Abstract
Super-resolution microscopy depends on steps that can contribute to the formation of image artifacts, leading to misinterpretation of biological information. We present NanoJ-SQUIRREL, an ImageJ-based analytical approach that provides quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same acquisition volume, this approach generates a quantitative map of super-resolution defects and can guide researchers in optimizing imaging parameters.
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Change history
19 October 2020
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Acknowledgements
We thank A. Knight (Holistx Ltd.) and S. Holden (Newcastle University) for critical reading of the manuscript; J. Ries (European Laboratory for Molecular Biology, Heidelberg) for provision of customized MATLAB software and critical reading of the manuscript; K. Tosheva (University College London) for critical reading of the manuscript and beta testing of the software; and B. Baum (University College London) for reagents. Many of the look-up tables used here are based on the open-source repository of D. Williamson at King′s College London. This work was funded by grants from the UK Biotechnology and Biological Sciences Research Council (BB/M022374/1; BB/P027431/1; BB/R000697/1) (R.H. and P.M.P.), MRC Programme Grant (MC_UU12018/7) (J.M.), the European Research Council (649101–UbiProPox) (J.M.), the UK Medical Research Council (MR/K015826/1) (R.H. and J.M.), the Wellcome Trust (203276/Z/16/Z) (S.C. and R.H.) and the Centre National de la Recherche Scientifique (CNRS ATIP-AVENIR program AO2016) (C.L.). D.A. is presently a Marie Curie fellow (Marie Sklodowska-Curie grant agreement No 750673). C.J. funded by a Commonwealth scholarship, funded by the UK government.
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Contributions
S.C. and R.H. devised the conceptual framework and derived theoretical results. S.C., D.A., C.L., J.M., and R.H. planned experiments. S.C. and R.H. wrote the algorithm. Simulations were done by S.C. Experimental data sets were acquired by S.C. (Fig. 1), D.A. (Fig. 2; Supplementary Notes 4 and 7), C.J. (Fig. 2 and Supplementary Note 9), P.M.P. (Fig. 3), and C.L. (Supplementary Notes 5, 10, and 11). Data were analyzed by S.C. and D.A.; and C.L., J.M., and R.H. provided research advice. The paper was written by S.C., D.A., J.M., and R.H. with editing contributions from all the authors.
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Supplementary information
Supplementary Text and Figures
Supplementary Notes 1–11 (PDF 32445 kb)
Supplementary Software
Binaries, source code and user manual for NanoJ-SQUIRREL (ZIP 13406 kb)
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Culley, S., Albrecht, D., Jacobs, C. et al. Quantitative mapping and minimization of super-resolution optical imaging artifacts. Nat Methods 15, 263–266 (2018). https://doi.org/10.1038/nmeth.4605
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DOI: https://doi.org/10.1038/nmeth.4605
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