Visualization of live cochlear stereocilia at a nanoscale resolution using hopping probe ion conductance microscopy

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

3 Scopus citations

Abstract

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3-4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette—which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier—is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface. Here, we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
Pages203-221
Number of pages19
DOIs
StatePublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1427
ISSN (Print)1064-3745

Bibliographical note

Funding Information:
This work was supported by the National Institute of Deafness and Other Communication Disorders grants R01 DC008861 and R01 DC014658.

Publisher Copyright:
© Springer Science+Business Media New York 2016.

Keywords

  • Hair cell
  • Live cell imaging
  • Nanopipette
  • Organ of Corti
  • Scanning ion conductance microscopy
  • Stereocilia

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Fingerprint

Dive into the research topics of 'Visualization of live cochlear stereocilia at a nanoscale resolution using hopping probe ion conductance microscopy'. Together they form a unique fingerprint.

Cite this