Precisely and accurately localizing single emitters in fluorescence microscopy

Nat Methods. 2014 Mar;11(3):253-66. doi: 10.1038/nmeth.2843.


Methods based on single-molecule localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of molecules and nanoparticles and contributed to the recent revolution in super-resolution localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized. We present a lucid synthesis of the developments on this localization precision and accuracy and their practical implications in order to guide the increasing number of researchers using single-particle tracking and super-resolution localization microscopy.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Fluorescent Dyes / chemistry*
  • Microscopy, Fluorescence / methods*
  • Microscopy, Fluorescence / standards*
  • Reproducibility of Results


  • Fluorescent Dyes