Abstract

Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1–2 d.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

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

  2. 2.

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

  3. 3.

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

  4. 4.

    et al. Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336 (2008).

  5. 5.

    et al. Comment on 'Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics'. Science 352, 527–527 (2016).

  6. 6.

    & Response to comment on 'Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics'. Science 352, 527–527 (2016).

  7. 7.

    & in Fluorescence Microscopy 213–225 (Elsevier, 2014).

  8. 8.

    , & Structured illumination microscopy for superresolution. Chem. Phys. Chem. 15, 566–576 (2014).

  9. 9.

    & Practical structured illumination microscopy. Methods Mol. Biol. 1251, 175–192 (2015).

  10. 10.

    in Quantitative Imaging in Cell Biology 123, 295–313 (Elsevier, 2014).

  11. 11.

    et al. Superresolution live imaging of plant cells using structured illumination microscopy. Nat. Protoc. 10, 1248–1263 (2015).

  12. 12.

    in Quantitative Imaging in Cell Biology 123, 315–333 (Elsevier, 2014).

  13. 13.

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

  14. 14.

    Image processing for structured illumination microscopy. in 1–3 (IEEE, 2015).

  15. 15.

    et al. Quantitative 3D structured illumination microscopy of nuclear structures. Nat. Protoc. .

  16. 16.

    , & A guide to structured illumination TIRF microscopy at high speed with multiple colors. J. Vis. Exp. (2016).

  17. 17.

    et al. Super-resolution imaging of the cytokinetic Z ring in live bacteria using fast 3D-structured illumination microscopy (f3D-SIM). J. Vis. Exp. e51469–e51469 (2014).

  18. 18.

    , , , & SIMToolbox: a MATLAB toolbox for structured illumination fluorescence microscopy. Bioinformatics 32, 318–320 (2015).

  19. 19.

    , , , & Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ. Nat. Commun. 7, 10980 (2016).

  20. 20.

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

  21. 21.

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

  22. 22.

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

  23. 23.

    & in Far-Field Optical Nanoscopy 3–26 (Springer, 2014).

  24. 24.

    , & STED microscopy and its applications: new insights into cellular processes on the nanoscale. Chem. Phys. Chem. 13, 1986–2000 (2012).

  25. 25.

    , , & Super-resolution 3D microscopy of live whole cells using structured illumination. Nat. Methods 8, 1044–1046 (2011).

  26. 26.

    et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

  27. 27.

    , & Imaging live-cell dynamics and structure at the single-molecule level. Mol. Cell 58, 644–659 (2015).

  28. 28.

    et al. Imaging cellular structures in super-resolution with SIM, STED and localisation microscopy: a practical comparison. Sci. Rep. 6, 27290 (2016).

  29. 29.

    , , , & Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination. Proc. Natl. Acad. Sci. USA 109, 5311 (2012).

  30. 30.

    et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3, 887–889 (2006).

  31. 31.

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

  32. 32.

    , , , & Simple buffers for 3D STORM microscopy. Biomed. Opt. Express 4, 885–899 (2013).

  33. 33.

    , & Does super-resolution fluorescence microscopy obsolete previous microscopic approaches to protein co-localization? Methods Mol. Biol. 1270, 255–275 (2015).

  34. 34.

    et al. Spatial separation of Xist RNA and polycomb proteins revealed by superresolution microscopy. Proc. Natl. Acad. Sci. USA 111, 2235–2240 (2014).

  35. 35.

    et al. DNA origami-based standards for quantitative fluorescence microscopy. Nat. Protoc. 9, 1367–1391 (2014).

  36. 36.

    et al. The evolution of structured illumination microscopy in studies of HIV. Methods 88, 20–27 (2015).

  37. 37.

    , & Structured illumination microscopy: artefact analysis and reduction utilizing a parameter optimization approach. J. Microsc. 216, 165–174 (2004).

  38. 38.

    , & OTF compensation in structured illumination superresolution images. Proc. SPIE 7094 (2008).

  39. 39.

    , , & Adaptive optics for structured illumination microscopy. Opt. Express 16, 9290–9305 (2008).

  40. 40.

    et al. Image filtering in structured illumination microscopy using the Lukosz bound. Opt. Express 21, 24431 (2013).

  41. 41.

    , , , & Phase optimisation for structured illumination microscopy. Opt. Express 21, 2032–2049 (2013).

  42. 42.

    et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  43. 43.

    , & NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

  44. 44.

    & Optimized approaches for optical sectioning and resolution enhancement in 2D structured illumination microscopy. Biomed. Optics Exp. 5, 2580–2590 (2014).

  45. 45.

    , , & Assessing resolution in super-resolution imaging. Methods 88, 3–10 (2015).

  46. 46.

    et al. OMERO: flexible, model-driven data management for experimental biology. Nat. Methods 9, 245–253 (2012).

  47. 47.

    et al. Measuring image resolution in optical nanoscopy. Nat. Methods 10, 557–562 (2013).

Download references

Acknowledgements

We acknowledge J. Neumann and M. Grange for technical support, and L. Ferrand of GE Healthcare for helpful discussion. The 2014 OMX User Meeting made notable contributions to the publication recommendations. We thank all colleagues who contributed to many discussions to shape this protocol. We are further indebted to H. Leonhard and I. Davis for their long-standing support. This work was funded by the Wellcome Trust Strategic Awards 091911 and 107457 supporting advanced microscopy at Micron Oxford. The OMX 3D-SIM system in the Rockefeller University Bio-Imaging Resource Center was funded by award no. S10RR031855 from the National Center for Research Resources. J.D. is supported by the NIH-Oxford-Cambridge Scholars Program. G.B. is supported by an MRC Next Generation Optical Microscopy Award (MR/K015869/1). A.M. is supported by JSPS KAKENHI grant nos. JP16H01440 (“resonance bio”), JP15K14500 and JP26292169.

Author information

Author notes

    • Graeme Ball

    Present address: Dundee Imaging Facility, School of Life Sciences, University of Dundee, Dundee, UK.

    • Justin Demmerle
    •  & Cassandravictoria Innocent

    These authors contributed equally to this work.

Affiliations

  1. Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, Oxford, UK.

    • Justin Demmerle
    • , Cassandravictoria Innocent
    • , Graeme Ball
    • , Ezequiel Miron
    • , Ian M Dobbie
    •  & Lothar Schermelleh
  2. Bio-Imaging Resource Center, The Rockefeller University, New York, New York, USA.

    • Alison J North
  3. Biomolecular Photonics Group, Faculty of Physics, Bielefeld University, Bielefeld, Germany.

    • Marcel Müller
  4. Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, Kobe, Japan.

    • Atsushi Matsuda
  5. Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

    • Atsushi Matsuda
  6. Department of Biological Chemistry, David Geffen School of Medicine, UCLA, Los Angeles, California, USA.

    • Yolanda Markaki

Authors

  1. Search for Justin Demmerle in:

  2. Search for Cassandravictoria Innocent in:

  3. Search for Alison J North in:

  4. Search for Graeme Ball in:

  5. Search for Marcel Müller in:

  6. Search for Ezequiel Miron in:

  7. Search for Atsushi Matsuda in:

  8. Search for Ian M Dobbie in:

  9. Search for Yolanda Markaki in:

  10. Search for Lothar Schermelleh in:

Contributions

C.I., J.D., A.J.N., M.M., E.M., Y.M. and L.S. collected data and created figures. A.M., G.B., M.M. and I.M.D. provided technical expertise and advice. All authors contributed to artifact documentation. J.D., C.I., A.J.N. and L.S. wrote the manuscript. A.J.N., Y.M. and L.S. conceived the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Lothar Schermelleh.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Figures and Table

    Supplementary Manual, Supplementary Table 1, Supplementary Method, and Supplementary Figures 1–10.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2017.019

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.