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Constructing a cost-efficient, high-throughput and high-quality single-molecule localization microscope for super-resolution imaging

Abstract

Single-molecule localization microscopy (SMLM) leverages the power of modern optics to unleash ultra-precise structural nanoscopy of complex biological machines in their native environments as well as ultra-sensitive and high-throughput medical diagnostics with the sensitivity of a single molecule. To achieve this remarkable speed and resolution, SMLM setups are either built by research laboratories with strong expertise in optical engineering or commercially sold at a hefty price tag. The inaccessibility of SMLM to life scientists for technical or financial reasons is detrimental to the progress of biological and biomedical discoveries reliant on super-resolution imaging. In this work, we present the NanoPro, an economic, high-throughput, high-quality and easy-to-assemble SMLM for super-resolution imaging. We show that our instrument performs similarly to the most expensive, best-in-class commercial microscopes and rivals existing open-source microscopes at a lower price and construction complexity. To facilitate its wide adoption, we compiled a step-by-step protocol, accompanied by extensive illustrations, to aid inexperienced researchers in constructing the NanoPro as well as assessing its performance by imaging ground-truth samples as small as 20 nm. The detailed visual instructions make it possible for students with little expertise in microscopy engineering to construct, validate and use the NanoPro in <1 week, provided that all components are available.

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Fig. 1: Schematic diagrams of open-source microscopes developed for super-resolution imaging.
Fig. 2: Schematic diagram of the NanoPro 1.0 assembly and its individual components.
Fig. 3: Assessing the performance of NanoPro 1.0 on ground-truth nanoruler samples.
Fig. 4: Images from good- and poor-quality alignment.
Fig. 5: Exemplary images of cellular and recombinant macromolecular complexes obtained by using NanoPro 1.0.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

Updated versions of the source code for NanoPro 1.0, as well as guiding instructions (visual assembly, alignment and operation guides and instructional videos), can be obtained from https://github.com/jdanial/NanoPro and are archived in Zenodo44. A compilation of NanoPro 1.0 for the Windows operating system is available in the GitHub repository.

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Acknowledgements

We thank M. Woolley, J. Prill and S. Impey from the mechanical workshop in the Yusuf Hamied Department of Chemistry at the University of Cambridge for fabricating the microscope assembly and A. Jayasinghe (https://www.fiverr.com/achinijayasingh) for illustrating the guides. We also thank E. Metzakopian and E. Wilson (UK DRI Cambridge) for providing us with the HeLa cells. This work was supported by a UK Medical Research Council (UK MRC)–funded World Class Labs capital equipment award from the UK Dementia Research Institute (UK DRI Ltd) to D.K. J.S.H.D. is funded by a postdoctoral fellowship from EISAI and the UK DRI Ltd pilot grant from the UK DRI Ltd and a research associateship from King’s College, University of Cambridge. J.Y.L.L. is funded by a scholarship from the Croucher Foundation Ltd (Hong Kong). D.K. is funded by a European Research Council (ERC) advanced grant (669237), the UK DRI Ltd funded by the UK MRC and the Royal Society (UK).

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Authors and Affiliations

Authors

Contributions

J.S.H.D. and D.K. conceived and designed the study. M.W. and J.S.H.D. designed the microscope. J.S.H.D., J.Y.L.L. and Y.W. assembled the microscope. J.S.H.D. wrote the NanoPro 1.0 software with input from J.Y.L.L. and Y.W. J.S.H.D., J.Y.L.L. and Y.W. performed the analysis. E.D. prepared all cellular samples. M.R.C., D.E., J.Y.L.L., Y.W. and J.S.H.D. revised the protocol. J.S.H.D. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to John S. H. Danial or David Klenerman.

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Nature Protocols thanks Benedict Diederich 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

Whiten, D. R. et al. ChemBioChem 19, 2033–2038 (2018): https://doi.org/10.1002/cbic.201800209

Sang, J. C. et al. Commun. Biol. 4, 1–11 (2021): https://doi.org/10.1038/s42003-021-02126-w

Sideris, D. I. et al. Brain Commun. 3, fcab147 (2021): https://doi.org/10.1093/braincomms/fcab147

Supplementary information

Supplementary Information

Supplementary Figs. 1–3, Table 1 and Note 1.

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Danial, J.S.H., Lam, J.Y.L., Wu, Y. et al. Constructing a cost-efficient, high-throughput and high-quality single-molecule localization microscope for super-resolution imaging. Nat Protoc 17, 2570–2619 (2022). https://doi.org/10.1038/s41596-022-00730-6

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