Ultrahigh Resolution Imaging of Biomolecules by Fluorescence Photoactivation Localization Microscopy

  • Samuel T. Hess
  • Travis J. Gould
  • Mudalige Gunewardene
  • Joerg Bewersdorf
  • Michael D. Mason
Part of the Methods in Molecular Biology™ book series (MIMB, volume 544)


Diffraction limits the biological structures that can be imaged by normal light microscopy. However, recently developed techniques are breaking the limits that diffraction poses and allowing imaging of biological samples at the molecular length scale. Fluorescence photoactivation localization microscopy (FPALM) and related methods can now image molecular distributions in fixed and living cells with measured resolution better than 30 nm. Based on localization of single photoactivatable molecules, FPALM uses repeated cycles of activation, localization, and photobleaching, combined with high-sensitivity fluorescence imaging, to identify and localize large numbers of molecules within a sample. Procedures and pitfalls for construction and use of such a microscope are discussed in detail. Representative images of cytosolic proteins, membrane proteins, and other structures, as well as examples of results during acquisition are shown. It is hoped that these details can be used to perform FPALM on a variety of biological samples, to significantly advance the understanding of biological systems.

Key words

FPALM PALM STORM Hemagglutinin Photoactivation EosFP Super-resolution PA-GFP 



The authors thank J. Zimmerberg and P. Blank for useful discussions and loaned equipment, G. Patterson for PA-GFP protein and constructs, J. Wiedenmann and U. Nienhaus for EosFP protein and constructs, J. Gosse for helpful discussions, V. Verkhusha for Dendra2 constructs, T. Tripp for machining services, and J. Lozier for molecular biology services. This work was supported in part by National Institutes of Health (NIH) grant K25-AI65459, National Science Foundation (NSF) grant CHE-0722759, and University of Maine startup funds.


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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Samuel T. Hess
    • 1
  • Travis J. Gould
    • 1
  • Mudalige Gunewardene
    • 1
  • Joerg Bewersdorf
    • 1
  • Michael D. Mason
    • 1
  1. 1.Department of Physics and Astronomy and Institute for Molecular BiophysicsUniversity of MaineOronoUSA

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