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Two-Photon STED Microscopy for Nanoscale Imaging of Neural Morphology In Vivo

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Super-Resolution Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1663))

Abstract

The advent of super-resolution microscopy offers to bridge the gap between electron and light microscopy. It has opened up the possibility of visualizing cellular structures and dynamic signaling events on the “mesoscale” well below the classic diffraction barrier of light microscopy (10–200 nm), while essentially retaining the advantages of fluorescence microscopy concerning multicolor labeling, detection sensitivity, signal contrast, live-cell imaging, and temporal resolution.

From among the new super-resolution techniques, STED microscopy stands out as a laser-scanning imaging modality, which enables nanoscale volume-metric imaging of cellular morphology. In combination with two-photon (2P) excitation, STED microscopy facilitates the visualization of the highly complex and dynamic morphology of neurons and glia cells deep inside living brain slices and in the intact brain in vivo.

Here, we present an overview of the principles and implementation of 2P-STED microscopy in vivo, providing the neurobiological context and motivation for this technique, and illustrating its capacity by showing images of dendritic spines and microglial processes obtained from living brain tissue.

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Acknowledgments

This work was supported by a PhD fellowship from the “European Neuroscience Campus” network (ENC) to MJTTV, a Boehringer Ingelheim PhD fellowship and a 4th-year extension grant from the “Fondation pour la Recherche Médicale” (FRM, FDT20160435677) to TP, and grants from the “Agence Nationale de la Recherche” (ANR) (ANR-13-BSV4-0007-01), France-BioImaging (ANR-10-INSB-04), and European Research Area Network NEURON II (ANR-12-NEUR-0007-03) to UVN. We thank Florian Levet (CNRS, University of Bordeaux) for the custom-made image analysis program.

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Author notes

  1. Mirelle J. T. ter Veer and Thomas Pfeiffer contributed equally to this work.

Authors and Affiliations

  1. Interdisciplinary Institute for Neuroscience, UMR 5297 CNRS, 146 rue Leo Saignat, 33077, Bordeaux, France

    Mirelle J. T. ter Veer, Thomas Pfeiffer & U. Valentin Nägerl

  2. Université de Bordeaux, Bordeaux, France

    Mirelle J. T. ter Veer, Thomas Pfeiffer & U. Valentin Nägerl

Authors
  1. Mirelle J. T. ter Veer
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  2. Thomas Pfeiffer
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  3. U. Valentin Nägerl
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Corresponding author

Correspondence to U. Valentin Nägerl .

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Editors and Affiliations

  1. High-Content Analysis of the Cell (HiCell) and Advanced Biological Screening Facility, BioQuant, Heidelberg University, Heidelberg, Germany

    Holger Erfle

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ter Veer, M.J.T., Pfeiffer, T., Nägerl, U.V. (2017). Two-Photon STED Microscopy for Nanoscale Imaging of Neural Morphology In Vivo. In: Erfle, H. (eds) Super-Resolution Microscopy. Methods in Molecular Biology, vol 1663. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7265-4_5

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  • DOI: https://doi.org/10.1007/978-1-4939-7265-4_5

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7264-7

  • Online ISBN: 978-1-4939-7265-4

  • eBook Packages: Springer Protocols

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