Histochemistry and Cell Biology

, Volume 141, Issue 6, pp 629–638

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

Original Paper

DOI: 10.1007/s00418-014-1192-3

Cite this article as:
Endesfelder, U., Malkusch, S., Fricke, F. et al. Histochem Cell Biol (2014) 141: 629. doi:10.1007/s00418-014-1192-3


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 σSMLM are obtained. Knowing σSMLM is a prerequisite for consistent visualization or any quantitative structural analysis, e.g., cluster analysis or colocalization studies.


Single-molecule localization microscopyFluorescence microscopyResolutionLocalization precisionSingle-molecule fluorescence

Supplementary material

418_2014_1192_MOESM1_ESM.docx (2.4 mb)
Supplementary material (DOCX 2453 kb)
418_2014_1192_MOESM2_ESM.zip (764 kb)
Supplementary software package (ZIP 763 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Institute of Physical and Theoretical ChemistryJohann Wolfgang Goethe University Frankfurt am MainFrankfurt am MainGermany