Abstract
The stability of fluorescent proteins (FPs) is crucial for imaging techniques such as live-cell imaging, super-resolution microscopy and correlative light and electron microscopy. Although stable green and yellow FPs are available, stable monomeric red FPs (RFPs) remain limited. Here we develop an extremely stable monomeric RFP named mScarlet3-H and determine its structure at a 1.5 Å resolution. mScarlet3-H exhibits remarkable resistance to high temperature, chaotropic conditions and oxidative environments, enabling efficient correlative light and electron microscopy imaging and rapid (less than 1 day) whole-organ tissue clearing. In addition, its high photostability allows long-term three-dimensional structured illumination microscopy imaging of mitochondrial dynamics with minimal photobleaching. It also facilitates dual-color live-cell stimulated emission depletion imaging 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 multimodality microscopy in cell cultures and model organisms, complementing green FPs for multiplexed imaging in zebrafish, mice and Nicotiana benthamiana.
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Data availability
The coordinates and structure factors for mScarlet-H and mScarlet3-H have been deposited in the Protein Data Bank with accession numbers 8ZXO and 8ZXH, respectively. The most essential raw datasets, including source files for supplementary figures and raw unprocessed images, are available on figshare at https://doi.org/10.6084/m9.figshare.28398170.v1 (ref. 50). The remaining files are available from the corresponding author upon request. All plasmids used in this study are available on WeKwikGene at https://wekwikgene.wllsb.edu.cn/. Source data are provided with this paper.
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Acknowledgements
We thank S. Papadaki from Westlake Laboratory for verifying all plasmid sequences and depositing them to WeKwikGene. We thank E. Snapp from Janelia Research campus for the help with the interpretation of OSER imaging results. This project was supported by the National Natural Science Foundation of China (grant no. 32201235 to Z.F.), the Natural Science Foundation of Fujian Province, China (grant nos. 2022J01287, 2023Y9272 and 2024J09036 to Z.F. and 2024J01074 to C.W.), the Research Foundation for Advanced Talents at Fujian Medical University, China (grant nos. XRCZX2021013 to Z.F. and XRCZX2022031 to C.W.), the Finance Special Science Foundation of Fujian Province, China (grant no. 22SCZZX002 to Z.F.), Foundation of NHC Key Laboratory of Technical Evaluation of Fertility Regulation for Non-human Primate, Fujian Maternity and Child Health Hospital (grant no. 2022-NHP-04 to Z.F.), Open Project Fund of Fujian Key Laboratory of Drug Target Discovery and Structural and Functional Research (grant no. FKLDSR-202102 to Z.F.), Foundation of Westlake University, Westlake Laboratory of Life Sciences and Biomedicine, National Natural Science Foundation of China (grant no. 32171093 to K.D.P.), and ‘Pioneer’ and ‘Leading Goose’ R&D Program of Zhejiang (grant no. 2024SSYS0031 to K.D.P.). We thank L. Zhou, M. Wu and X. Lin at the Public Technology Service Center, Fujian Medical University for support with EM sample preparation and EM imaging.
Author information
Authors and Affiliations
Contributions
Z.F. conceived and supervised the whole project. H.X., Q.C. and C.W. engineered mScarlet3-H and measured its properties. H.X., P.L. and D.Q. did STED imaging. Q.C. and M. Liu did SIM imaging. H.Z. and J.D. performed rapid tissue clearing. S.W. did CLEM imaging. K.D.P. and W.Z. tested the photostability of mScarlet3-H. P.L. and Y.W. measured the pKa of mScarlet3-H. Y. Cui and Yanbin Li performed experiments on mScarlet3-H’s performance in plants. F.Z. did experiments on mScarlet3-H’s performance in zebrafish. Y.W. and G.F. helped to do EM sample preparation. Yiwei Yang and Y. Chen cultured cells. C.Y. conducted the MD simulation. J.X. analyzed the images from rapid tissue clearing. D.L., T.J. and W.F. helped with SIM imaging and analyzing the images of 3D-SIM imaging. F.H., Y.X. and R.Y. helped to purify mScarlet3-H. Q.Z., S.F., M. Li, Yu Li and Yufeng Yang solved the crystal structures of mScarlet-H and mScarlet3-H. Z.F., K.D.P. and Q.Z. wrote the paper. All authors reviewed the paper.
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Competing interests
A Chinese patent application (no. 202410568362.4) covering the use of mScarlet3-H for CLEM, rapid tissue clearing, expansion microscopy and fluorescent microscopy has been filed in which the Fujian Medical University is the applicant and Z.F., Y.W., H.X., Q.C., C.W., S.W. and Yiwei Yang are the inventors. The other authors declare no competing interests.
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Nature Methods thanks Benjamin Campbel, Takeharu Nagai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.
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Supplementary information
Supplementary Information
Supplementary Figs. 1–21 and Tables 1–21.
Supplementary Video 1
Long-term super-resolution imaging of the ER and microtubule dynamics in live HeLa cells acquired with CSU-W1 SoRa imaging setup (×100 NA 1.41, total duration 01:18 h:min; plays at 100 f.p.s.). ER: mScarlet3-H; EMTB: mBaoJin.
Supplementary Video 2
Long-term super-resolution imaging of mitochondria and EB3 dynamics in live HeLa cells acquired with CSU-W1 SoRa imaging setup (×100 NA 1.41, total duration 50:45 m:s; plays at 100 f.p.s.). Mito: mScarlet3-H; EB3: mBaoJin.
Supplementary Video 3
Long-term super-resolution imaging of mitochondria and lifeact labeled actin dynamics in live HeLa cells acquired with CSU-W1 SoRa imaging setup (x100 NA 1.41, total duration 58:29 m:s; plays at 20 f.p.s.). Lifeact: mScarlet3-H; Mito: mBaoJin.
Supplementary Video 4
Long-term super-resolution imaging of ER and mitochondria dynamics in live HeLa cells acquired with CSU-W1 SoRa imaging setup (×100 NA 1.41, total duration 08:15 m:s; plays at 10 f.p.s.). ER: mScarlet3-H; Mito: mBaoJin.
Supplementary Video 5
Long-term imaging of cell mitosis of zebrafish larva labeled by mScarlet3-H using a STELLARIS 8 FALCON confocal microscope.
Supplementary Video 6
Long-term imaging of H2B-mScarlet3-H in a developing zebrafish larva using a STELLARIS 8 FALCON confocal microscope.
Supplementary Video 7
Video showing the fluorescence dynamics of HeLa cells expressing H2B-mScarlet3-H alternately treated with PBS and 5 M GdnHCl.
Supplementary Video 8
Long-term super-resolution imaging of the dynamics of mitochondria labeled by mScarlet3-H in live COS-7 cells using a 3D-SIM imaging setup (×100 NA 1.49, total duration 2 h).
Supplementary Video 9
Long-term super-resolution imaging of the dynamics of ER sheet labeled by mScarlet3-H in live COS-7 cells using a STED imaging setup (×100 NA 1.45, total duration 09:00 min:s).
Supplementary Video 10
Long-term super-resolution imaging of the dynamics of ER sheet fusion labeled by mScarlet3-H in live COS-7 cells using a STED imaging setup (×100 NA 1.45, total duration 10.8 s).
Supplementary Video 11
Long-term super-resolution imaging of the dynamics of ER sheet fission labeled by mScarlet3-H in live COS-7 cells using a STED imaging setup (×100 NA 1.45, total duration 3:18 m:s).
Supplementary Video 12
Long-term super-resolution imaging of the dynamics of ER ball labeled by mScarlet3-H in live COS-7 cells using a STED imaging setup (×100 NA 1.45, total duration 14.4 s).
Supplementary Video 13
Long-term super-resolution imaging of the dynamics of ER and mitochondria in live COS-7 cells using a STED imaging setup (×100 NA 1.45, total duration 5:32 m:s). ER: mScarlet3-H; Mito: HBmito Crimson.
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Xiong, H., Chang, Q., Ding, J. et al. A highly stable monomeric red fluorescent protein for advanced microscopy. Nat Methods 22, 1288–1298 (2025). https://doi.org/10.1038/s41592-025-02676-5
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DOI: https://doi.org/10.1038/s41592-025-02676-5
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