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Triangle-beam interference structured illumination microscopy

Nature Photonics volume 19, pages 1122–1131 (2025)Cite this article

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Abstract

Structured illumination microscopy (SIM) is a powerful tool for live-cell super-resolution imaging. Conventional two-dimensional (2D)-SIM uses one-dimensional stripe patterns and rotates them at three angles to achieve uniform resolution. Here, to alleviate photobleaching and improve the temporal resolution of 2D-SIM, we develop triangle-beam interference SIM (3I-SIM), which generates a 2D lattice pattern based on radially polarized beam interference. The radial polarization enhances the signal-to-noise ratio of the high-frequency components. Compared with conventional 2D-SIM, 3I-SIM reduces photobleaching and improves the temporal resolution to 242 Hz. Benefiting from unidirectional phase shift, 3I-SIM provides threefold higher rolling frame rate than conventional 2D-SIM to visualize fast biological dynamics. We further developed 3I-Net, a deep neural network with a co-supervised training scheme, to enhance the performance of 3I-SIM under an extremely low signal intensity. Its higher sensitivity enables the consecutive acquisition of over 100,000 time points at a spatial resolution of 100 nm. We continuously monitor the fine morphological changes in neuronal growth cones for up to 13 h, as well as the transient signals from actin filaments regulating endoplasmic reticulum dynamics. We believe 3I-SIM will offer a suitable platform to study complex and rapid biological processes with high data throughput.

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Fig. 1: Principle of 3I-SIM.
Fig. 2: Evaluation of super-resolution and optical sectioning imaging capability of 3I-SIM.
Fig. 3: 3I-SIM enables faster and gentler live organelle imaging.
Fig. 4: Observation of the interaction between LDs and ER network using 3I-SIM.
Fig. 5: DL improved 3I-SIM imaging capabilities in photon-limited conditions.
Fig. 6: Super-resolution visualization of ER-associated actin dynamics at millisecond scales.

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Data availability

The fluorescence data demonstrating the efficacy of the 3I-SIM analytical reconstruction algorithm are available via Figshare at https://figshare.com/articles/dataset/3I-SIM_dataset/26334118. The DL training dataset of the four typical organelles for 3I-SIM reconstruction is available via Zenodo at https://zenodo.org/records/14969641 (ref. 51). Other data are available from the corresponding authors upon reasonable request.

Code availability

The code of the analytical reconstruction algorithm, user-interactive reconstruction software, DL 3I-Net model and pretrained models on the four typical subcellular structures are available via GitHub at https://github.com/YiweiHou/3I-SIMReconstruction.

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Acknowledgements

P.X. acknowledges funding support from the National Key R&D Program of China (2022YFC3401100) and the National Natural Science Foundation of China (62025501, 31971376 and 92150301). M.L. acknowledges the National Natural Science Foundation of China (62335008 and 62405010). We thank the National Center for Protein Sciences at Peking University in Beijing, China, for assistance with Oxford Instruments Imaris software. We thank the Optical Imaging Facility at the Chinese Institute for Brain Research in Beijing, China, for assistance with ZEISS Elyra 7.

Author information

Author notes

  1. These authors contributed equally: Yunzhe Fu, Yiwei Hou.

Authors and Affiliations

  1. Department of Biomedical Engineering, National Biomedical Imaging Center, College of Future Technology, Peking University, Beijing, China

    Yunzhe Fu, Yiwei Hou, Qianxi Liang, Xin Chen, Boya Jin, Meiqi Li & Peng Xi

  2. Airy Technologies Co. Ltd, Beijing, China

    Wenyi Wang

  3. Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, China

    Jing Ling

  4. Peking-Tsinghua Center For Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Qiuchen Gu & Pengli Zheng

  5. School of Electrical and Electronic Engineering, Yonsei University, Seoul, Korea

    Donghyun Kim

  6. School of Life Sciences, Peking University, Beijing, China

    Pengli Zheng & Meiqi Li

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Contributions

P.X., M.L. and Y.F. conceived the 3I-SIM imaging system. P.X. and M.L. supervised this project. Y.F. designed the optical path and built the 3I-SIM system. Y.H. and X.C. developed the analytical 3I-SIM reconstruction framework and codes. Y.H. developed the DL reconstruction framework and codes. Y.F. performed all the imaging experiments on biological structures. Q.L. helped deduce the polarization engineering. J.L. helped in preparing the hippocampal neurons. Q.G. and P.Z. helped in preparing the AC–ER sample and aided relevant discussions. D.K. and B.J. helped with the data analysis. Y.F. and Y.H. composed all the figures and videos. Y.F., Y.H., M.L. and P.X. wrote the manuscript with input from all authors. All authors are involved in the discussions of the results.

Corresponding authors

Correspondence to Meiqi Li or Peng Xi.

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Competing interests

P.X., Y.F., Y.H., M.L. and Q.X. have filed a Chinese patent application (CN119310725A) on the presented framework. Also, P.X., Y.H., Y.F. and M.L. have filed a Chinese patent application (CN119477724A). The other authors declare no competing interests.

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Nature Photonics thanks Hui Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–21, Notes 1–4 and Tables 1–3.

Reporting Summary

Supplementary Video 1

Long-term imaging of actin filaments in a HUVEC cell for over an hour, continuously recording 5,900 time points at a temporal resolution of 1 Hz.

Supplementary Video 2

Continuous recording of the dynamic motion of the ER network at 14,000 time points with an ultrahigh spatiotemporal resolution of 1,697 Hz.

Supplementary Video 3

Role of ER during continuous movement and mutual contact with LEs or Lysos.

Supplementary Video 4

Dynamic interactions of microtubule and ER networks.

Supplementary Video 5

Visualizing the dynamic movement of LDs.

Supplementary Video 6

Long-term observation of hippocampal neurons.

Supplementary Video 7

Visualization of ER-associated actin dynamics at millisecond scales.

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Fu, Y., Hou, Y., Liang, Q. et al. Triangle-beam interference structured illumination microscopy. Nat. Photon. 19, 1122–1131 (2025). https://doi.org/10.1038/s41566-025-01730-0

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