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3D d STORM Imaging of Fixed Brain Tissue

  • Frank Herrmannsdörfer
  • Benjamin Flottmann
  • Siddarth Nanguneri
  • Varun Venkataramani
  • Heinz Horstmann
  • Thomas Kuner
  • Mike Heilemann
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1538)

Abstract

Central nervous system tissue contains a high density of synapses each composed of an intricate molecular machinery mediating precise transmission of information. Deciphering the molecular nanostructure of pre- and postsynaptic specializations within such a complex tissue architecture poses a particular challenge for light microscopy. Here, we describe two approaches suitable to examine the molecular nanostructure of synapses at 20–30 nm lateral and 50–70 nm axial resolution within an area of 500 μm × 500 μm and a depth of 0.6 μm to several micrometers. We employ single-molecule localization microscopy (SMLM) on immunolabeled fixed brain tissue slices. tomoSTORM utilizes array tomography to achieve SMLM in 40 nm thick resin-embedded sections. dSTORM of cryo-sectioned slices uses optical sectioning in 0.1–4 μm thick hydrated sections. Both approaches deliver 3D nanolocalization of two or more labeled proteins within a defined tissue volume. We review sample preparation, data acquisition, analysis, and interpretation.

Key words

Super-resolution microscopy dSTORM tomoSTORM Brain tissue fixation Thin sectioning STORM-data postprocessing 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Frank Herrmannsdörfer
    • 1
  • Benjamin Flottmann
    • 1
    • 2
  • Siddarth Nanguneri
    • 1
  • Varun Venkataramani
    • 1
  • Heinz Horstmann
    • 1
  • Thomas Kuner
    • 1
  • Mike Heilemann
    • 1
    • 2
  1. 1.Department of Functional Neuroanatomy, Institute for Anatomy and Cell BiologyHeidelberg UniversityHeidelbergGermany
  2. 2.Single Molecule Biophysics, Institute of Physical and Theoretical ChemistryGoethe-University FrankfurtFrankfurtGermany

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