Protocol

Micro and Nano Technologies in Bioanalysis

Volume 544 of the series Methods in Molecular Biology™ pp 483-522

Date:

Ultrahigh Resolution Imaging of Biomolecules by Fluorescence Photoactivation Localization Microscopy

  • Samuel T. HessAffiliated withDepartment of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine Email author 
  • , Travis J. GouldAffiliated withDepartment of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine
  • , Mudalige GunewardeneAffiliated withDepartment of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine
  • , Joerg BewersdorfAffiliated withDepartment of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine
  • , Michael D. MasonAffiliated withDepartment of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine

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Summary

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