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Nanoscale Imaging pp 339–359Cite as

Correlative Atomic Force and Single-Molecule Fluorescence Microscopy of Nucleoprotein Complexes

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1814))

Abstract

Correlative imaging by fluorescence and atomic force microscopy provides a versatile tool to extract orthogonal information on structurally heterogeneous biomolecular assemblies. In this chapter, we describe an integrated setup for correlative fluorescence and force microscopy. We present factors influencing data quality, as well as step-by-step protocols for sample preparation, data acquisition, and data processing that yield nanoscale topographic resolution, high image registration accuracy, and single-fluorophore sensitivity. We demonstrate the capabilities of the approach through simultaneous characterization of mesoscale geometry and composition in a multipart nucleoprotein complex.

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Acknowledgments

We thank Dr. Wolfgang Wende for kindly providing the EcoRV (C21S/K58C) expression plasmid. We acknowledge funding from KU Leuven through the IDO program for financial support; WF, SR, and WV like to thank Fonds Wetenschappelijk Onderzoek (FWO) for personal fellowships.

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Author notes

  1. Herlinde De Keersmaecker and Wout Frederickx contributed equally to this work.

Authors and Affiliations

  1. Division of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven-University of Leuven, Leuven, Belgium

    Herlinde De Keersmaecker, Wout Frederickx, Yasuhiko Fujita, Steven De Feyter, Hiroshi Uji-i, Susana Rocha & Willem Vanderlinden

  2. Research Institute for Electronic Science, Nanomaterials and Nanoscopy, Hokkaido University, Sapporo, Japan

    Hiroshi Uji-i

  3. Department of Physics, Nanosystems Initiative Munich, and Center for NanoScience, LMU Munich, Munich, Germany

    Willem Vanderlinden

Authors
  1. Herlinde De Keersmaecker
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  2. Wout Frederickx
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  3. Yasuhiko Fujita
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  4. Steven De Feyter
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  5. Hiroshi Uji-i
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  6. Susana Rocha
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  7. Willem Vanderlinden
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Corresponding author

Correspondence to Willem Vanderlinden .

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Editors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA

    Yuri L. Lyubchenko

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De Keersmaecker, H. et al. (2018). Correlative Atomic Force and Single-Molecule Fluorescence Microscopy of Nucleoprotein Complexes. In: Lyubchenko, Y. (eds) Nanoscale Imaging. Methods in Molecular Biology, vol 1814. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8591-3_20

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  • DOI: https://doi.org/10.1007/978-1-4939-8591-3_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8590-6

  • Online ISBN: 978-1-4939-8591-3

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