Quantitative Single-Molecule Localization Microscopy (qSMLM) of Membrane Proteins Based on Kinetic Analysis of Fluorophore Blinking Cycles

  • Franziska Fricke
  • Joel Beaudouin
  • Sebastian Malkusch
  • Roland Eils
  • Mike HeilemannEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1663)


Photoswitchable or photoactivatable fluorophores are the key in single-molecule localization microscopy. Next to providing fluorescence images with subdiffraction spatial resolution, additional information is available from observing single fluorophores over time. This includes the characteristic photophysical phenomenon of “blinking” that is exhibited by single fluorescent proteins or fluorophores and follows well-defined kinetic laws. Analyzing the kinetics of “blinking” allows determining the number of fluorophores in a multi-molecular complex. As such, quantitative information at the molecular level can be extracted, representing a tremendously useful extension of single-molecule super-resolution microscopy. This concept is in particular useful to study homo- and heterooligomeric signaling protein complexes in the plasma membrane of an intact cell with molecular resolution. Here, we provide an experimental framework for deciphering the stoichiometry of membrane proteins on the basis of SMLM and photoswitching statistics.

Key words

SMLM Membrane proteins Molecular counting Cluster analysis 


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Franziska Fricke
    • 1
  • Joel Beaudouin
    • 2
    • 3
    • 6
  • Sebastian Malkusch
    • 1
  • Roland Eils
    • 2
    • 3
  • Mike Heilemann
    • 1
    • 4
    • 5
    Email author
  1. 1.Institute of Physical and Theoretical ChemistryGoethe-University FrankfurtFrankfurt am MainGermany
  2. 2.Department for Bioinformatics and Functional Genomics, Bioquant and Institute of Pharmacy and Molecular BiotechnologyHeidelberg UniversityHeidelbergGermany
  3. 3.Division of Theoretical BioinformaticsGerman Cancer Research Center (DKFZ)HeidelbergGermany
  4. 4.Single Molecule BiologyBioquant, Heidelberg UniversityHeidelbergGermany
  5. 5.Institute for Anatomy and Cell BiologyHeidelberg UniversityHeidelbergGermany
  6. 6.Institut de Biologie StructuraleUniversité Grenoble Alpes, Centre National de la Recherche Scientifique, Commissariat à l’Energie Atomique et aux Energies AlternativesGrenobleFrance

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