Quantitative localization microscopy: effects of photophysics and labeling stoichiometry

PLoS One. 2015 May 20;10(5):e0127989. doi: 10.1371/journal.pone.0127989. eCollection 2015.

Abstract

Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or generally per fluorescently labeled site from the build-up of spatial image correlation during acquisition. To this end we employ a model for the interplay between the statistics of activation, bleaching, and labeling stoichiometry. We validated our method using single fluorophore labeled DNA oligomers and multiple-labeled neutravidin tetramers where we find a counting error of less than 17% without any calibration of transition rates. Furthermore, we demonstrated our quantification method on nanobody- and antibody-labeled biological specimens.

Publication types

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

MeSH terms

  • Animals
  • Calibration
  • Chlorocebus aethiops
  • Cluster Analysis
  • Fluorescent Dyes / chemistry
  • Light*
  • Microscopy, Fluorescence / methods*
  • Rats
  • Staining and Labeling*
  • Vero Cells

Substances

  • Fluorescent Dyes