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Superresolution Microscopy for Visualization of Physical Contacts Between Chromosomes at Nanoscale Resolution

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Chromatin

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2458))

Abstract

This protocol describes the fluorescence in situ hybridization (FISH) of DNA probes on mitotic chromosome spreads optimized for two super-resolution microscopy approaches—structured illumination microscopy (SIM) and stimulated emission depletion (STED). It is based on traditional DNA FISH methods that can be combined with immunofluorescence labeling (Immuno-FISH). This technique previously allowed us to visualize ribosomal DNA linkages between human acrocentric chromosomes and provided information about the activity status of linked rDNA loci. Compared to the conventional wide-field and confocal microscopy, the quality of SIM and STED data depends a lot more on the optimal specimen preparation, choice of fluorophores, and quality of the fluorescent labeling. This protocol highlights details that make specimens suitable for super-resolution microscopy and tips for good imaging practices.

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Acknowledgments

We are thankful to the Microscopy core facility at the Stowers Institute for enabling super-resolution experiments. We thank Patrina Pellett, Jay Unruh, and Sean McKinney for conceptual help, and Brian Slaughter for the critical review of the manuscript. We thank Jennifer Gerton for mentorship and members of the Gerton lab for discussions. We are grateful to Scott Rider for assistance with labeled probes and to Martha Stampfer for HMECs. We thank Christophe Leterrier for making his collection of colorblind-friendly LUTs publicly available. This study was supported by funding from Stowers Institute for Medical Research.

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Authors and Affiliations

  1. Stowers Institute for Medical Research, Kansas City, MO, USA

    Zulin Yu & Tamara A. Potapova

Authors
  1. Zulin Yu
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  2. Tamara A. Potapova
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Corresponding author

Correspondence to Tamara A. Potapova .

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Editors and Affiliations

  1. Department of Pathology Dunedin School of Medicine, University of Otago, Dunedin, New Zealand

    Julia Horsfield

  2. Cardiology, Division Heart & Lungs, University Medical Center Utrecht, Utrecht, The Netherlands

    Judith Marsman

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Yu, Z., Potapova, T.A. (2022). Superresolution Microscopy for Visualization of Physical Contacts Between Chromosomes at Nanoscale Resolution. In: Horsfield, J., Marsman, J. (eds) Chromatin. Methods in Molecular Biology, vol 2458. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2140-0_20

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  • DOI: https://doi.org/10.1007/978-1-0716-2140-0_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2139-4

  • Online ISBN: 978-1-0716-2140-0

  • eBook Packages: Springer Protocols

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