • Protein Engineering / methods*
  • Protein Structure, Secondary
  • Zebrafish


  • Channelrhodopsins
  • Indicators and Reagents
  • Calcium