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Single-Molecule Localization Microscopy of Subcellular Protein Distribution in Neurons

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Fluorescent Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2440))

Abstract

Over the past years several forms of superresolution fluorescence microscopy have been developed that offer the possibility to study cellular structures and protein distribution at a resolution well below the diffraction limit of conventional fluorescence microscopy (<200 nm). A particularly powerful superresolution technique is single-molecule localization microscopy (SMLM). SMLM enables the quantitative investigation of subcellular protein distribution at a spatial resolution up to tenfold higher than conventional imaging, even in live cells. Not surprisingly, SMLM has therefore been used in many applications in biology, including neuroscience. This chapter provides a step-by-step SMLM protocol to visualize the nanoscale organization of endogenous proteins in dissociated neurons but can be extended to image other adherent cultured cells. We outline a number of methods to visualize endogenous proteins in neurons for live-cell and fixed application, including immunolabeling, the use of intrabodies for live-cell SMLM, and endogenous tagging using CRISPR/Cas9.

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Acknowledgments

This work was supported by the Netherlands Organization for Scientific Research (ALW-VIDI 171.029 to H.D.M.) and the European Research Council (ERC-StG 716011 to H.D.M.).

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Author notes

  1. Jelmer Willems and Manon Westra contributed equally.

Authors and Affiliations

  1. Division of Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands

    Jelmer Willems, Manon Westra & Harold D. MacGillavry

Authors
  1. Jelmer Willems
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  2. Manon Westra
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  3. Harold D. MacGillavry
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Corresponding author

Correspondence to Harold D. MacGillavry .

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Editors and Affiliations

  1. Department of Microbiology and Immunology, Center for Human Immunology, The University of Western Ontario, London, ON, Canada

    Bryan Heit

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Willems, J., Westra, M., MacGillavry, H.D. (2022). Single-Molecule Localization Microscopy of Subcellular Protein Distribution in Neurons. In: Heit, B. (eds) Fluorescent Microscopy. Methods in Molecular Biology, vol 2440. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2051-9_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2051-9_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2050-2

  • Online ISBN: 978-1-0716-2051-9

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