Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome

Sebastian Matthias Markert; Sebastian Britz; Sven Proppert; Marietta Lang; Daniel Witvliet; Ben Mulcahy; Markus Sauer; Mei Zhen; Jean-Louis Bessereau; Christian Stigloher

doi:10.1117/1.NPh.3.4.041802

4 May 2016 Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome

Sebastian Matthias Markert, Sebastian Britz, Sven Proppert, Marietta Lang, Daniel Witvliet, Ben Mulcahy, Markus Sauer, Mei Zhen, Jean-Louis Bessereau, Christian Stigloher

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Neurophotonics, Vol. 3, Issue 4, 041802 (May 2016). https://doi.org/10.1117/1.NPh.3.4.041802

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.

CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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Sebastian Matthias Markert, Sebastian Britz, Sven Proppert, Marietta Lang, Daniel Witvliet, Ben Mulcahy, Markus Sauer, Mei Zhen, Jean-Louis Bessereau, and Christian Stigloher "Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome," Neurophotonics 3(4), 041802 (4 May 2016). https://doi.org/10.1117/1.NPh.3.4.041802

Published: 4 May 2016

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