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Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles
Hair cells—the sensory cells of the vertebrate inner ear—bear at their apical surfaces a bundle of actin-filled protrusions called stereocilia, which mediate the cells’ mechanosensitivity. Hereditary deafness is often associated with morphological disorganization of stereocilia bundles, with the abs...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8569945/ https://www.ncbi.nlm.nih.gov/pubmed/34746139 http://dx.doi.org/10.3389/fcell.2021.744248 |
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author | Ivanchenko, Maryna V. Indzhykulian, Artur A. Corey, David P. |
author_facet | Ivanchenko, Maryna V. Indzhykulian, Artur A. Corey, David P. |
author_sort | Ivanchenko, Maryna V. |
collection | PubMed |
description | Hair cells—the sensory cells of the vertebrate inner ear—bear at their apical surfaces a bundle of actin-filled protrusions called stereocilia, which mediate the cells’ mechanosensitivity. Hereditary deafness is often associated with morphological disorganization of stereocilia bundles, with the absence or mislocalization within stereocilia of specific proteins. Thus, stereocilia bundles are closely examined to understand most animal models of hereditary hearing loss. Because stereocilia have a diameter less than a wavelength of light, light microscopy is not adequate to reveal subtle changes in morphology or protein localization. Instead, electron microscopy (EM) has proven essential for understanding stereocilia bundle development, maintenance, normal function, and dysfunction in disease. Here we review a set of EM imaging techniques commonly used to study stereocilia, including optimal sample preparation and best imaging practices. These include conventional and immunogold transmission electron microscopy (TEM) and scanning electron microscopy (SEM), as well as focused-ion-beam scanning electron microscopy (FIB-SEM), which enables 3-D serial reconstruction of resin-embedded biological structures at a resolution of a few nanometers. Parameters for optimal sample preparation, fixation, immunogold labeling, metal coating and imaging are discussed. Special attention is given to protein localization in stereocilia using immunogold labeling. Finally, we describe the advantages and limitations of these EM techniques and their suitability for different types of studies. |
format | Online Article Text |
id | pubmed-8569945 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-85699452021-11-06 Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles Ivanchenko, Maryna V. Indzhykulian, Artur A. Corey, David P. Front Cell Dev Biol Cell and Developmental Biology Hair cells—the sensory cells of the vertebrate inner ear—bear at their apical surfaces a bundle of actin-filled protrusions called stereocilia, which mediate the cells’ mechanosensitivity. Hereditary deafness is often associated with morphological disorganization of stereocilia bundles, with the absence or mislocalization within stereocilia of specific proteins. Thus, stereocilia bundles are closely examined to understand most animal models of hereditary hearing loss. Because stereocilia have a diameter less than a wavelength of light, light microscopy is not adequate to reveal subtle changes in morphology or protein localization. Instead, electron microscopy (EM) has proven essential for understanding stereocilia bundle development, maintenance, normal function, and dysfunction in disease. Here we review a set of EM imaging techniques commonly used to study stereocilia, including optimal sample preparation and best imaging practices. These include conventional and immunogold transmission electron microscopy (TEM) and scanning electron microscopy (SEM), as well as focused-ion-beam scanning electron microscopy (FIB-SEM), which enables 3-D serial reconstruction of resin-embedded biological structures at a resolution of a few nanometers. Parameters for optimal sample preparation, fixation, immunogold labeling, metal coating and imaging are discussed. Special attention is given to protein localization in stereocilia using immunogold labeling. Finally, we describe the advantages and limitations of these EM techniques and their suitability for different types of studies. Frontiers Media S.A. 2021-10-22 /pmc/articles/PMC8569945/ /pubmed/34746139 http://dx.doi.org/10.3389/fcell.2021.744248 Text en Copyright © 2021 Ivanchenko, Indzhykulian and Corey. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cell and Developmental Biology Ivanchenko, Maryna V. Indzhykulian, Artur A. Corey, David P. Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles |
title | Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles |
title_full | Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles |
title_fullStr | Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles |
title_full_unstemmed | Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles |
title_short | Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles |
title_sort | electron microscopy techniques for investigating structure and composition of hair-cell stereociliary bundles |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8569945/ https://www.ncbi.nlm.nih.gov/pubmed/34746139 http://dx.doi.org/10.3389/fcell.2021.744248 |
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