title: 3D d STORM Imaging of Fixed Brain Tissue.
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.
authors.affiliation: Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, 69120, Heidelberg, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Herrmannsdörfer
authors.affiliation: Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, 69120, Heidelberg, Germany.|Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Flottmann
authors.affiliation: Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, 69120, Heidelberg, Germany.
authors.forename: Siddarth
authors.initials: S
authors.lastname: Nanguneri
authors.affiliation: Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, 69120, Heidelberg, Germany.
authors.forename: Varun
authors.initials: V
authors.lastname: Venkataramani
authors.affiliation: Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, 69120, Heidelberg, Germany.
authors.forename: Heinz
authors.initials: H
authors.lastname: Horstmann
authors.affiliation: Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, 69120, Heidelberg, Germany. kuner@uni-heidelberg.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kuner
authors.affiliation: Department of Functional Neuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg University, Im Neuenheimer Feld 307, 69120, Heidelberg, Germany.|Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany.
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
mesh_terms: D015415:Biomarkers; D001921:Brain / Q000000981:diagnostic imaging* / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D000072760:Single Molecule Imaging / Q000379:methods*; D014054:Tomography / Q000379:methods*
chemical_list: D015415:Biomarkers
keywords: Brain tissue fixation; STORM-data postprocessing; Super-resolution microscopy; Thin sectioning; dSTORM; tomoSTORM