Measuring image resolution in optical nanoscopy

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Abstract

Resolution in optical nanoscopy (or super-resolution microscopy) depends on the localization uncertainty and density of single fluorescent labels and on the sample's spatial structure. Currently there is no integral, practical resolution measure that accounts for all factors. We introduce a measure based on Fourier ring correlation (FRC) that can be computed directly from an image. We demonstrate its validity and benefits on two-dimensional (2D) and 3D localization microscopy images of tubulin and actin filaments. Our FRC resolution method makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, to optimize and rank different emitter localization and labeling strategies, to define a stopping criterion for data acquisition, to describe image anisotropy and heterogeneity, and even to estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, showing instead how the best image resolution can be achieved as fast as possible.

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Figure 1: The FRC principle and trade-off between localization uncertainty and labeling density.
Figure 2: The effect of localization density and data processing on resolution.
Figure 3: Spurious correlations from a two-color localization microscopy image.
Figure 4: 3D resolution.

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Acknowledgements

We thank K. Jalink for encouragement and support; S. Schwartz, F. Huang, J. Byars and S. Liu for assistance with experiments; and V. van Ravesteijn and P. Kruit for providing scanning electron microscope data. We appreciate the thoughtful comments of T. Young and L. van Vliet. R.P.J.N. and D.L.P. are supported by the Dutch Technology Foundation STW, which is part of the Netherlands Organisation for Scientific Research (NWO) and which is partly funded by the Ministry of Economic Affairs, Agriculture and Innovation. K.A.L. was supported by US National Science Foundation CAREER Award #0954836 and US National Institutes of Health grant P50GM085273.

Author information

R.P.J.N., S.S. and B.R. devised the conceptual framework and derived theoretical results. Simulations were done by R.P.J.N. Experimental data sets were acquired by R.P.J.N. (Fig. 2a–i), D.L.P. (Fig. 2j–n), K.A.L. (Figs. 2a–i and 4) and M.B. (Fig. 3). Data were analyzed by R.P.J.N., M.B., S.S. and B.R. D.G. provided research advice. The paper was written by R.P.J.N., D.G., S.S. and B.R.

Correspondence to Sjoerd Stallinga or Bernd Rieger.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16 and Supplementary Notes 1–7 (PDF 5383 kb)

Supplementary Software

Matlab code and ImageJ plugin to compute FRC resolution (ZIP 15962 kb)

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Nieuwenhuizen, R., Lidke, K., Bates, M. et al. Measuring image resolution in optical nanoscopy. Nat Methods 10, 557–562 (2013) doi:10.1038/nmeth.2448

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