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Build and operation of a custom 3D, multicolor, single-molecule localization microscope

Nature Protocols (2024)Cite this article

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Abstract

Single-molecule localization microscopy (SMLM) enables imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations of SMLM are capable of three-dimensional, multicolor and high-throughput imaging and can yield key biological insights. However, widespread access to these technologies is limited, primarily by the cost of commercial options and complexity of de novo development of custom systems. Here we provide a comprehensive guide for interested researchers who wish to establish a high-end, custom-built SMLM setup in their laboratories. We detail the initial configuration and subsequent assembly of the SMLM, including the instructions for the alignment of all the optical pathways, the software and hardware integration, and the operation of the instrument. We describe the validation steps, including the preparation and imaging of test and biological samples with structures of well-defined geometries, and assist the user in troubleshooting and benchmarking the system’s performance. Additionally, we provide a walkthrough of the reconstruction of a super-resolved dataset from acquired raw images using the Super-resolution Microscopy Analysis Platform. Depending on the instrument configuration, the cost of the components is in the range US$95,000–180,000, similar to other open-source advanced SMLMs, and substantially lower than the cost of a commercial instrument. A builder with some experience of optical systems is expected to require 4–8 months from the start of the system construction to attain high-quality three-dimensional and multicolor biological images.

Key points

  • The authors describe the complete configuration and assembly of a custom-built single-molecule localization microscope, including optical alignment, software and hardware integration, validation steps and benchmarking of the system’s performance.

  • The microscope is optimized for ultra-stable three-dimensional imaging performance and multichannel functionalities, while remaining substantially less expensive than similar commercial systems.

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Fig. 1: A full rendering of the EMBL-SMLM.
Fig. 2: Schematic of the optical and motion control aspects of the EMBL–SMLM.
Fig. 3: The µManager and htSMLM GUIs.
Fig. 4: The fully assembled inverted microscope body.
Fig. 5: The fully assembled multicolor, 3D imaging capable emission path.
Fig. 6: The fully assembled illumination paths showing the different optional modules.
Fig. 7: The fully assembled focus lock path.
Fig. 8: 3D calibration of the microscope based on fluorescent bead z-stacks.
Fig. 9: 2D imaging of the NPC.
Fig. 10: 3D imaging of the NPC.

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Data availability

All CAD parts and assemblies, mechanical drawings and electronic board files are available from the project repository: https://github.com/ries-lab/3DSMLM. All materials provided in the repository are licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (CC-BY-NC-ND-4.0). The sequences of raw images that were localized and rendered to produce Figs. 9 and 10 can be provided on request.

Code availability

The user interface, FPGA firmware and modified µManager device adapters are available from the project repository (https://github.com/ries-lab/3DSMLM). All materials provided in the repository are licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (CC-BY-NC-ND-4.0). The analysis software, SMAP which is used used throughout the Protocol, is available at https://github.com/jries/SMAP.

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Acknowledgements

We thank C. Kieser (EMBL Electronic Workshop) for help with construction of and documentation for the microFPGA. We thank A. Milberger (EMBL Mechanical Workshop) for providing all mechanical drawings. We thank J. Deschamps (Human Technopole, Milan, Italy) for providing the EMU htSMLM user interface and continued support in various aspects of microscope control. We thank A. Roy for assistance in testing the protocol. This work was supported by the European Research Council (CoG-724489) and the European Molecular Biology Laboratory. We acknowledge the access and services provided by the Imaging Centre at the European Molecular Biology Laboratory, generously supported by the Boehringer Ingelheim Foundation.

Author information

Authors and Affiliations

  1. EMBL Imaging Centre, EMBL Heidelberg, Heidelberg, Germany

    Rory M. Power & Timo Zimmermann

  2. Cell Biology and Biophysics Unit, EMBL Heidelberg, Heidelberg, Germany

    Aline Tschanz, Timo Zimmermann & Jonas Ries

  3. Max Perutz Labs, Vienna Biocenter Campus, Vienna, Austria

    Jonas Ries

  4. University of Vienna, Center for Molecular Biology, Department of Structural and Computational Biology, Vienna, Austria

    Jonas Ries

  5. University of Vienna, Faculty of Physics, Vienna, Austria

    Jonas Ries

Authors
  1. Rory M. Power
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  4. Jonas Ries
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Contributions

R.M.P. and J.R. designed and developed the microscope hardware. A.T. and J.R. performed sample preparation. R.M.P., A.T. and J.R. performed imaging and data analysis. T.Z. and J.R. provided project supervision. All authors wrote the manuscript and designed the protocol.

Corresponding authors

Correspondence to Rory M. Power or Jonas Ries.

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Nature Protocols thanks Carlas Smith and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Li, Y. et al. Nat. Methods 15, 367–369 (2018): https://doi.org/10.1038/nmeth.4661

Diekmann, R. et al. Nat. Methods 17, 909–912 (2020): https://doi.org/10.1038/s41592-020-0918-5

Deschamps, J. et al. HardwareX 13, e00407 (2023): https://doi.org/10.1016/j.ohx.2023.e00407

Thevathasan, J. V. et al. Nat. Methods 16, 1045–1053 (2019): https://doi.org/10.1038/s41592-019-0574-9

Wu, Y.-L. et al. Nat. Methods 20, 139–148 (2023): https://doi.org/10.1038/s41592-022-01676-z

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Supplementary Notes 1–26, Protocols 1–8, Troubleshooting, Figs. 1–67 and References.

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Power, R.M., Tschanz, A., Zimmermann, T. et al. Build and operation of a custom 3D, multicolor, single-molecule localization microscope. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00989-x

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