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Methods Mol Biol. 2017;1538:169-184.

3D d STORM Imaging of Fixed Brain Tissue.

Author information

1
Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, 69120, Heidelberg, Germany.
2
Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany.
3
Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, 69120, Heidelberg, Germany. kuner@uni-heidelberg.de.

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.

KEYWORDS:

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

PMID:
27943191
DOI:
10.1007/978-1-4939-6688-2_13
[Indexed for MEDLINE]

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