Measuring localization performance of super-resolution algorithms on very active samples

Opt Express. 2011 Apr 11;19(8):7020-33. doi: 10.1364/OE.19.007020.


Super-resolution fluorescence imaging based on single-molecule localization relies critically on the availability of efficient processing algorithms to distinguish, identify, and localize emissions of single fluorophores. In multiple current applications, such as three-dimensional, time-resolved or cluster imaging, high densities of fluorophore emissions are common. Here, we provide an analytic tool to test the performance and quality of localization microscopy algorithms and demonstrate that common algorithms encounter difficulties for samples with high fluorophore density. We demonstrate that, for typical single-molecule localization microscopy methods such as dSTORM and the commonly used rapidSTORM scheme, computational precision limits the acceptable density of concurrently active fluorophores to 0.6 per square micrometer and that the number of successfully localized fluorophores per frame is limited to 0.2 per square micrometer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Fluorescent Dyes / chemistry
  • Image Processing, Computer-Assisted
  • Markov Chains
  • Microscopy, Fluorescence / methods*
  • Normal Distribution
  • Poisson Distribution
  • Software
  • Stochastic Processes
  • Time Factors


  • Fluorescent Dyes