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Ultrahigh Resolution Imaging of Biomolecules by Fluorescence Photoactivation Localization Microscopy

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Book cover Micro and Nano Technologies in Bioanalysis

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 544))

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.

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Acknowledgments

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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  1. Department of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine, 313 Bennett Hall, 04469, Orono, ME, USA

    Samuel T. Hess, Travis J. Gould, Mudalige Gunewardene, Joerg Bewersdorf & Michael D. Mason

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  1. Samuel T. Hess
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  2. Travis J. Gould
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  3. Mudalige Gunewardene
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Correspondence to Samuel T. Hess .

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

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    Hess, S.T., Gould, T.J., Gunewardene, M., Bewersdorf, J., Mason, M.D. (2009). Ultrahigh Resolution Imaging of Biomolecules by Fluorescence Photoactivation Localization Microscopy. In: Foote, R., Lee, J. (eds) Micro and Nano Technologies in Bioanalysis. Methods in Molecular Biology™, vol 544. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-483-4_32

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    • DOI: https://doi.org/10.1007/978-1-59745-483-4_32

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