Dual color localization microscopy of cellular nanostructures

Authors

  • Manuel Gunkel,

    1. Kirchhoff-Institute for Physics & BioQuant Center University of Heidelberg, Heidelberg, Germany
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  • Fabian Erdel,

    1. Deutsches Krebsforschungszentrum & BioQuant Center University of Heidelberg, Research Group Genome Organization & Function, Heidelberg, Germany
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  • Karsten Rippe,

    1. Deutsches Krebsforschungszentrum & BioQuant Center University of Heidelberg, Research Group Genome Organization & Function, Heidelberg, Germany
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  • Paul Lemmer,

    1. Kirchhoff-Institute for Physics & BioQuant Center University of Heidelberg, Heidelberg, Germany
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  • Rainer Kaufmann,

    1. Kirchhoff-Institute for Physics & BioQuant Center University of Heidelberg, Heidelberg, Germany
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  • Christoph Hörmann,

    1. Kirchhoff-Institute for Physics & BioQuant Center University of Heidelberg, Heidelberg, Germany
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  • Roman Amberger,

    1. Kirchhoff-Institute for Physics & BioQuant Center University of Heidelberg, Heidelberg, Germany
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  • Christoph Cremer Professor

    Corresponding author
    1. Kirchhoff-Institute for Physics & BioQuant Center University of Heidelberg, Heidelberg, Germany
    2. Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany
    3. Institute for Molecular Biophysics, The Jackson Laboratory, Bar Harbor, ME, USA
    • Institute for Molecular Biophysics, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA, Fax: +49-6221-54-9271
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Abstract

The dual color localization microscopy (2CLM) presented here is based on the principles of spectral precision distance microscopy (SPDM) with conventional autofluorescent proteins under special physical conditions. This technique allows us to measure the spatial distribution of single fluorescently labeled molecules in entire cells with an effective optical resolution comparable to macromolecular dimensions. Here, we describe the application of the 2CLM approach to the simultaneous nanoimaging of cellular structures using two fluorochrome types distinguished by different fluorescence emission wavelengths. The capabilities of 2CLM for studying the spatial organization of the genome in the mammalian cell nucleus are demonstrated for the relative distributions of two chromosomal proteins labeled with autofluorescent GFP and mRFP1 domains. The 2CLM images revealed quantitative information on their spatial relationships down to length-scales of 30 nm.

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