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Fluorescence In Situ Hybridization Applications for Super-Resolution 3D Structured Illumination Microscopy

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Nanoimaging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 950))

Abstract

Fluorescence in situ hybridization on three-dimensionally preserved cells (3D-FISH) is an efficient tool to analyze the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level. 3D reconstructions from serial optical sections obtained by confocal laser scanning microscopy (CLSM) have long been considered the gold standard for 3D-FISH analyses. Recent super-resolution techniques circumvent the diffraction-limit of optical resolution and have defined a new state-of-the-art in bioimaging. Three-dimensional structured illumination microscopy (3D-SIM) represents one of these technologies. Notably, 3D-SIM renders an eightfold improved volumetric resolution over conventional imaging, and allows the simultaneous visualization of differently labeled target structures. These features make this approach highly attractive for the analysis of spatial relations and substructures of nuclear targets that escape detection by conventional light microscopy. Here, we focus on the application of 3D-SIM for the visualization of subnuclear 3D-FISH preparations. In comparison with conventional fluorescence microscopy, the quality of 3D-SIM data is dependent to a much greater extent on the optimal sample preparation, labeling and acquisition conditions. We describe typical problems encountered with super-resolution imaging of in situ hybridizations in mammalian tissue culture cells and provide optimized DNA-/(RNA)-FISH protocols including combinations with immunofluorescence staining (Immuno-FISH) and DNA replication labeling using click chemistry.

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Acknowledgments

The authors are indebted to H. Leonhardt and the BioImaging Network Munich for generous support. We thank J. Neumann for technical help and A. Nemeth for critical comments on the manuscript. This work was supported by DFG grants SCHE1596/2-1 and CR59/28-1 to M.C. and L.S and SFB 684 to Y.M.

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Authors and Affiliations

  1. Biocenter, Department of Biology II, Ludwig Maximilians University Munich(LMU), Munich, Germany

    Yolanda Markaki, Daniel Smeets & Marion Cremer

  2. Department of Biochemistry, University of Oxford, Oxford, UK

    Lothar Schermelleh

Authors
  1. Yolanda Markaki
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  2. Daniel Smeets
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  3. Marion Cremer
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  4. Lothar Schermelleh
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Corresponding author

Correspondence to Lothar Schermelleh .

Editor information

Editors and Affiliations

  1. Nat. Inst. Biomedical Imaging & Bioengin, National Institutes of Health, South Drive 13, Bethesda, 20892, Maryland, USA

    Alioscka A. Sousa

  2. , National Cancer Institute, National Institutes of Health, Center Drive 10, Bethesda, 20892, USA

    Michael J. Kruhlak

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Markaki, Y., Smeets, D., Cremer, M., Schermelleh, L. (2013). Fluorescence In Situ Hybridization Applications for Super-Resolution 3D Structured Illumination Microscopy. In: Sousa, A., Kruhlak, M. (eds) Nanoimaging. Methods in Molecular Biology, vol 950. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-137-0_4

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  • DOI: https://doi.org/10.1007/978-1-62703-137-0_4

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-136-3

  • Online ISBN: 978-1-62703-137-0

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