Abstract
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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Data availability
Software and hardware plans are available from online repositories. The CAD file repository (Autodesk Inventor) can be found at https://github.com/4Pi-SMS-consortium/CAD-files. The complete parts list is available at https://github.com/4Pi-SMS-consortium/CAD-files/blob/master/PartsList.xlsx and as Supplementary Table 1. The microscope control software can be downloaded from https://github.com/Gurdon-Super-Res-Lab/Microscope-Control. The raw image files used to create Fig. 25a–c and Fig. 25d–f are available via the Zenodo online repositories: https://zenodo.org/record/3929647#.X6Q-31NKgdU and https://zenodo.org/record/4022827#.X6Q_GVNKgdU, respectively.
Code availability
The data analysis software referenced in this paper is available online at https://github.com/4Pi-SMS-consortium/4Pi-SMS-analysis.
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Acknowledgements
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
Contributions
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.
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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): https://doi.org/10.1016/j.cell.2016.06.016
Zhang, Y. et al. Nat. Methods 17, 225–231 (2020): https://doi.org/10.1038/s41592-019-0676-4
Zhang, Y. et al. Proc. Natl Acad. Sci. USA 114, 6098–6103 (2017): https://doi.org/10.1073/pnas.1705823114
Supplementary information
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). https://doi.org/10.1038/s41596-020-00428-7
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DOI: https://doi.org/10.1038/s41596-020-00428-7
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