Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy.
Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy.
复制标题
使用跳跃探针离子电导显微镜以纳米级分辨率可视化活体耳蜗立体纤毛。
DOI：
10.1007/978-1-4939-3615-1_12
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Frolenkov,GregoryI
中科院分区：
文献类型：
--
作者：
Vélez-Ortega,ACatalina;Frolenkov,GregoryI
检测耳蜗中声音引起的振动的机械感觉装置位于听觉感觉毛细胞的顶端，它由充满肌动蛋白的突起组成，称为静纤毛。幼年啮齿动物听毛细胞的静纤毛束由3-4排高度递减、粗细不等的静纤毛组成。活毛细胞中的听觉静纤毛束的形态学研究一直具有挑战性，因为每个静纤毛的直径接近或低于光学显微镜的分辨率极限。从理论上讲，扫描探针显微镜技术，如原子力显微镜，可以在纳米级分辨率下观察活细胞的表面。然而，它们用于毛细胞成像的实现在很大程度上是不成功的，因为探针通常在成像期间损坏束并破坏束的内聚性。我们克服了这些限制，使用跳跃探针离子电导显微镜（HPICM），非接触式扫描探针技术，非常适合活细胞的成像与复杂的地形。Corti器官外植体放置在生理溶液中，然后将玻璃纳米移液管连接到3D定位压电系统和膜片钳放大器，用于以纳米分辨率扫描活毛细胞的表面，而无需接触细胞表面。在这里，我们提供了一个详细的协议，用于小鼠或大鼠静纤毛束在活听觉毛细胞中使用HPICM成像。我们提供了有关纳米移液管的制造，HPICM设置的校准，我们已经优化了活静纤毛束成像的参数，最后，一些基本的图像后处理操作的信息。
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.