A highly photostable monomeric red fluorescent protein for dual-color 3D STED and time-lapse 3D SIM imaging

Highly photostable red fluorescent proteins (RFPs) are invaluable for dual-color fluorescence microscopy, including super-resolution microscopy. Here we present mScarlet3‑S2, an RFP that exhibits a 29-fold improvement in photostability over its predecessor, mScarlet3, and outperforms other existing RFPs. This high photostability enables prolonged 2D and 3D imaging using both structured illumination microscopy and stimulated emission depletion microscopy. Using mScarlet3‑S2, we achieved over 150 Z-stacks in 3D STED imaging, revealing the architecture of the endoplasmic reticulum (ER) in detail. Key findings facilitated by mScarlet3‑S2 include nonplanar ER junctions, nuclear envelope (NE) invaginations, 3D maps of ER-NE contacts, diverse contact morphotypes (punctate, ribbon-like and branched) and polarized ER-NE junction distributions. These findings redefine our structural understanding of the ER-NE interface and demonstrate the value of mScarlet3‑S2 in revealing subcellular complexity.