Orange/far-red hybrid voltage indicators with reduced phototoxicity enable reliable long-term imaging in neurons and cardiomyocytes

Author:

Liu Shuzhang12,Ling Jing34ORCID,Chen Peng56ORCID,Cao Chang1ORCID,Peng Luxin1,Zhang Yuan4,Ji Guangshen7,Guo Yingna7,Chen Peng R.13ORCID,Zou Peng1238ORCID,Chen Zhixing3456ORCID

Affiliation:

1. College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of the Ministry of Education, Peking University, Beijing 100871, China

2. IDG/McGovern Institute for Brain Research at Peking University, Beijing 100871, China

3. Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China

4. College of Future Technology, Institute of Molecular Medicine, National Biomedical Imaging Center, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Peking University, Beijing 100871, China

5. Peking University-Nanjing Institute of Translational Medicine, Nanjing 211800, China

6. Genvivo Biotech, Nanjing 211800, China

7. State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China

8. Chinese Institute for Brain Research, Beijing 102206, China

Abstract

Hybrid voltage indicators (HVIs) are chemogenetic sensors that combines the superior photophysical properties of organic dyes and the genetic targetability of protein sensors to report transient membrane voltage changes. They exhibit boosted sensitivity in excitable cells such as neurons and cardiomyocytes. However, the voltage signals recorded during long-term imaging are severely diminished or distorted due to phototoxicity and photobleaching issues. To capture stable electrophysiological activities over a long time, we employ cyanine dyes conjugated with a cyclooctatetraene (COT) molecule as the fluorescence reporter of HVI. The resulting orange-emitting HVI–COT–Cy3 enables high-fidelity voltage imaging for up to 30 min in cultured primary neurons with a sensitivity of ~ −30% ΔF/F 0 per action potential (AP). It also maximally preserves the signal of individual APs in cardiomyocytes. The far-red-emitting HVI–COT–Cy5 allows two-color voltage/calcium imaging with GCaMP6s in neurons and cardiomyocytes for 15 min. We leverage the HVI–COT series with reduced phototoxicity and photobleaching to evaluate the impact of drug candidates on the electrophysiology of excitable cells.

Funder

Ministry of Science and Technology of the People's Republic of China

Beijing Municipal Science and Technology Commission

MOST | National Natural Science Foundation of China

Publisher

Proceedings of the National Academy of Sciences

Subject

Multidisciplinary

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