Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging.

Yong Qian,Sarah Aufmkolk,Danielle M Orozco Cosio,Abhi Aggarwal,Edward S Boyden,Wan-Chi Su,Robert E Campbell,Mitchell H Murdock,Edward S Ruthazer,Yu-Fen Chang,Anne Schohl,Kiryl D Piatkevich,Orhan T Celiker,Paul W Wiseman,Polina V Lishko

doi:10.1371/journal.pbio.3000965

Abstract

Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.

Highlights

Fluorescence imaging of intracellular calcium ion (Ca2+) transients using genetically encoded Ca2+ indicators (GECIs) is a powerful and effective technique to monitor in vivo neuron activity in model organisms [1,2,3,4]
Based on ample precedent from the development of the green fluorescent protein (GFP)-based genetically encoded calcium ion indicator (GECI) GCaMP series [1,3] and the red fluorescent protein (RFP)-based R-GECO series [2,11], we reasoned that NIR-GECO1 was likely to be amenable to further improvement by protein engineering and directed molecular evolution
NIR-GECO2G is improved relative to NIR-GECO1 in terms of both overall cellular brightness and sensitivity

Summary

Introduction

Fluorescence imaging of intracellular calcium ion (Ca2+) transients using genetically encoded Ca2+ indicators (GECIs) is a powerful and effective technique to monitor in vivo neuron activity in model organisms [1,2,3,4]. Over a time frame spanning 2 decades [5,6], tremendous effort has been invested in the development of visible wavelength GECIs based on green and red fluorescent proteins (GFP and RFP, respectively).

Methods

Results

Conclusion