Abstract
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 (https://projects.gwdg.de/projects/csdism-2020) 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.
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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 https://doi.org/10.25625/WTE3OI, and the raw data files for Fig. 4 can be found at https://doi.org/10.25625/QVWCNT.
Code availability
The presented software for CSD-ISM acquisition and reconstruction, including demo data, is available at https://projects.gwdg.de/projects/csdism-2020.
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Acknowledgements
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.
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Contributions
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.
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The authors declare no competing interests.
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Peer review information Nature Protocols thanks Lucien Weiss and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Related links
Key references using this protocol
Schulz, O. et al. Proc. Natl Aad. Sci. USA 110, 21000–21005 (2013): https://doi.org/10.1073/pnas.1315858110
Müller, C. B. & Enderlein, J. Phys. Rev. Lett. 104, 198101 (2010): https://doi.org/10.1103/PhysRevLett.104.198101
Gregor, I. et al. Nat. Methods 14, 1087–1089 (2017): https://doi.org/10.1038/nmeth.4467
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.
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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). https://doi.org/10.1038/s41596-020-00408-x
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DOI: https://doi.org/10.1038/s41596-020-00408-x
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