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Photoswitching Proteins pp 239–260Cite as

Photoactivatable Fluorescent Proteins for Super-resolution Microscopy

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1148))

Abstract

Super-resolution fluorescence microscopy techniques such as simulated emission depletion (STED) microscopy and photoactivated localization microscopy (PALM) allow substructures, organelles or even proteins within a cell to be imaged with a resolution far below the diffraction limit of ~200 nm. The development of advanced fluorescent proteins, especially photoactivatable fluorescent proteins of the GFP family, has greatly contributed to the successful application of these techniques to live-cell imaging. Here, we will illustrate how two fluorescent proteins with different photoactivation mechanisms can be utilized in high resolution dual color PALM imaging to obtain insights into a cellular process that otherwise would not be accessible. We will explain how to set up and perform the experiment and how to use our latest software “a-livePALM” for fast and efficient data analysis.

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) and the State of Baden-Württemberg through the Center for Functional Nanostructures (CFN) and by DFG grant Ni 291/9.

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Authors and Affiliations

  1. Institute of Applied Physics and Center for Functional Nanostructures (CFN), Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Str. 1, Karlsruhe, 76131, Germany

    Yuji Ishitsuka, Karin Nienhaus & G. Ulrich Nienhaus

  2. Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, 76344, Germany

    G. Ulrich Nienhaus

  3. Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, IL, 61801, USA

    G. Ulrich Nienhaus

Authors
  1. Yuji Ishitsuka
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  2. Karin Nienhaus
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  3. G. Ulrich Nienhaus
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Corresponding author

Correspondence to G. Ulrich Nienhaus .

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Editors and Affiliations

  1. Institute of Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany

    Sidney Cambridge

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Ishitsuka, Y., Nienhaus, K., Nienhaus, G.U. (2014). Photoactivatable Fluorescent Proteins for Super-resolution Microscopy. In: Cambridge, S. (eds) Photoswitching Proteins. Methods in Molecular Biology, vol 1148. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0470-9_16

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  • DOI: https://doi.org/10.1007/978-1-4939-0470-9_16

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  • Publisher Name: Humana Press, New York, NY

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