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活哺乳动物听觉毛细胞的纳米分辨率立体纤毛束成像。

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells.

机构信息

Department of Physiology, College of Medicine, University of Kentucky; Universidad Nacional de Colombia.

Department of Physiology, College of Medicine, University of Kentucky;

出版信息

J Vis Exp. 2021 Jan 21(167). doi: 10.3791/62104.

Abstract

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

摘要

内耳毛细胞检测到声音引起的位移,并将这些刺激转化为毛束中的电信号,毛束由排列成逐渐增加高度的行的纤毛组成。当纤毛偏转时,它们会拉动连接纤毛的微小(直径约为 5nm)细胞外尖端连接,将力传递到机械敏感转导通道。尽管几十年来一直在活体毛细胞中研究机械转导,但纤毛尖端机械转导机制的功能重要超微结构细节(例如尖端连接的动态或转导依赖性纤毛重塑)仍然只能通过电子显微镜在死细胞中进行研究。从理论上讲,扫描探针技术(如原子力显微镜)具有足够的分辨率来可视化纤毛的表面。然而,无论成像模式如何,即使原子力显微镜探针与纤毛束的最轻微接触通常也会损坏纤毛束。在这里,我们提出了一种用于活的啮齿动物听觉毛细胞的跳跃探针离子电导显微镜(HPICM)成像的详细方案。这种非接触式扫描探针技术允许使用具有复杂形貌的活细胞(如毛细胞)进行时间推移成像,具有单纳米分辨率,并且不与样品进行物理接触。HPICM 使用通过玻璃纳米管的电流来检测靠近管的细胞表面,而 3D 定位压电系统扫描表面并生成其图像。通过 HPICM,我们能够在不造成明显损坏的情况下,对活听觉毛细胞中的纤毛束和连接纤毛的连接进行数小时的成像。我们预计,HPICM 的使用将允许直接探索活毛细胞纤毛的超微结构变化,以更好地理解其功能。

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