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.
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Acknowledgments
We thank Yuri E. Korchev, Pavel Novak and Andrew I. Shevchuk for providing advice on all stages of this project and for the guidance on the use of the HPICM Control and ScanIC Image software, Oleg A. Belov and Samir A. Rawashdeh, for their help with software manipulations, and Julián Vergara-Rojas, for his assistance during the HPICM imaging of rat inner hair cell stereocilia bundles. This work was supported by the National Institute of Deafness and Other Communication Disorders grants R01 DC008861 and R01 DC014658.
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Vélez-Ortega, A.C., Frolenkov, G.I. (2016). Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy. In: Sokolowski, B. (eds) Auditory and Vestibular Research. Methods in Molecular Biology, vol 1427. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3615-1_12
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DOI: https://doi.org/10.1007/978-1-4939-3615-1_12
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