title: Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome.
abstract: Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.
authors.affiliation: University of Würzburg , Biocenter, Division of Electron Microscopy, Am Hubland, Würzburg 97074, Germany.
authors.forename: Sebastian Matthias
authors.initials: SM
authors.lastname: Markert
authors.affiliation: University of Würzburg , Biocenter, Division of Electron Microscopy, Am Hubland, Würzburg 97074, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Britz
authors.affiliation: University of Würzburg, Department of Biotechnology and Biophysics, Am Hubland, Würzburg 97074, Germany; University of Würzburg, Department of Neurophysiology, Institute of Physiology, Röntgenring 9, Würzburg 97070, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Proppert
authors.affiliation: University of Würzburg , Biocenter, Division of Electron Microscopy, Am Hubland, Würzburg 97074, Germany.
authors.forename: Marietta
authors.initials: M
authors.lastname: Lang
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada; University of Toronto, Department of Molecular Genetics, Physiology and Institute of Medical Science, 1 King's College Cir, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Witvliet
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada; University of Toronto, Department of Molecular Genetics, Physiology and Institute of Medical Science, 1 King's College Cir, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Ben
authors.initials: B
authors.lastname: Mulcahy
authors.affiliation: University of Würzburg , Department of Biotechnology and Biophysics, Am Hubland, Würzburg 97074, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada; University of Toronto, Department of Molecular Genetics, Physiology and Institute of Medical Science, 1 King's College Cir, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Mei
authors.initials: M
authors.lastname: Zhen
authors.affiliation: Institut NeuroMyoGene, Université Claude Bernard Lyon 1 , CNRS UMR 5310, INSERM U1217, 16 rue R. Dubois, Villeurbanne Cedex F-69622, France.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Bessereau
authors.affiliation: University of Würzburg , Biocenter, Division of Electron Microscopy, Am Hubland, Würzburg 97074, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Stigloher
keywords: Caenorhabditis elegans; correlative light and electron microscopy; direct stochastic optical reconstruction microscopy; gap junction; structured illumination microscopy; super-resolution microscopy