A highly stable monomeric red fluorescent protein for advanced microscopy

Authors:

Haiyan Xiong, Qiyuan Chang, Jiayi Ding, Shuyuan Wang, Wenhao Zhang, Yu Li, Yaochen Wu, Pengyan Lin, Chengyu Yang, Miaoxing Liu, Guicun Fang, Yiwei Yang, Jiongfang Xie, Dong Qi, Tao Jiang, Wenfeng Fu, Fen Hu, Yiming Chen, Rongcai Yue, Yanbin Li, Yong Cui, Min Li, Shilong Fan, Yufeng Yang, Yunlu Xu, Dong Li, Fenghua Zhang, Hu Zhao, Congxian Wu, Qingbing Zheng, Kiryl D. Piatkevich, Zhifei Fu

Abstract:

The stability of fluorescent proteins (FPs) is crucial for imaging techniques such as live-cell imaging, super-resolution microscopy and correlative light and electron microscopy. Although stable green and yellow FPs are available, stable monomeric red FPs (RFPs) remain limited. Here we develop an extremely stable monomeric RFP named mScarlet3-H and determine its structure at a 1.5 Å resolution. mScarlet3-H exhibits remarkable resistance to high temperature, chaotropic conditions and oxidative environments, enabling efficient correlative light and electron microscopy imaging and rapid (less than 1 day) whole-organ tissue clearing. In addition, its high photostability allows long-term three-dimensional structured illumination microscopy imaging of mitochondrial dynamics with minimal photobleaching. It also facilitates dual-color live-cell stimulated emission depletion imaging with a high signal-to-noise ratio and strong specificity. Systematic benchmarking against high-performing RFPs established mScarlet3-H as a highly stable RFP for multimodality microscopy in cell cultures and model organisms, complementing green FPs for multiplexed imaging in zebrafish, mice and Nicotiana benthamiana.

< Back to publications

Full article >