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

Please always quote using this URN: urn:nbn:de:bvb:20-opus-187292
  • 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 protocolsCorrelating 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.show moreshow less

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Metadaten
Author: Sebastian Matthias Markert, Sebastian Britz, Sven Proppert, Marietta Lang, Daniel Witvliet, Ben Mulcahy, Markus Sauer, Mei Zhen, Jean-Louis Bessereau, Christian Stigloher
URN:urn:nbn:de:bvb:20-opus-187292
Document Type:Journal article
Faculties:Medizinische Fakultät / Physiologisches Institut
Fakultät für Biologie / Theodor-Boveri-Institut für Biowissenschaften
Language:English
Parent Title (English):Neurophotonics
Year of Completion:2016
Volume:3
Issue:4
Pagenumber:041802
Source:Neurophotonics (2016) 3:4, 041802. https://doi.org/10.1117/1.NPh.3.4.041802
DOI:https://doi.org/10.1117/1.NPh.3.4.041802
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Tag:caenorhabditis elegans; correlative light and electron microscopy; direct stochasticoptical reconstruction microscopy; fluorescent-probes; gap junction; image data; innexins; junction proteins; localization micoscopy; nervous-system; neural circuits; reconstruction; resolution limit; structured illumination microscopy; super-resolution microscopy
Release Date:2020/06/10
Licence (German):License LogoCC BY: Creative-Commons-Lizenz: Namensnennung