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Artifact-free high-density localization microscopy analysis

Abstract

High-density analysis methods for localization microscopy increase acquisition speed but produce artifacts. We demonstrate that these artifacts can be eliminated by the combination of Haar wavelet kernel (HAWK) analysis with standard single-frame fitting. We tested the performance of this method on synthetic, fixed-cell, and live-cell data, and found that HAWK preprocessing yielded reconstructions that reflected the structure of the sample, thus enabling high-speed, artifact-free super-resolution imaging of live cells.

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Fig. 1: HAWK processing improves results and removes artifacts in localization microscopy.
Fig. 2: HAWK analysis of live-cell localization yields results closer to ground truth and better reproduction of less intense features.

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Acknowledgements

We thank D. Matthews for assistance with the SIM and iSIM systems in the Nikon Imaging Centre at King’s College London, E. Rosten for helpful discussions, and M. Parsons for critical reading of the manuscript. mEOS3.2 sequence was amplified from a template provided by D. Owen (King’s College London, London, UK). This work was supported by the Royal Society (University Research Fellowship to S.C.), the Leverhulme Trust (RPG-2015-105 to S.C.), the Human Frontier Science Program (RGP0035/2016 to S.C.), BBSRC (BB/K01563X/1 and BB/N022696/1 to S.C.), the MRC (Next Generation Optical Imaging grant MR/K015664/1 to G.E.J., M.G., and S.C.; G1100041 to G.E.J.), and the British Heart Foundation (RG/15/8/31480 to M.G.).

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Authors

Contributions

R.J.M. and S.C. conceived and designed the analysis and experiments. L.M.H. created the mEOS3.2–talin construct with assistance and advice from G.E.J. and prepared the podosome samples, P.B. prepared the psoas myofibril samples, M.G. prepared the cardiac myofibril samples, and K.P. prepared the focal adhesion samples. R.J.M. carried out the analysis and experiments. R.J.M., G.E.J., and S.C. discussed the results and prepared the manuscript, and all authors contributed to revision of the manuscript.

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Correspondence to Susan Cox.

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King’s College London has filed a patent (UK Patent Application No. 1800026.5) that includes portions of the research described in this article, the value of which may be affected by this publication.

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Supplementary text and figures

Supplementary Figures 1–15, Supplementary Table 1 and Supplementary Note 1

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Supplementary Software

HAWK plugin

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Marsh, R.J., Pfisterer, K., Bennett, P. et al. Artifact-free high-density localization microscopy analysis. Nat Methods 15, 689–692 (2018). https://doi.org/10.1038/s41592-018-0072-5

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