Single-Molecule Localization Microscopy of Subcellular Protein Distribution in Neurons

Jelmer Willems; Manon Westra; Harold D MacGillavry

doi:10.1007/978-1-0716-2051-9_16

Single-Molecule Localization Microscopy of Subcellular Protein Distribution in Neurons

Jelmer Willems
, Manon Westra
, Harold D MacGillavry

Cell Biology, Neurobiology and Biophysics

Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic › peer-review

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.

Original language	English
Title of host publication	Fluorescent Microscopy
Editors	Bryan Heit
Place of Publication	New York
Publisher	Humana Press
Pages	271-288
Number of pages	18
Edition	1
ISBN (Electronic)	978-1-0716-2051-9
ISBN (Print)	978-1-0716-2050-2, 978-1-0716-2053-3
DOIs	https://doi.org/10.1007/978-1-0716-2051-9_16
Publication status	Published - 27 Feb 2022

Publication series

Name	Methods in Molecular Biology
Publisher	Humana Press
Volume	2440
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Bibliographical note

Funding Information:
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.).

Publisher Copyright:
© 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

Neuron
Photoactivated localization microscopy
Single-molecule localization microscopy
Stochastic optical reconstruction microscopy
Superresolution microscopy
Synapse

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10.1007/978-1-0716-2051-9_16

978-1-0716-2051-9_16Final published version, 600 KBLicence: Taverne

Cite this

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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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Single-Molecule Localization Microscopy of Subcellular Protein Distribution in Neurons. / Willems, Jelmer; Westra, Manon; MacGillavry, Harold D.
Fluorescent Microscopy. ed. / Bryan Heit. 1. ed. New York: Humana Press, 2022. p. 271-288 (Methods in Molecular Biology; Vol. 2440).

Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic › peer-review

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Single-Molecule Localization Microscopy of Subcellular Protein Distribution in Neurons

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