Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics

Jasper Akerboom, Nicole Carreras Calderón, Lin Tian, Sebastian Wabnig, Matthias Prigge, Johan Tolö, Andrew Gordus, Michael B. Orger, Kristen E. Severi, John J. Macklin, Ronak Patel, Stefan R. Pulver, Trevor J. Wardill, Elisabeth Fischer, Christina Schüler, Tsai Wen Chen, Karen S. Sarkisyan, Jonathan S. Marvin, Cornelia I. Bargmann, Douglas S. KimSebastian Kügler, Leon Lagnado, Peter Hegemann, Alexander Gottschalk, Eric R. Schreiter, Loren L. Looger

Research output: Contribution to journalArticlepeer-review

519 Scopus citations


vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

Original languageEnglish (US)
JournalFrontiers in Molecular Neuroscience
Issue numberFEB
StatePublished - Feb 11 2013
Externally publishedYes


  • Calcium imaging
  • Genetically encoded calcium indicator
  • Multi-color imaging
  • Optogenetics
  • Protein engineering


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