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A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment

Abstract

The localization precision is a crucial and important parameter for single-molecule localization microscopy (SMLM) and directly influences the achievable spatial resolution. It primarily depends on experimental imaging conditions and the registration potency of the algorithm used. We propose a new and simple routine to estimate the average experimental localization precision in SMLM, based on the nearest neighbor analysis. By exploring different experimental and simulated targets, we show that this approach can be generally used for any 2D or 3D SMLM data and that reliable values for the localization precision σ SMLM are obtained. Knowing σ SMLM is a prerequisite for consistent visualization or any quantitative structural analysis, e.g., cluster analysis or colocalization studies.

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Acknowledgments

We thank Steve Wolter for helpful discussions. We acknowledge funding by the German Ministry of Education and Research (Grants 0315262 and 0316170D) and the cluster of excellence “Macromolecular Complexes” (CEF, DFG cluster of excellence (EXC 115)).

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Correspondence to Ulrike Endesfelder or Mike Heilemann.

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Endesfelder, U., Malkusch, S., Fricke, F. et al. A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment. Histochem Cell Biol 141, 629–638 (2014). https://doi.org/10.1007/s00418-014-1192-3

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Keywords

  • Single-molecule localization microscopy
  • Fluorescence microscopy
  • Resolution
  • Localization precision
  • Single-molecule fluorescence