Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP

Wenyan Guo; Xiuli Liu; Yurong Liu; Yadong Gang; Xiaobin He; Yao Jia; Fangfang Yin; Pei Li; Fei Huang; Hongfu Zhou; Xiaojun Wang; Hui Gong; Qingming Luo; Fuqiang Xu; Shaoqun Zeng

doi:10.1364/BOE.8.003281

Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP

Biomed Opt Express. 2017 Jun 15;8(7):3281-3288. doi: 10.1364/BOE.8.003281. eCollection 2017 Jul 1.

Authors

Wenyan Guo^{1

2}, Xiuli Liu^{1

2}, Yurong Liu^{1

2}, Yadong Gang^{1

2}, Xiaobin He³, Yao Jia^{1

2}, Fangfang Yin^{1

2}, Pei Li^{1

2}, Fei Huang^{1

2}, Hongfu Zhou^{1

2}, Xiaojun Wang^{1

2}, Hui Gong^{1

2}, Qingming Luo^{1

2}, Fuqiang Xu^{1

2

3}, Shaoqun Zeng^{1

2}

Affiliations

¹ Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, China.
² Department of Biomedical Engineering, Key Laboratory for Biomedical Photonics of Ministry of Education, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, China.
³ State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Brain Research Center, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, West No.30 Xiao Hong Shan, Wuhan 430071, China.

Abstract

The pH-sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EGFP or EYFP is good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is an urgent need. Here a pH-sensitive red fluorescent protein, pHuji, is selected and verified to remain pH-sensitive in HM20 resin. We observe 183% fluorescence intensity of pHuji in resin-embeded mouse brain and 29.08-fold fluorescence intensity of reactivated pHuji compared to the quenched state. pHuji and EGFP can be quenched and chemically reactivated simultaneously in resin, thus enabling simultaneous two-color micro-optical sectioning tomography of resin-embedded mouse brain. This method may greatly facilitate the visualization of neuronal morphology and neural circuits to promote understanding of the structure and function of the brain.

Keywords: (110.4190) Multiple imaging; (160.2540) Fluorescent and luminescent materials; (170.3880) Medical and biological imaging; (180.2520) Fluorescence microscopy.