On August 17, 2023, the team led by Zhixing Chen (Peking-Tsinghua Center for Life Science, Peking University) and Peng Zou (College of Chemistry and Molecular Engineering, Peking University) published their latest collaborative work with the title “Orange/far-red hybrid voltage indicators with reduced phototoxicity enable reliable long-term imaging in neurons and cardiomyocytes”in Proc. Natl. Acad. Sci. U.S.A.
Voltage imaging is an advanced method to measure and interpret the electrical communications between cells, and continuous or cumulative voltage recordings for a long time are valuable for studying synaptic plasticity, animal behavioral rhythms, or characterizing pharmacological toxicity. Hybrid voltage indicators (HVIs) based on the voltage-sensitive domain and fluorescent dyes significantly outperform in voltage sensitivityand cell specificity. However, in order to obtain a high signal-to-noise ratio, voltage imaging requires higher excitation intensity, which can lead to more severe phototoxicity and photobleaching(Nat. Chem. 2021, 13, 472-479). In 2020, Zhixing Chen's group demonstrated that the cyclooctatetraene(COT) conjugation strategy could reduce the phototoxicity of fluorescent probes in live cells (Chem. Sci. 2020, 11, 8506-8516). To develop a new generation of voltage indicators with minimized phototoxicity, they synthesized highly photostable COT-modified cyanine dyes and combinedthem with HVI (Fig. 1).
Fig. 1 Scheme of gentle hybrid voltage indicator HVI-COT.
Among the HVI-COT-Cyanines, HVI-COT-Cy3 showed excellent voltage sensitivity (−33.4±0.8% ΔF/F0 per action potential), which make it the most sensitive orange-emitting voltage indicator. Additionally, the photostability of HVI-COT-Cy3 is 40-fold better than that of Voltron2525. With remarkable photostability and minimal phototoxicity, HVI-COT-Cy3 could continuously record for 30 min in neurons and could detect the effects on firing rate from various drugs treatments (Fig. 2).
Fig. 2 The mechanisms of fluorophore labeling, voltage sensing, photobleaching, phototoxicity, and long-term voltage imaging (720,000 frames) of HVI-COT in neurons.
Furthermore, the far-red indicatorHVI-COT-Cy5 was coupled with the green fluorescent calcium indicator GCaMP6s to achieve simultaneous detection of voltage and calcium signals in neurons or cardiomyocytes for 15 min (Fig. 3).
Fig. 3 Simultaneous imaging with HVI-COT-Cy5 and GCaMP6s for 15 min in neurons or cardiomyocytes.
Prof. Chen Zhixing (Peking-Tsinghua Center for Life Science, Peking University) and Prof. Peng Zou (College of Chemistry and Molecular Engineering, Peking University) are co-corresponding authors. Ph.D. students Shuzhang Liu (College of Chemistry and Molecular Engineering, Peking University) and Jing Ling (Peking-Tsinghua Center for Life Science, Peking University) are co-first authors of the paper.
The research was jointly supported by the Ministry of Science and Technology, the Beijing Municipal Science & Technology Commission, the National Natural Science Foundation of China, the Bayer Investigator Award, and the Beijing National Laboratory for Molecular Sciences in China.
Link of the article:https://www.pnas.org/doi/10.1073/pnas.2306950120.
