Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles
Open Access
- 22 October 2021
- journal article
- research article
- Published by Frontiers Media SA in Frontiers in Cell and Developmental Biology
Abstract
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.Keywords
Funding Information
- National Institute on Deafness and Other Communication Disorders (DC017166, DC002281, DC016932)
This publication has 86 references indexed in Scilit:
- Scanning Electron MicroscopyCurrent Protocols in Microbiology, 2012
- Striated organelle, a cytoskeletal structure positioned to modulate hair-cell transductionProceedings of the National Academy of Sciences of the United States of America, 2012
- Network anatomy and in vivo physiology of visual cortical neuronsNature, 2011
- Stereocilin connects outer hair cell stereocilia to one another and to the tectorial membraneJournal of Comparative Neurology, 2010
- Asymmetric distribution of cadherin 23 and protocadherin 15 in the kinocilial links of avian sensory hair cellsJournal of Comparative Neurology, 2010
- Tip links in hair cells: molecular composition and role in hearing lossCurrent Opinion in Otolaryngology & Head and Neck Surgery, 2009
- Harmonin Mutations Cause Mechanotransduction Defects in Cochlear Hair CellsNeuron, 2009
- Localization of inner hair cell mechanotransducer channels using high-speed calcium imagingNature Neuroscience, 2009
- Three-dimensional Architecture of Hair-bundle Linkages Revealed by Electron-microscopic TomographyJournal of the Association for Research in Otolaryngology, 2008
- The deaf mouse mutant whirler suggests a role for whirlin in actin filament dynamics and stereocilia developmentCell Motility, 2007