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  • Perspective
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Faster, sharper, and deeper: structured illumination microscopy for biological imaging

Nature Methods volume 15, pages 1011–1019 (2018)Cite this article

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An Author Correction to this article was published on 29 December 2018

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Abstract

Structured illumination microscopy (SIM) allows rapid, super-resolution (SR) imaging in live specimens. We review recent technical advances in SR-SIM, with emphasis on imaging speed, resolution, and depth. Since its introduction decades ago, the technique has grown to offer myriad implementations, each with its own strengths and weaknesses. We discuss these, aiming to provide a practical guide for biologists and to highlight which approach is best suited to a given application.

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Fig. 1: Principles underlying traditional and spot-scanning SR-SIM, and comparison of their optical transfer functions.
Fig. 2: Planar dynamics of cellular organelles and protein distributions imaged with traditional high-NA TIRF-SIM, instant TIRF-SIM, traditional GI-SIM, and 2D Hessian-SIM.
Fig. 3: 3D biology revealed with traditional 3D SR-SIM, CSD-ISM, and ISIM.
Fig. 4: Alternative SR-SIM implementations for better penetration depth and higher spatial resolution.
Fig. 5: Comparison of SR-SIM implementations.

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Change history

  • 29 December 2018

    In the version of this Perspective originally published, Fig. 4g included an incorrect inset adapted from a different figure than the main image in the panel. This error has been corrected in the PDF and HTML versions of the paper.

References

  1. Sahl, S. J., Hell, S. W. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat. Rev. Mol. Cell Biol. 18, 685–701 (2017).

    Article  CAS  Google Scholar 

  2. Strtohl, F. & Kaminski, C. F. Frontiers in structured illumination microscopy. Optica 3, 667–677 (2016).

    Article  Google Scholar 

  3. Heintzmann, R. & Huser, T. Super-resolution structured illumination microscopy. Chem. Rev. 117, 13890–13908 (2017).

    Article  CAS  Google Scholar 

  4. Li, D. et al. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, aab3500 (2015).

    Article  Google Scholar 

  5. Neil, M. A. A., Juskaitis, R. & Wilson, T. Method of obtaining optical sectioning by using structured light in a conventional microscope. Opt. Lett. 22, 1905–1907 (1997).

    Article  CAS  Google Scholar 

  6. Wicker, K. & Heintzmann, R. Resolving a misconception about structured illumination. Nat. Photonics 8, 342–344 (2014).

    Article  CAS  Google Scholar 

  7. Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    Article  CAS  Google Scholar 

  8. Heintzmann, R. & Cremer, C. Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. Proc. SPIE 3568, 185–196 (1999).

    Article  Google Scholar 

  9. Heintzmann, R. & Gustafsson, M. G. L. Subdiffraction resolution in continuous samples. Nat. Photonics 3, 362–364 (2009).

    Article  CAS  Google Scholar 

  10. Gustafsson, M. G. L., Agard, D. A. & Sedat, J. W. Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination. Proc. SPIE 3919, 141–150 (2000).

    Article  Google Scholar 

  11. Demmerle, J. et al. Strategic and practical guidelines for successful structured illumination microscopy. Nat. Protoc. 12, 988–1010 (2017).

    Article  CAS  Google Scholar 

  12. Müller, C. B. & Enderlein, J. Image scanning microscopy. Phys. Rev. Lett. 104, 198101 (2010).

    Article  Google Scholar 

  13. York, A. G. et al. Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy. Nat. Methods 9, 749–754 (2012).

    Article  CAS  Google Scholar 

  14. Sheppard, C. J. R. Super-resolution in confocal imaging. Optik (Stuttg.) 80, 53–54 (1988).

    Google Scholar 

  15. Huff, J. The Airyscan detector from ZEISS: confocal imaging with improved signal-to-noise ratio and super-resolution. Nat. Methods 12, 1205 (2015).

    Article  Google Scholar 

  16. Schulz, O. et al. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy. Proc. Natl. Acad. Sci. USA 110, 21000–21005 (2013).

    Article  CAS  Google Scholar 

  17. Winter, P. W. & Shroff, H. Faster fluorescence microscopy: advances in high speed biological imaging. Curr. Opin. Chem. Biol. 20, 46–53 (2014).

    Article  CAS  Google Scholar 

  18. Roth, S., Sheppard, C. J. R., Wicker, K. & Heintzmann, R. Optical photon reassignment microscopy (OPRA). Opt. Nanoscopy 2, 5 (2013).

    Article  Google Scholar 

  19. De Luca, G. M. R. et al. Re-scan confocal microscopy: scanning twice for better resolution. Biomed. Opt. Express 4, 2644–2656 (2013).

    Article  Google Scholar 

  20. Azuma, T. & Kei, T. Super-resolution spinning-disk confocal microscopy using optical photon reassignment. Opt. Express 23, 15003–15011 (2015).

    Article  CAS  Google Scholar 

  21. Shroff, H. & York, A. Multi-focal structured illumination microscopy systems and methods. US patent 9696534 (2017).

  22. York, A. G. et al. Instant super-resolution imaging in live cells and embryos via analog image processing. Nat. Methods 10, 1122–1126 (2013).

    Article  CAS  Google Scholar 

  23. Dan, D. et al. DMD-based LED-illumination super-resolution and optical sectioning microscopy. Sci. Rep. 3, 1116 (2013).

    Article  Google Scholar 

  24. Cheng, L. C. et al. Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device. Biomed. Opt. Express 5, 2526–2536 (2014).

    Article  Google Scholar 

  25. Yeh, L. H., Tian, L. & Waller, L. Structured illumination microscopy with unknown patterns and a statistical prior. Biomed. Opt. Express 8, 695–711 (2017).

    Article  Google Scholar 

  26. Gustafsson, M. G. L. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008).

    Article  CAS  Google Scholar 

  27. Sahl, S. J. et al. Comment on “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 352, 527 (2016).

    Article  CAS  Google Scholar 

  28. Li, D. & Betzig, E. Response to Comment on “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics”. Science 352, 527 (2016).

    CAS  PubMed  Google Scholar 

  29. Ball, G. et al. SIMcheck: a toolbox for successful super-resolution structured illumination microscopy. Sci. Rep. 5, 15915 (2015).

    Article  CAS  Google Scholar 

  30. Zheng, W. et al. Adaptive optics improves multiphoton super-resolution imaging. Nat. Methods 14, 869–872 (2017).

    Article  CAS  Google Scholar 

  31. Guo, M. et al. Single-shot super-resolution total internal reflection fluorescence microscopy. Nat. Methods 15, 425–428 (2018).

    Article  CAS  Google Scholar 

  32. Visitech International Ltd. Scanning device, system and method. UK patent application GB1806845.2 (2018).

  33. Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L. & Gustafsson, M. G. L. Super-resolution video microscopy of live cells by structured illumination. Nat. Methods 6, 339–342 (2009).

    Article  CAS  Google Scholar 

  34. Brunstein, M., Wicker, K., Hérault, K., Heintzmann, R. & Oheim, M. Full-field dual-color 100-nm super-resolution imaging reveals organization and dynamics of mitochondrial and ER networks. Opt. Express 21, 26162–26173 (2013).

    Article  Google Scholar 

  35. Fiolka, R., Beck, M. & Stemmer, A. Structured illumination in total internal reflection fluorescence microscopy using a spatial light modulator. Opt. Lett. 33, 1629–1631 (2008).

    Article  Google Scholar 

  36. Förster, R. et al. Simple structured illumination microscope setup with high acquisition speed by using a spatial light modulator. Opt. Express 22, 20663–20677 (2014).

    Article  Google Scholar 

  37. Sochacki, K. A., Dickey, A. M., Strub, M. P. & Taraska, J. W. Endocytic proteins are partitioned at the edge of the clathrin lattice in mammalian cells. Nat. Cell Biol. 19, 352–361 (2017).

    Article  CAS  Google Scholar 

  38. Nixon-Abell, J. et al. Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER. Science 354, aaf3928 (2016).

    Article  Google Scholar 

  39. Huang, X. et al. Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy. Nat. Biotechnol. 36, 451–459 (2018).

    Article  CAS  Google Scholar 

  40. Shao, L., Kner, P., Rego, E. H. & Gustafsson, M. G. Super-resolution 3D microscopy of live whole cells using structured illumination. Nat. Methods 8, 1044–1046 (2011).

    Article  CAS  Google Scholar 

  41. Fiolka, R., Shao, L., Rego, E. H., Davidson, M. W. & Gustafsson, M. G. L. Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination. Proc. Natl. Acad. Sci. USA 109, 5311–5315 (2012).

    Article  CAS  Google Scholar 

  42. Lesterlin, C., Ball, G., Schermelleh, L. & Sherratt, D. J. RecA bundles mediate homology pairing between distant sisters during DNA break repair. Nature 506, 249–253 (2014).

    Article  CAS  Google Scholar 

  43. Ingaramo, M. et al. Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue. Proc. Natl. Acad. Sci. USA 111, 5254–5259 (2014).

    Article  CAS  Google Scholar 

  44. Winter, P. W. et al. Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples. Optica 1, 181–191 (2014).

    Article  Google Scholar 

  45. Gregor, I. et al. Rapid nonlinear image scanning microscopy. Nat. Methods 14, 1087–1089 (2017).

    Article  CAS  Google Scholar 

  46. Wang, K. et al. Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue. Nat. Commun. 6, 7276 (2015).

    Article  CAS  Google Scholar 

  47. Booth, M. J. Wavefront sensorless adaptive optics for large aberrations. Opt. Lett. 32, 5–7 (2007).

    Article  Google Scholar 

  48. Thomas, B., Wolstenholme, A., Chaudhari, S. N., Kipreos, E. T. & Kner, P. Enhanced resolution through thick tissue with structured illumination and adaptive optics. J. Biomed. Opt. 20, 26006 (2015).

    Article  Google Scholar 

  49. Power, R. M. & Huisken, J. A guide to light-sheet fluorescence microscopy for multiscale imaging. Nat. Methods 14, 360–373 (2017).

    Article  CAS  Google Scholar 

  50. Breuninger, T., Greger, K. & Stelzer, E. H. K. Lateral modulation boosts image quality in single plane illumination fluorescence microscopy. Opt. Lett. 32, 1938–1940 (2007).

    Article  Google Scholar 

  51. Keller, P. J. et al. Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat. Methods 7, 637–642 (2010).

    Article  CAS  Google Scholar 

  52. Planchon, T. A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).

    Article  CAS  Google Scholar 

  53. Gao, L. et al. Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens. Cell 151, 1370–1385 (2012).

    Article  CAS  Google Scholar 

  54. Chen, B. C. et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).

    Article  Google Scholar 

  55. Chang, B. J., Perez Meza, V. D. & Stelzer, E. H. K. csiLSFM combines light-sheet fluorescence microscopy and coherent structured illumination for a lateral resolution below 100 nm. Proc. Natl. Acad. Sci. USA 114, 4869–4874 (2017).

  56. Gustafsson, M. G. L. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. USA 102, 13081–13086 (2005).

  57. Heintzmann, R., Jovin, T. M. & Cremer, C. Saturated patterned excitation microscopy—a concept for optical resolution improvement. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 19, 1599–1609 (2002).

    Article  Google Scholar 

  58. Rego, E. H. et al. Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution. Proc. Natl. Acad. Sci. USA 109, E135–E143 (2012).

  59. 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).

    Article  CAS  Google Scholar 

  60. Curd, A. et al. Construction of an instant structured illumination microscope. Methods 88, 37–47 (2015).

    Article  CAS  Google Scholar 

  61. Young, L. J., Ströhl, F. & Kaminski, C. F. A guide to structured illumination TIRF microscopy at high speed with multiple colors. J. Vis. Exp. https://doi.org/10.3791/53988 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Laissue, P. P., Alghamdi, R. A., Tomancak, P., Reynaud, E. G. & Shroff, H. Assessing phototoxicity in live fluorescence imaging. Nat. Methods 14, 657–661 (2017).

    Article  CAS  Google Scholar 

  63. Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015).

    Article  CAS  Google Scholar 

  64. Weigert, M. et al. Content-aware image restoration: pushing the limits of fluorescence microscopy. bioRxiv Preprint at https://www.biorxiv.org/content/early/2018/07/03/236463 (2018).

  65. Ouyang, W., Aristov, A., Lelek, M., Hao, X. & Zimmer, C. Deep learning massively accelerates super-resolution localization microscopy. Nat. Biotechnol. 36, 460–468 (2018).

    Article  CAS  Google Scholar 

  66. Christiansen, E. M. et al. In silico labeling: predicting fluorescent labels in unlabeled images. Cell 173, 792–803 (2018).

    Article  CAS  Google Scholar 

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Acknowledgements

Support for this work was provided by the Intramural Research Programs of the National Institute of Biomedical Imaging and Bioengineering. We thank J. Giannini, W. Zheng, T. Lambert, A. North, S. Coleman, D. Li, Y. Su, R. Christensen, C. Smith, P. La Riviere, G. Patterson, and H. Eden for useful discussion and feedback on the manuscript. We also thank P. Shah and Z. Bao for performing the instant SIM imaging presented in Fig. 3h. Disclaimer: The NIH and its staff do not recommend or endorse any company, product, or service.

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  1. Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, USA

    Yicong Wu & Hari Shroff

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Correspondence to Yicong Wu.

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H.S. is co-inventor on US patent 9,696,534, owned by NIH and licensed to VisiTech International and Yokogawa Electric Corporation, describing multifocal and analog implementations of SR-SIM. He and his laboratory receive a share of royalties.

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Supplementary Figure 1 Real-space explanation of wide-field microscopy and SR-SIM.

a, Wide-field. b, SR-SIM. The sample is represented by two white dots spaced within the diffraction limit, the illumination by the red color (left column), and horizontal line profiles through the sample with yellow curves. In wide-field illumination, multiplying the sample with unvarying illumination (middle column) and blurring with emission point spread function (right column) fails to resolve the two dots, even after deconvolution. In contrast, when using sharp, phase-shifted illumination patterns, fluorescence from each point can be better isolated, implying that additional information about the sample can be retrieved (middle, right columns). Combining the three images appropriately resolves the two points.

Supplementary Figure 2 Conceptual and illumination schemes in spot-scanning SR-SIM.

a, Two equivalent schemes for achieving super-resolution in spot-scanning SR-SIM. Top: Four excitation foci are shown with inter‐focus distance x and diameter d. Bottom-left: foci are shrunk without altering the distance between them (e.g., ISM, MSIM, ISIM12,13,22). Bottom-right: the inter‐foci distance is extended to 2x, while leaving the size of the foci unchanged (e.g., OPRA, RCM, 2P-ISIM18,19,45). Either method produces an equivalent result, as the only difference between the output images is a global scaling factor. Image reproduced from ref. 45 with permission. b, Schematic of various spot-scanning SR-SIM techniques. In ISM, a detector array is used to record the entire shape of the fluorescence spot at each scan position. In MSIM, parallelizing acquisition by using multiple foci instead of a single focus dramatically boosts the imaging speed. In OPRA/RCM, the fluorescence reassignment is performed optically instead of digitally to produce a super-resolved image directly on the camera. In ISIM, the use of multifocal excitation patterns in combination with optical processing offers video-rate super-resolution imaging. Image reproduced from ref. 2 with permission.

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Wu, Y., Shroff, H. Faster, sharper, and deeper: structured illumination microscopy for biological imaging. Nat Methods 15, 1011–1019 (2018). https://doi.org/10.1038/s41592-018-0211-z

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