A highly stable monomeric red fluorescent protein for advanced microscopy

The stability of fluorescent proteins (FPs) is critical for imaging techniques such as live-cell imaging, super-resolution microscopy, and correlative light and electron microscopy (CLEM). While stable green and yellow FPs are available, stable monomeric red fluorescent proteins (RFPs) remain limited. Here, we developed an extremely stable monomeric RFP named mScarlet3-H and determined its structure at 1.5 Å resolution. mScarlet3-H exhibits remarkable resistance to high temperature, chaotropic conditions, and oxidative environments, enabling efficient CLEM imaging and rapid (less than 1 day) whole-organ tissue clearing. Additionally, its high photostability allows long-term 3D structured illumination microscopy (SIM) imaging of mitochondrial dynamics with minimal photobleaching. It also facilitates dual-color live-cell stimulated emission depletion (STED) microscopy 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 multi-modality microscopy in cell cultures and model organisms, complementing green FPs for multiplexed imaging in zebrafish, mice, and Nicotiana benthamiana.