Localization accuracy in single-molecule microscopy

Biophys J. 2004 Feb;86(2):1185-200. doi: 10.1016/S0006-3495(04)74193-4.


One of the most basic questions in single-molecule microscopy concerns the accuracy with which the location of a single molecule can be determined. Using the Fisher information matrix it is shown that the limit of the localization accuracy for a single molecule is given by, lambda(em)/2pi n(a) square root of gammaAt, where lambda(em), n(a), gamma, A, and t denote the emission wavelength of the single molecule, the numerical aperture of the objective, the efficiency of the optical system, the emission rate of the single molecule and the acquisition time, respectively. Using Monte Carlo simulations it is shown that estimation algorithms can come close to attaining the limit given in the expression. Explicit quantitative results are also provided to show how the limit of the localization accuracy is reduced by factors such as pixelation of the detector and noise sources in the detection system. The results demonstrate what is achievable by single-molecule microscopy and provide guidelines for experimental design.

Publication types

  • Evaluation Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Biopolymers / analysis
  • Biopolymers / chemistry*
  • Biopolymers / metabolism*
  • Computer Simulation
  • Image Enhancement / methods*
  • Image Interpretation, Computer-Assisted / methods*
  • Microscopy, Fluorescence / methods*
  • Models, Biological*
  • Models, Chemical*
  • Models, Statistical
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Stochastic Processes
  • Tissue Distribution


  • Biopolymers