Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Doubling the resolution of a confocal spinning-disk microscope using image scanning microscopy


Fluorescence microscopy has become an indispensable tool for cell biology. Recently, super-resolution methods have been developed to overcome the diffraction limit of light and have shown living cells in unprecedented detail. Often, these methods come at a high cost and with complexity in terms of instrumentation and sample preparation, thus calling for the development of low-cost, more accessible methods. We previously developed image scanning microscopy (ISM), which uses structured illumination to double the resolution and quadruple the contrast of a confocal microscope. Implementing this technique into a confocal spinning-disk (CSD) microscope allows recording ISM images with up to ~1 frame per second, making it ideal for imaging dynamic biological processes. Here we present a step-by-step protocol describing how to convert any existing commercial CSD microscope into a CSD-ISM, with only moderate changes to the hardware and at low cost. Operation of the CSD-ISM is realized with a field programmable gate array using the software environment Micro-Manager, a popular open-source platform for microscopy. The provided software ( takes care of all algorithmic complexities and numerical workload of the CSD-ISM, including hardware synchronization and image reconstruction. The hardware modifications described here result in a theoretical maximum increase in resolution of √2 ≈ 1.41, which can be further improved by deconvolution to obtain a theoretical maximum twofold increase. An existing CSD setup can be upgraded in ~3 d by anyone with basic knowledge in optics, electronics and microscopy software.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Working principle of a CSD microscope.
Fig. 2: Software GUIs for the CSD-ISM system.
Fig. 3: Calibration image.
Fig. 4: ISM example images of beads and cells.

Data availability

The data presented in Fig. 3 (calibration images), Fig. 4 (ISM example images of beads and cells) and Supplementary Fig. 7 were generated for this protocol. The raw data files for Fig. 3 can be found at, and the raw data files for Fig. 4 can be found at

Code availability

The presented software for CSD-ISM acquisition and reconstruction, including demo data, is available at


  1. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    Article  CAS  Google Scholar 

  2. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. 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 

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

    Google Scholar 

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

    Article  Google Scholar 

  7. 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 

  8. Stuurman, N., Amdodaj, N. & Vale, R. μManager: open source software for light microscope imaging. Microsc. Today 15, 42–43 (2007).

    Article  Google Scholar 

  9. Edelstein, A. D., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using µManager. Curr. Protoc. Mol. Biol. 92, 14.20.1–14.20.17 (2010).

    Article  Google Scholar 

  10. Edelstein, A. D. et al. Advanced methods of microscope control using µManager software. J. Biol. Methods 1, e10 (2014).

    Article  Google Scholar 

  11. Dertinger, T., Colyer, R., Iyer, G., Weiss, S. & Enderlein, J. Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc. Natl Acad. Sci. USA 106, 22287–22292 (2009).

    Article  CAS  Google Scholar 

  12. Hayashi, S. Resolution doubling using confocal microscopy via analogy with structured illumination microscopy. Jpn. J. Appl. Phys. 55, 082501 (2016).

    Article  Google Scholar 

  13. Sheppard, C. J., Mehta, S. B. & Heintzmann, R. Superresolution by image scanning microscopy using pixel reassignment. Opt. Lett. 38, 2889–2892 (2013).

    Article  Google Scholar 

  14. Sheppard, C. J., Castello, M., Tortarolo, G., Vicidomini, G. & Diaspro, A. Image formation in image scanning microscopy, including the case of two-photon excitation. JOSA A 34, 1339–1350 (2017).

    Article  Google Scholar 

  15. Van den Eynde, R. et al. Quantitative comparison of camera technologies for cost-effective super-resolution optical fluctuation imaging (SOFI). J. Phys. Photonics 1, 044001 (2019).

    Article  Google Scholar 

  16. Toomre, D. & Pawley, J. B.. in Handbook of Biological Confocal Microscopy 3rd ed (ed Pawley, J.) 221–237 (Springer, 2006).

  17. Nakano, A. Spinning-disk confocal microscopy—a cutting-edge tool for imaging of membrane traffic. Cell Struct. Funct. 27, 349–355 (2002).

    Article  Google Scholar 

Download references


We thank E. Butkevich for preparation of the cell samples and A. Chizhik for the design of Fig. 1. We thank M. Schönekeß from our institute’s electronics workshop for providing Supplementary Fig. 4. S.Q. acknowledges funding from the European Research Council via its Horizon 2020 Framework Programme (675512, BE-OPTICAL). S.I. acknowledges financial support from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) via project A11 of SFB 937. J.E. acknowledges funding from DFG under Germany’s Excellence Strategy - EXC 2067/1-390729940.

Author information

Authors and Affiliations



J.E. conceived the project and designed the experiments. S.Q. developed the software. S.I. and I.G. performed experiments. S.Q. and S.I. analyzed the data. S.Q. and S.I. wrote the manuscript with the input of all other authors, J.E. revised and performed final edits to the manuscript.

Corresponding author

Correspondence to Jörg Enderlein.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Lucien Weiss and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Schulz, O. et al. Proc. Natl Aad. Sci. USA 110, 21000–21005 (2013):

Müller, C. B. & Enderlein, J. Phys. Rev. Lett. 104, 198101 (2010):

Gregor, I. et al. Nat. Methods 14, 1087–1089 (2017):

Supplementary information

Supplementary Information

Supplementary Note 1, Supplementary Figs. 1–7 and Supplementary Table 1.

Supplementary Video 1

Screen-captured video explaining the use of the Micro-Manager plugin.

Supplementary Video 2

Screen-captured video explaining the use of the reconstruction software.

Supplementary Video 3

This video illustrates the data acquisition and the ISM reconstruction of the bead image in Fig. 4a,b.

Supplementary Video 4

This video illustrates the data acquisition and the ISM reconstruction of the cell image in Fig. 4g,h.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Qin, S., Isbaner, S., Gregor, I. et al. Doubling the resolution of a confocal spinning-disk microscope using image scanning microscopy. Nat Protoc 16, 164–181 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


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.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing