mYongHong: a structure-guided design of stable and monomeric red fluorescent protein

The stability of fluorescent proteins (FPs) is crucial for many imaging techniques, including long-term live cell imaging, correlative light and electron microscopy (CLEM), and tissue clearing. While several stable green and yellow FPs exist, the options for stable monomeric red fluorescent proteins (RFPs), commonly used as spectral counterparts for dual-color imaging, are significantly limited. Here, we applied structure-guided mutagenesis to develop an extremely stable monomeric RFP named mYongHong and report its structure at 1.5 Å resolution. Remarkably, mYongHong exhibited exceptional resistance to high temperature, chaotropic conditions, and highly oxidative environments, facilitating its efficient applications in CLEM imaging of various organelles and rapid (less than 1 day) whole organ tissue clearing. Additionally, our findings indicate that mYongHong’s thermal and chemical stability was associated with its high photostability, enabling the tracking of the fusion and fission of mitochondria using 3D structured illumination microscopy for up to 2 hours with minimal photobleaching. More importantly, the outstanding photostability made mYongHong a unique RFP for stimulated emission depletion microscopy, allowing it to capture the dynamics of the endoplasmic reticulum with a high signal-to-background ratio and strong specificity. Systematic benchmarking of mYongHong against high-performing RFPs established it as a highly stable FP suitable for multi-modality microscopy of cell cultures and model organisms (zebrafish, mice, and Nicotiana benthamiana plant), complementing existing GFPs for multiplexed imaging.