Improved Orange and Red Ca2+ Indicators and Photophysical Considerations for Optogenetic Applications

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• 作者：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
• 刊名：ACS Chemical Neuroscience
• 出版年：2013
• 出版时间：June 19, 2013
• 年：2013
• 卷：4
• 期：6
• 页码：963-972
• 全文大小：575K
• 年卷期：v.4,no.6(June 19, 2013)
• ISSN：1948-7193

文摘

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.

Keywords:

Ca²⁺ imaging; fluorescent proteins; Ca²⁺ indicators; channelrhodopsin; photoactivation