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Selective-plane-activation structured illumination microscopy

Nature Methods (2024)Cite this article

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Abstract

The background light from out-of-focus planes hinders resolution enhancement in structured illumination microscopy when observing volumetric samples. Here we used selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. Theoretical investigation of the imaging properties and experimental demonstrations show that selective plane activation is beneficial for imaging dense microstructures in cells and cell spheroids.

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Fig. 1: Theoretical estimation of imaging properties.
Fig. 2: Imaging of single cells and a spheroid with single-photon and two-photon SPA-SIM.
Fig. 3: Evaluation of the practical spatial resolution in cell and spheroid imaging.

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Data availability

The data underlying the results presented in this paper are available at https://doi.org/10.5281/zenodo.10652642.

Code availability

The MATLAB-based SIM reconstruction code and homemade software written in C# are available from the authors upon request. The MATLAB codes for the theoretical calculation of imaging properties are available at https://doi.org/10.5281/zenodo.10652642.

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Acknowledgements

The authors thank S. Kawano (Osaka University) for help in developing the control system for the SPA-SIM setup; T. Yoshimori (Osaka University) for providing facilities and cells necessary for the establishment of the Skylan-NS–LC3B expressing MEF; and S. Yamaoka (Tokyo Medical and Dental University) and T. Kitamura (The University of Tokyo) for providing the pMRX-IRES-puro retroviral vector and the Plat-E retroviral packaging cells, respectively. This work was partially supported by Core Research for Evolutionary Science and Technology by Japan Science and Technology Agency (grant no. JPMJCR15N3 to T.N., JPMJCR1925 and JPMJPF2009 to K. Fujita). This work was also partially supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (grant no. 18H05410 to T.N.). Part of this work was performed under the Research Program of ‘Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials’ in ‘Network Joint Research Center for Materials and Devices’ to K. Fujita and T.N. R.H. acknowledges support by the Collaborative Research Center SFB 1278 (Poly Target, project C04) funded by the Deutsche Forschungsgemeinschaft and the Leibniz science campus Infecto-Optics, project HotAim 2.0.

Author information

Author notes

  1. These authors contributed equally: Kenta Temma, Ryosuke Oketani.

Authors and Affiliations

  1. Department of Applied Physics, Osaka University, Osaka, Japan

    Kenta Temma, Ryosuke Oketani, Toshiki Kubo, Kazuki Bando, Shunsuke Maeda & Katsumasa Fujita

  2. Advanced Photonics and Biosensing Open Innovation Laboratory, AIST-Osaka University, Osaka, Japan

    Kenta Temma & Katsumasa Fujita

  3. Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka, Japan

    Kenta Temma, Tatsuya Kaminishi, Koki Fukuda, Takeharu Nagai & Katsumasa Fujita

  4. Department of Chemistry, Kyushu University, Fukuoka, Japan

    Ryosuke Oketani

  5. SANKEN (The Institute of Scientific and Industrial Research), Osaka University, Osaka, Japan

    Kazunori Sugiura, Tomoki Matsuda & Takeharu Nagai

  6. Leibniz Institute of Photonic Technology, Jena, Germany

    Rainer Heintzmann

  7. Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University Jena, Jena, Germany

    Rainer Heintzmann

  8. Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan

    Tatsuya Kaminishi & Maho Hamasaki

  9. Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

    Koki Fukuda & Maho Hamasaki

  10. Research Institute for Electronic Science, Hokkaido University, Hokkaido, Japan

    Takeharu Nagai

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Contributions

K.T. and R.O. contributed equally to this study. K. Fujita supervised the project and established the SPA-SIM concept. R.O. built the numerical calculation programs, and K.T. modified the program and performed the calculations. R.O., K.T., K.B., T.Ku. and S.M. designed the optical setup. R.O. and K.T. constructed the setup. T.Ku. developed part of the control system. K.T. and R.O. performed the experiments. K.T. and T.Ku. prepared the single-cell and cell spheroid samples. K.S., T.M. and T.N. prepared reversibly photoswitchable fluorescent proteins for SPA-SIM measurements. K. Fukuda, T.Ka. and M.H. prepared MEF for autophagosome imaging. R.H. built the reconstruction program. K.T., R.O. and T.Ku. performed reconstruction. K.T., R.O. and K. Fujita wrote the paper. All of the authors reviewed the paper.

Corresponding author

Correspondence to Katsumasa Fujita.

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Competing interests

K.T., R.O. and K. Fujita filed a patent application (26 May 2023) for the proposed method. The other authors have no competing interests.

Peer review

Peer review information

Nature Methods thanks Yonghong Shao 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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Extended data

Extended Data Fig. 1 Comparison of imaging properties and spatial resolution.

A variety of illuminations and corresponding fluorescence excitation patterns, effective PSFs and simulated bead images in SPA-SIM. For comparison, Fig. 1(b), (c), (d), (e), and (f) are the same as (a), (b), (c), (d), and (g) in this figure, respectively. In addition, the imaging properties under high-NA light sheet activation, single-photon Bessel sheet activation and SPA-3DSIM are shown in (e), (f), (h), and (i). The images of excitation patterns, PSFs and beads are shown for aperture ratio (AR) 0.6. AR is effective NA used for structured illumination and related to its period, which is expressed as the ratio to the full NA. The definition of AR is depicted in Supplementary Fig. 14. The NAs for excitation and detection were assumed to be 1.1. The FWHMs of PSFs in lateral (x) and axial (z) directions are shown in the left column for AR 0.6 and 0.8.

Extended Data Fig. 2 Imaging conditions for acquired images in Fig.2 and Supplementary Figures.

AR (aperture ratio) is effective NA used for structured illumination, which is expressed as the ratio to the full NA.

Supplementary information

Supplementary Information

Supplementary Note 1, Supplementary Figs. 1–14.

Reporting Summary

Peer Review File

Supplementary Video 1

Time-lapse images of actin filament movement in a living HeLa cell labeled with Skylan-NS obtained with SPIM and SPA-SIM shown in Fig. 2g. The acquisition rate was 0.6 frame s−1 with an interval of 7 s to visualize slow actin motion.

Supplementary Video 2

Time-lapse images of mitochondria in a living HeLa cell labeled with Skylan-NS obtained using SPIM and SPA-SIM at 1 frame s−1 with an interval of 4 s. Several images at different time points and enlarged views are shown in Fig. 2h.

Supplementary Video 3

3D images of mitochondria in a living HeLa cell labeled with Skylan-NS obtained using 3DSIM and SPA-SIM. The images from a single viewpoint are shown in Fig. 2i,j. The imaging volumes were 71 × 71 × 25 µm3.

Supplementary Video 4

3D images of mitochondria in living HeLa cells labeled with Skylan-NS obtained using conventional SIM and SPA-SIM. The images from a single viewpoint are shown in Supplementary Fig. 8. The imaging volumes were 71 × 71 × 25 µm3.

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Temma, K., Oketani, R., Kubo, T. et al. Selective-plane-activation structured illumination microscopy. Nat Methods (2024). https://doi.org/10.1038/s41592-024-02236-3

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