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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
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.
References
Wilson, T. & Sheppard, C. J. R. Theory and Practice of Scanning Optical Microscopy (Academic Press, 1984).
Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).
Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E. H. K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).
Fujita, K., Kobayashi, M., Kawano, S., Yamanaka, M. & Kawata, S. High-resolution confocal microscopy by saturated excitation of fluorescence. Phys. Rev. Lett. 99, 228105 (2007).
Marriott, G. et al. Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells. Proc. Natl Acad. Sci. USA 105, 17789–17794 (2008).
Vettenburg, T., Corral, A., Rodríguez-Pulido, A., Flors, C. & Ripoll, J. Photoswitching-enabled contrast enhancement in light sheet fluorescence microscopy. ACS Photonics 4, 424–428 (2017).
Kubo, T. et al. Visible-wavelength multiphoton activation confocal microscopy. ACS Photonics 8, 2666–2673 (2021).
Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).
Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).
Heintzmann, R. & Cremer, C. G. Laterally modulated excitation microscopy; improvement of resolution by using a diffraction grating. Opt. Biopsies Microsc. Tech. III 3568, 185–196 (1999).
Kim, J., Koo, B. K. & Knoblich, J. A. Human organoids: model systems for human biology and medicine. Nat. Rev. Mol. Cell Biol. 21, 571–584 (2020).
Friedrich, J., Seidel, C., Ebner, R. & Kunz-Schughart, L. A. Spheroid-based drug screen: considerations and practical approach. Nat. Protoc. 4, 309–324 (2009).
Cella Zanacchi, F. et al. Live-cell 3D super-resolution imaging in thick biological samples. Nat. Methods 8, 1047–1050 (2011).
Bon, P. et al. Self-interference 3D super-resolution microscopy for deep tissue investigations. Nat. Methods 15, 449–454 (2018).
Chen, B. C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).
Chen, B. et al. Resolution doubling in light-sheet microscopy via oblique plane structured illumination. Nat. Methods 19, 1419–1426 (2022).
Zhang, X. et al. Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy. Proc. Natl Acad. Sci. USA 113, 10364–10369 (2016).
Gustafsson, M. G. L. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008).
Gao, L., Shao, L., Chen, B. C. & Betzig, E. 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy. Nat. Protoc. 9, 1083–1101 (2014).
Shinoda, H. et al. Acid-tolerant reversibly switchable green fluorescent protein for super-resolution imaging under acidic conditions. Cell Chem. Biol. 26, 1469–1479 (2019).
Descloux, A., Grußmayer, K. S. & Radenovic, A. Parameter-free image resolution estimation based on decorrelation analysis. Nat. Methods 16, 918–924 (2019).
Velasco, M. G. M. et al. 3D super-resolution deep-tissue imaging in living mice. Optica 8, 442–450 (2021).
Lu, C. H. et al. Lightsheet localization microscopy enables fast, large-scale, and three-dimensional super-resolution imaging. Commun. Biol. 2, 177 (2019).
Mudry, E. et al. Structured illumination microscopy using unknown speckle patterns. Nat. Photonics 6, 312–315 (2012).
Débarre, D., Botcherby, E. J., Booth, M. J. & Wilson, T. Adaptive optics for structured illumination microscopy. Opt. Express 16, 9290–9305 (2008).
Žurauskas, M. et al. IsoSense: frequency enhanced sensorless adaptive optics through structured illumination. Optica 6, 370–379 (2019).
Lin, R., Kipreos, E. T., Zhu, J., Khang, C. H. & Kner, P. Subcellular three-dimensional imaging deep through multicellular thick samples by structured illumination microscopy and adaptive optics. Nat. Commun. 12, 3148 (2021).
Fahrbach, F. O., Gurchenkov, V., Alessandri, K., Nassoy, P. & Rohrbach, A. Self-reconstructing sectioned Bessel beams offer submicron optical sectioning for large fields of view in light-sheet microscopy. Opt. Express 21, 11425–11440 (2013).
Yamanaka, M. et al. Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging. J. Biomed. Opt. 20, 101202 (2015).
Müller, M., Mönkemöller, V., Hennig, S., Hübner, W. & Huser, T. Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ. Nat. Commun. 7, 10980 (2016).
Wicker, K., Mandula, O., Best, G., Fiolka, R. & Heintzmann, R. Phase optimisation for structured illumination microscopy. Opt. Express 21, 2032–2049 (2013).
Heintzmann, R. et al. Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes. Micron 34, 293–300 (2003).
Shinoda, H. et al. Acid-tolerant monomeric GFP from Olindias formosa. Cell Chem. Biol. 25, 330–338 (2018).
Fu, C., Donovan, W. P., Shikapwashya-Hasser, O., Ye, X. & Cole, R. H. Hot fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase. PLoS One 9, e115318 (2014).
Fujita, N. et al. An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol. Biol. Cell 19, 4651–4659 (2008).
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
Authors and Affiliations
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
Ethics declarations
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.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Temma, K., Oketani, R., Kubo, T. et al. Selective-plane-activation structured illumination microscopy. Nat Methods 21, 889–896 (2024). https://doi.org/10.1038/s41592-024-02236-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41592-024-02236-3