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Improved Orange and Red Ca2+ Indicators and Photophysical Considerations for Optogenetic Applications #MMPMID23452507
Wu J; Liu L; Matsuda T; Zhao Y; Rebane A; Drobizhev M; Chang YF; Araki S; Arai Y; March K; Hughes TE; Sagou K; Miyata T; Nagai T; Li Wh; Campbell RE
ACS Chem Neurosci 2013[Jun]; 4 (6): 963-72 PMID23452507show ga
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.