• bleaching steps;
  • protein assembly;
  • single-molecule imaging;
  • stoichiometry;
  • subunit counting


The limit of subdiffraction imaging with fluorescent proteins currently lies at 20 nm, and therefore most protein complexes are too small (2–5 nm) to spatially resolve their individual subunits by optical means. However, the number and stoichiometry of subunits within an immobilized protein complex can be resolved by the observation of photobleaching steps of individual fluorophores or co-localization of single-molecule fluorescence emission in multiple colors. We give an overview of the proteins that have been investigated by this approach and the different techniques that can be used to immobilize and label the proteins. This minireview should serve as a guideline for scientists who want to employ single-molecule subunit counting for their research.