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Implementation of a 4Pi-SMS super-resolution microscope


The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available numerical aperture, and coupling this with interferometric detection has demonstrated 3D resolution down to 10 nm over entire cellular volumes. The aim of this protocol is to enable interested researchers to establish 4Pi-SMS super-resolution microscopy in their laboratories. We describe in detail how to assemble the optomechanical components of a 4Pi-SMS instrument, align its optical beampath and test its performance. The protocol further provides instructions on how to prepare test samples of fluorescent beads, operate this instrument to acquire images of whole cells and analyze the raw image data to reconstruct super-resolution 3D data sets. Furthermore, we provide a troubleshooting guide and present examples of anticipated results. An experienced optical instrument builder will require ~12 months from the start of ordering hardware components to acquiring high-quality biological images.

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Fig. 1: 4Pi-SMS detection and excitation path optical overview.
Fig. 2: 4Pi-SMS system overview.
Fig. 3: Detection path beam-alignment tool.
Fig. 4: Waveplate orientation and example calibration data.
Fig. 5: QWP calibration setup.
Fig. 6: Microscope tower assembly and stage stack.
Fig. 7: NPBS and wedge assembly.
Fig. 8: DM installation.
Fig. 9: Alignment laser setup.
Fig. 10: Labeled diagram of the 4Pi-SMS emission path.
Fig. 11: DM control pattern used for centering.
Fig. 12: Objective pupil plane location and alignment camera setup.
Fig. 13: DM conjugation.
Fig. 14: Excitation path diagram with labels used throughout the alignment procedure.
Fig. 15: Sample mounting in custom holder FAB-P0035.
Fig. 16: Image layout on data-acquisition camera.
Fig. 17: Example interference scan data.
Fig. 18: Interference variation across the field of view.
Fig. 19: Example quartz wedge calibration data for setting the phase between the four interference images.
Fig. 20: Fluorescent bead with and without astigmatism above and below the focal plane.
Fig. 21: Reduced moment versus Z position and phase calibration data example.
Fig. 22: Objective lock NIR laser focus.
Fig. 23: Example environmental vibration data.
Fig. 24: DM surface interference images.
Fig. 25: Example 4Pi-SMS images from immunostained microtubules and Nup96-SNAP labeled nuclear pore complexes.

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Data availability

Software and hardware plans are available from online repositories. The CAD file repository (Autodesk Inventor) can be found at The complete parts list is available at and as Supplementary Table 1. The microscope control software can be downloaded from The raw image files used to create Fig. 25a–c and Fig. 25d–f are available via the Zenodo online repositories: and, respectively.

Code availability

The data analysis software referenced in this paper is available online at


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We thank Fang Huang for advice, discussions and original code for the analysis software; Jacopo Antonello for advice, discussions and help with setting up the DMs; Chao He for assembling the DM calibration tool; David Miguel Susano Pinto for help and discussions with the analysis code; Andrew Barentine and Zach Marin for help and discussion about the analysis code; and David Baddeley for imaging advice, helpful discussions and help with the analysis code. J.W., M.A.P. and I.M.D. were supported by John Fell Fund award 141/144 and Wellcome Trust awards 105605/Z/14/Z and 107457/Z/15/Z. E.S.A. was supported by Wellcome Trust awards 095927/B/11/Z and 203285/Z/16/Z. G.S. was supported by Wellcome Trust awards 095927/B/11/Z and 203144/Z/16/Z. Y.Z., K.H., M.D.L. and J.B. were supported by Wellcome Trust awards 095927/A/11/Z and 203285/B/16/Z and National Institutes of Health (NIH) award R01 GM118486. K.H. was additionally supported by NIH award T32EB019941. R.D. was supported by an award from the Engelhorn Foundation. J.R. and Y.L. were supported by European Research Council award ERC CoG-724489, funding from the EMBL and the 4D Nucleome/4DN NIH Common Fund award U01 EB021223. Y.L. was additionally supported by the EMBL Interdisciplinary Postdoc Programme (EIPOD) under Marie Curie Actions COFUND and a start-up grant from the Southern University of Science and Technology, China. M.J.B. was supported by European Research Council award AdOMIS 695140 and Wellcome Trust award 203285/C/16/Z.

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Authors and Affiliations



Hardware development: J.B., M.J.B., Y.Z., M.A.P., J.W., Y.L., E.S.A. and G.S.; software development: Y.Z., Y.L. and E.S.A.; specimen/imaging protocols: Y.Z. and M.D.L.; alignment protocols: E.S.A., G.S., J.W., Y.L., Y.Z. and K.H.; index matching protocol: R.D., J.R. and Y.L.; project supervision: J.B., M.J.B., J.R. and I.M.D.; writing and editing of the manuscript: all authors.

Corresponding authors

Correspondence to Ian M. Dobbie, Jonas Ries, Martin J. Booth or Joerg Bewersdorf.

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Competing interests

J.B. has financial interests in Bruker Corp. and Hamamatsu Photonics. J.B. is co-inventor of a US patent application (US20170251191A1) related to the 4Pi-SMS system and image analysis used in this work. Y.Z. and J.B. have filed a US patent application about the salvaged fluorescence multicolor imaging method described in this work.

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

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Key references using this protocol

Huang, F. et al. Cell 166, 1028–1040 (2016):

Zhang, Y. et al. Nat. Methods 17, 225–231 (2020):

Zhang, Y. et al. Proc. Natl Acad. Sci. USA 114, 6098–6103 (2017):

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Supplementary Information

Supplementary Methods and Supplementary Table 1.

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Wang, J., Allgeyer, E.S., Sirinakis, G. et al. Implementation of a 4Pi-SMS super-resolution microscope. Nat Protoc 16, 677–727 (2021).

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