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.

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Publication Details

Journal
Nature Methods
Published
2025-11-26
DOI
https://doi.org/10.1038/s41592-025-02962-2
Citations
3
Primary Topic
Advanced Fluorescence Microscopy Techniques
Type
article
Field-Weighted Citation Impact
4.90
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article

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

Ke Zheng, Liangyi Chen, Zhixing Chen, Kunhao Wang et al.
3 citations
Nature Methods
Advanced Fluorescence Microscopy Techniques
4.90
article

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

Ke Zheng, Liangyi Chen, Zhixing Chen, Kunhao Wang, Fudong Xue, Leiting Pan, Pingyong Xu, Ya Ding, Shiqun Zhao, Tianyi Li, Qi Dong, Peng Xia, Lin Yuan, Wenting He
article en
3 citations

Abstract

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.

Nature MethodsVol. 23(1)
Chinese Academy of Sciences (CN), Peking University (CN), Nankai University (CN), Beijing Institute of Optoelectronic Technology (CN), Beijing Founder Electronics (China) (CN), Institute of Biophysics (CN), University of Chinese Academy of Sciences (CN), Zhejiang University (CN)
Openalex Percentile: Top 5%
Advanced Fluorescence Microscopy Techniques
4.90
DOIOpen Access PDFOpenAlex
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