title: Improved orange and red Ca²± indicators and photophysical considerations for optogenetic applications.
abstract: 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.
authors: Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors: Jiahui
authors: J
authors: Wu
authors: Lin
authors: L
authors: Liu
authors: Tomoki
authors: T
authors: Matsuda
authors: Yongxin
authors: Y
authors: Zhao
authors: Aleksander
authors: A
authors: Rebane
authors: Mikhail
authors: M
authors: Drobizhev
authors: Yu-Fen
authors: YF
authors: Chang
authors: Satoko
authors: S
authors: Araki
authors: Yoshiyuki
authors: Y
authors: Arai
authors: Kelsey
authors: K
authors: March
authors: Thomas E
authors: TE
authors: Hughes
authors: Ken
authors: K
authors: Sagou
authors: Takaki
authors: T
authors: Miyata
authors: Takeharu
authors: T
authors: Nagai
authors: Wen-Hong
authors: WH
authors: Li
authors: Robert E
authors: RE
authors: Campbell
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002540:Cerebral Cortex / Q000737:chemistry / Q000378:metabolism; D000075402:Channelrhodopsins; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D051379:Mice; D062308:Optogenetics / Q000379:methods*; D009924:Organ Culture Techniques; D055668:Photochemical Processes; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D015027:Zebrafish
chemical_list: D000075402:Channelrhodopsins; D007202:Indicators and Reagents; D002118:Calcium