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

Nature Methodsvolume 15pages10111019 (2018) | Download Citation

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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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.

Author information

Affiliations

  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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Competing interests

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.

Corresponding author

Correspondence to Yicong Wu.

Integrated supplementary information

  1. 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.

  2. 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.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1 and 2 and Supplementary Table 1

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https://doi.org/10.1038/s41592-018-0211-z