A highly stable monomeric red fluorescent protein for advanced microscopy

The stability of fluorescent proteins (FPs) is crucial for many imaging techniques, including long-term. live cell imaging, super-resolution microscopy, correlative light and electron microscopy (CLEM), and tissue clearing. Although several stable green and yellow FPs are available, the options for stable monomeric red fluorescent proteins (RFPs), commonly used as spectral counterparts for dual-color imaging, are significantly limited. Here, we developed an extremely stable monomeric RFP named mScarlet3-H and determined its structure at 1.5 Å resolution. Remarkably, mScarlet3-H 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. Furthermore, our findings indicate that mScarlet3-H’s thermal and chemical stabilities were accompanied by 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. Importantly, the outstanding photostability of mScarlet3-H makes it a unique RFP for stimulated emission depletion microscopy (STED), allowing dual-color STED imaging of the dynamic interactions between endoplasmic reticulum and mitochondria with a high signal-to-noise ratio and strong specificity. Systematic benchmarking of mScarlet3-H against high-performing RFPs established it as a highly stable monomeric RFP suitable for multi-modality microscopy of cell cultures and model organisms, such as zebrafish, mice, and Nicotiana benthamiana plant, complementing existing green FPs for multiplexed imaging.

Fu’s lab is dedicated to the development and application of super-resolution correlative light and electron microscopy (SR-CLEM), with the focus on creating novel fluorescent proteins, optimizing super-resolution algorithms, and improving SR-CLEM imaging techniques. Related articles were published in Nature Methods (2020 and 2025), Nano Letters (2020), Journal of Visualized Experiments (2024), Sleep (2022), and Journal of Cell Biology, etc. Currently, his lab is utilizing SR-CLEM and volume electron microscopy (VEM) to map neural circuits.)