Name: stereocilia bundle imaging with nanoscale resolution in live mammalian auditory hair cells
Uploaded: 2021-01-21T14:00:00
Description: Watch this Scientific Journal Video about Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells at JoVE.com

我们感谢Yuri Korchev教授（英国帝国理工学院）在项目的所有阶段提供的长期支持和建议。我们还感谢Pavel Novak博士和Andrew Shevchuk博士（英国帝国理工学院）以及Oleg Belov博士（俄罗斯国家听力学研究中心）在软件开发方面的帮助。该研究得到了NIDCD / NIH的支持（R01 DC008861和R01 DC014658至G.I.F.）。

该研究是根据美国国立卫生研究院《实验动物护理和使用指南》中的建议进行的。所有动物程序均由肯塔基大学机构动物护理和使用委员会（IACUC）批准（协议00903M2005）。
1.纳米塑胶的制造和测试
<ol><li>在微量移液器拉拔器中创建一个程序，以获得电阻在200至400 MΩ之间的移液器，其对应于约50-70nm的内吸头直径。参数将取决于微量移液器拉拔器。要获得带有非柔...

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为了获得成功的HPICM图像，用户需要建立一个低噪音和低振动的系统，并制造合适的移液器。我们强烈建议在尝试进行任何活细胞成像之前使用AFM校准标准品来测试系统的稳定性。一旦测试了系统的分辨率，用户就可以考虑对Corti样本的固定器官进行成像，以熟悉成像设置，然后再尝试任何活细胞成像。
成像的最佳设定点因移液器而异，这取决于其吸头的各个形状（在电子显?...

尽管听觉毛细胞中的立体纤毛束足够大，可以通过光学显微镜观察，并在膜片钳实验中在活细胞中偏转，但转导机制的基本结构组件（例如尖端链接）只能用死细胞中的电子显微镜成像。在哺乳动物听觉毛细胞中，转导机械位于尖端链接的下端，即在短行立体纤毛1的尖端，并通过立体纤毛2，3的尖端的信号传导局部调节。然而，由于立体纤毛细胞的小尺寸，活毛细胞中该位置的表面结构的无标记成像是不可能的。
哺乳动物耳蜗有两种类型的听觉感觉细胞：内毛细胞和外毛细胞。在内毛细胞中，与外毛细胞中的发丝相比，立体纤毛更长，更厚4。第一排和第二排立体纤毛在小鼠或大鼠内毛细胞中的直径为300-500nm。由于光的衍射，使用无标记光学显微镜可以达到的最大分辨率约为200nm。因此，使用光学显微镜观察内毛细胞束第一排和第二排内的单个立体纤毛相对容易。相反，内毛细胞中较短的行立体纤毛和外毛细胞的所有立体纤毛的直径约为100-200nm，并且无法用光学显微镜5可视化。尽管最近在超分辨率成像方面取得了进展，但这一基本限制在任何光学无标记成像中都存在。目前市售的所有超分辨技术都需要某种荧光分子6，这限制了它们的应用。除了由于需要特定的荧光标记分子而受到限制之外，暴露在强光照射下已被证明会诱导细胞损伤并可能影响细胞过程，这在研究活细胞时是一个很大的缺点7。
我们目前对毛细胞立体纤毛束的超微结构细节的了解主要是通过各种电子显微镜（EM）技术获得的，例如扫描电子显微镜（SEM），透射电子显微镜（TEM），冷冻断裂EM，以及最近使用3D技术，例如使用聚焦离子束或冷冻电镜断层扫描8，9，10，11，12，13， 14，15，16 。不幸的是，所有这些EM技术都需要对样品进行化学或冷冻固定。根据现象的时间尺度，这一要求使得对立体纤毛尖端的动态过程的研究要么不可能，要么非常劳动密集型。
用原子力显微镜（AFM）对活毛细胞的毛束进行成像的努力有限，17，18。由于AFM在生理溶液中起作用，因此从理论上讲，它可以可视化活毛细胞立体纤毛束随时间变化的动态变化。问题在于高分辨率AFM的原理，这意味着AFM探头和样品之间存在一定的物理接触，即使在破坏性最小的"攻丝"模式19中也是如此。当AFM探针遇到立体纤毛时，它通常会撞到它，破坏发束的结构。因此，这种技术不适合可视化活的，甚至是固定的毛细胞束17，18。通过使用仅对样品20表面施加流体动力的大型球形AFM探头，可以部分缓解该问题。然而，即使这样的探针非常适合测试样品21的机械性能，它在对Corti22的器官成像时仅提供亚微米级的分辨率，并且仍然对样品施加对于高度敏感的立体纤毛束来说可能是巨大的力。
扫描离子电导显微镜（SICM）是扫描探针显微镜的一种版本，它使用填充有导电溶液23的玻璃移液器探针。当移液器接近细胞并且通过移液器的电流减少时，SICM会检测表面。由于这在接触细胞之前就已经发生，因此SICM非常适合在生理溶液24中对活细胞进行非接触式成像。SICM的最佳分辨率约为单纳米，这允许在活细胞25的质膜上解析单个蛋白质复合物。然而，与其他扫描探针技术类似，SICM只能对相对平坦的表面进行成像。我们通过发明跳跃探针离子电导显微镜（HPICM）26克服了这一限制，其中纳米移液器在每个成像点接近样品（图1A）。使用HPICM，我们能够以纳米级分辨率27对活听觉毛细胞中的立体纤毛束进行成像。
这种技术的另一个基本优点是HPICM不仅仅是一种成像工具。与其他扫描探针技术相比，HPICM / SICM探针是一种广泛用于细胞生理学的电极，用于各种刺激的电记录和局部传递。离子通道活性通常不会干扰HPICM成像，因为通过HPICM探针的总电流比最大离子通道25产生的细胞外电流大几个数量级。然而，HPICM允许在感兴趣的结构上精确定位纳米移液器，以及随后从该结构28进行单通道膜片钳记录。这就是我们如何获得外毛细胞立体纤毛29尖端的单通道活动的第一个初步记录。值得一提的是，由于细胞外介质的巨大分流，即使通过纳米移液管的大电流也无法在质膜上产生电位的显着变化。然而，单个离子通道可以通过液体流过纳米移液器30 或通过局部施用激动剂31以化学方式被机械激活。
在HPICM中，当纳米移液器在一个点上依次接近样品，缩回，然后向横向移动以重复接近时，就会生成图像（图1A）。膜片钳放大器不断向移液器中的AgCl线施加电压（图1B），以在浴液中产生~1 nA的电流。当移液器远离电池表面时，该电流的值被确定为参考电流（Iref，图1C）。然后，移液器在Z轴上移动以接近样品，直到电流减少用户预定义的量（设定值），通常为Iref的0.2%-1%（图1C，顶部迹线）。然后，系统将此时的Z值保存为样品的高度，以及该成像点的X和Y坐标。然后，移液器以用户定义的速度（通常为700-900 nm / ms）从表面缩回（图1C，底部迹线）。在缩回后，移液器（或者，在我们的例子中，样品 - 见图1B）被横向移动到下一个成像点，获得新的参考电流值，移液器再次接近样品，重复该过程。移液器的X-Y运动在直置显微镜设置中是首选，通常用于记录毛细胞机械转导电流。在这种情况下，HPICM探针不是从顶部以32的角度接近毛细胞束。然而，HPICM成像的最佳分辨率是在倒置显微镜设置中实现的（图1A，B），其中样品在X-Y方向上的运动与纳米移液器的Z运动脱耦合，从而消除了潜在的机械伪影。
使用HPICM，我们获得了小鼠和大鼠内外毛细胞立体纤毛束的地形图像，甚至可视化了直径约为5nm的立体纤毛之间的联系26，27。使用这种技术进行毛细胞束成像的成功取决于几个因素。首先，纳米移液器电流的噪声（方差）应尽可能小，以便为HPICM成像提供尽可能低的设定点。低设定值允许HPICM探针在更远的距离和与探针方法的任何角度"感知"立体纤毛表面，并且令人惊讶的是，提高了HPICM成像的X-Y分辨率（参见讨论）。其次，系统中的振动和漂移应减少到小于10nm，因为它们直接导致成像伪影。最后，即使HPICM探头和样品台通过具有单个纳米或更高精度的校准反馈控制压电致动器在Z轴和X-Y轴上移动，纳米移液器尖端的直径也决定了电流的扩散（传感体积）和分辨率（图1A）。因此，在对活毛细胞进行成像之前，至关重要的是要拉动足够的移液器，用校准样品达到所需的分辨率，并在记录系统中实现低噪声。
至少几十年来，SICM技术尚未商业化，世界上只有少数几个实验室与帝国理工学院（英国）的Korchev教授的领先实验室一起开发。最近，几种SICM系统开始商业化（见 材料表），所有这些都基于原始的HPICM原理。然而，对毛细胞中的立体纤毛束进行成像需要进行一些定制修改，这些修改在封闭的"准备就绪"系统中在技术上具有挑战性（甚至是不可能的）。因此，需要一些组件集成。由于HPICM设置代表了一个具有更严格的振动和漂移要求以及HPICM探头和样品的压电驱动运动的膜片钳装置（图1D），因此对于任何精通膜片钳的研究人员来说，这种集成都相对容易。然而，一个没有适当背景的科学家肯定首先需要一些电生理学培训。尽管仍然存在挑战，例如提高成像速度（参见 讨论），但我们已经能够以纳米级分辨率对活毛细胞中的立体纤毛束进行成像而不会损坏它们。
本文提出了一个详细的方案，使用我们的定制系统对年轻的产后大鼠或小鼠耳蜗外植体中的活听觉毛细胞束进行成功的HPICM成像。集成组件列在 材料表。本文还介绍了可能遇到的常见问题以及如何排除故障。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analog oscilloscope</td>
			<td>B&#38;K Precision</td>
			<td>2160C</td>
			<td>Analog oscilloscope for real-time monitoring of nanopipette current and Z-axis approach</td>
		</tr>
		<tr>
			<td>AFM calibration standards</td>
			<td>TED PELLA Inc</td>
			<td>HS-100MG; HS-20MG</td>
			<td>These 100 and 20 nm calibration standards are used to test the performance of HPICM system</td>
		</tr>
		<tr>
			<td>Benchtop vibration Isolator</td>
			<td>AMETEK/TMC</td>
			<td>Everstill K-400</td>
			<td>Active vibration isolation</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments (WPI)</td>
			<td>1B100F-4</td>
			<td>Borosilicate glass capillaries for the nanopipettes</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>To be added to the bath solution to adjust osmolarity</td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>National Instruments Corporation</td>
			<td>PCI-6221</td>
			<td>Multi-channel input/output digitizer</td>
		</tr>
		<tr>
			<td>Fast analog Proportional-Integral-Derivative (PID) control for Z movement</td>
			<td>Standford Research Systems</td>
			<td>SIM900, SIM960, SIM980</td>
			<td>Instrumentation modules integrated in an external PID controller for Z movement. It requires a fast response that is usually not implemented in commercial piezo amplifiers.</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>AMETEK/TMC</td>
			<td>Type II</td>
			<td>Required to shield electromagnetic interference</td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>World Precision Instruments (WPI)</td>
			<td>FD5040-100</td>
			<td>Used as the dish for the chamber for the tissue</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>14025092</td>
			<td>Extracellular (bath) solution</td>
		</tr>
		<tr>
			<td>Instrumentation amplifier</td>
			<td>Brownlee Precision</td>
			<td>Model 440</td>
			<td>Instrumentation amplifier provides required offsets, filtering, and secondary magnification or attenuation</td>
		</tr>
		<tr>
			<td>Laser-based micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-2000/G</td>
			<td>Micropipette puller to fabricate the nanopipettes. Laser is needed for sharp quartz pipettes.</td>
		</tr>
		<tr>
			<td>Lebovitz&#39;s L-15, without phenol red</td>
			<td>Gibco, Thermo Fisher Scientific</td>
			<td>21083027</td>
			<td>Extracellular (bath) solution</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Scientifica</td>
			<td>PatchStar</td>
			<td>Used for &#34;course&#34; positioning of the Z piezo actuator</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td>Inverted optical microscope</td>
		</tr>
		<tr>
			<td>Patch amplifier</td>
			<td>Molecular Devices</td>
			<td>Axopatch 200B</td>
			<td>The patch clamp amplifier measures the current through the nanopipette</td>
		</tr>
		<tr>
			<td>Piezo amplifier (XY axes)</td>
			<td>Physik Instrumente (PI)</td>
			<td>E-500.00, E-505.00, E-509.C2A</td>
			<td>Amplification and PID control for XY piezo translation stage</td>
		</tr>
		<tr>
			<td>Piezo amplifier (Z axis)</td>
			<td>Piezosystem jena</td>
			<td>ENT 400 &#38; 800</td>
			<td>Custom amplifier consisting of ENT 400 power supply and two ENT 800 amplifiers in parallel to achieve max current of 1.6 nA</td>
		</tr>
		<tr>
			<td>Plastic Coverslips</td>
			<td>TED PELLA Inc</td>
			<td>26028</td>
			<td>Used in the fabrication of the chambers for the tissue&#160;</td>
		</tr>
		<tr>
			<td>SICM controller &#38; software*</td>
			<td>Ionscope, UK (ionscope.com)</td>
			<td>N/A</td>
			<td>Custom controller based on SBC6711 digital signal processing board from Innovative Integration Ltd</td>
		</tr>
		<tr>
			<td>Silicone elastomer (Sylgard)</td>
			<td>World Precision Instruments (WPI)</td>
			<td>SYLG184</td>
			<td>Used to attach the flexible glass fibers to the chamber for the tissue</td>
		</tr>
		<tr>
			<td>Silicon glue</td>
			<td>The Dow Chemical Company</td>
			<td>734</td>
			<td>Used to glue the different parts of the chamber for the tissue</td>
		</tr>
		<tr>
			<td>Tungsten rod</td>
			<td>A-M Systems</td>
			<td>717500</td>
			<td>Used for holding the dental floss strands in the chamber for the tissue</td>
		</tr>
		<tr>
			<td>XY piezo nanopositioner</td>
			<td>Physik Instrumente (PI)</td>
			<td>P-733.2DD</td>
			<td>XY translation stage with capacitive sensors</td>
		</tr>
		<tr>
			<td>Z piezo nanopositioner</td>
			<td>Piezosystem jena</td>
			<td>RA 12/24 SG</td>
			<td>Ring piezoactuator with a strain gage sensor</td>
		</tr>
		<tr>
			<td colspan="4">*Ionscope does not sell separate SICM controllers anymore. There are few other commercial systems:&#160; NX12-Bio and NX10 SICM,&#160;</td>
		</tr>
		<tr>
			<td colspan="4">Park Systems, Korea and SICM modules from ICAPPIC Limited, UK (icappic.com). All these systems are based on the original&#160;</td>
		</tr>
		<tr>
			<td colspan="4">HPICM principles. However, imaging stereocilia bundles in the hair cells requires several custom modifications that are technically&#160;</td>
		</tr>
		<tr>
			<td colspan="4">challenging (or even impossible) in the closed &#8220;ready-to-go&#8221; systems such as Ionscope or NX12-Bio/NX10. Currently, there is only one&#160;</td>
		</tr>
		<tr>
			<td colspan="4">modular system (ICAPPIC) that has the flexibility to suit any SICM/HPICM experiment but requires some component integration.&#160;</td>
		</tr>
	</tbody>
</table>

stereocilia bundle imaging with nanoscale resolution in live mammalian auditory hair cells

内耳毛细胞检测声音诱导的位移，并将这些刺激转化为发束中的电信号，该发束由立体纤毛组成，这些发束排列成高度增加的行。当立体纤毛偏转时，它们会拉扯微小的（直径约5nm）细胞外尖端连接，这些细胞外尖端链接相互连接立体纤毛，从而将力传递到机械敏感转导通道。尽管机械纵促在活毛细胞中已经研究了几十年，但立体纤毛尖端的机电转导机制在功能上重要的超微结构细节（例如尖端链接动力学或转导依赖性立体纤毛重塑）仍然只能在具有电子显微镜的死细胞中进行研究。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立体纤毛的表面。然而，与成像模式无关，即使原子力显微镜探针与立体纤毛束最轻微的接触通常也会损坏束。在这里，我们提出了活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对具有复杂形貌的活细胞表面进行延时成像，如毛细胞，具有单纳米分辨率，并且不与样品进行物理接触。HPICM使用通过玻璃纳米移液器的电流来检测移液器附近的细胞表面，而3D定位压电系统扫描表面并生成其图像。使用HPICM，我们能够对立体纤毛束和连接活听觉毛细胞中的立体纤毛束和连接立体纤毛细胞数小时，而不会造成明显的损伤。我们预计，HPICM的使用将允许直接探索活毛细胞立体纤毛的超微结构变化，以更好地了解其功能。

本文中提出的方案可用于可视化具有复杂地形的任何活细胞。按照这些步骤，我们常规获得活大鼠听觉毛细胞束的图像（图6B，D）。尽管与SEM图像相比，我们的X-Y分辨率较低，但我们的HPICM图像可以成功地分辨出立体纤毛的不同行，立体纤毛尖端的形状，甚至是连接相邻立体纤毛的小链接（直径约5nm）（图6F）。此外，HPICM图像具有SEM图像?...

Watch this Scientific Journal Video about Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells at JoVE.com

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

在这里，我们提出了跳跃探针离子电导显微镜（HPICM）的方案，这是一种非接触式扫描探针技术，允许对活听觉毛细胞中的立体纤毛束进行纳米级成像。

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia ...

这种实用性可以帮助我们研究在毛细胞立体纤毛束表面的机械电转导机械的纳米结构组分的动态变化。该技术的主要优点是在单个纳米分辨率下对具有复杂形貌的活细胞表面进行延时成像，并且不与样品进行物理接触。这种技术几乎可以应用于任何活细胞。我们之前在My Image Bacteria Soon中匹配了感染病毒的肺上皮细胞系的表面，肌肉细胞。在每次成像开始时测试纳米颗粒。制造纳米移液器后，检查气泡，去除任何气泡（如果存在），然后将纳米移液器安装到跳跃探针离子电导显微镜移液器支架上。在四毫升的浴液中溶液到腔室中，并将腔室置于HPICM级上。将接地电极引入浴液，并确保通过膜片钳放大器施加到移液器的电压为零。将微型机械手置于垂直位置后，将移液器移至Z中，直到其接触液体并将放大器偏移设置为零，然后加100毫伏以检查移液器电流。使用硅胶将AFM校准标准品连接到腔室，并用四毫升HBSS覆盖样品。使用双面胶带将腔室固定到HPICM设置的X Y级，并如图所示将新的纳米移液器加载到支架上。测试纳米移液器电阻和直径后，将电流设置为一纳安培。使用粗糙的膜片钳机械手将纳米移液器定位在校准标准中心上方，并增加设定点，同时实时监测示波器上Z压电致动器传感器的信号。在建立稳定的可重复Z进近循环后，将设定点降低到刚好高于不稳定点的值，并以大约每秒5微米的速度向下移动移液器，直到它到达样品。实时Z定位信号的底部水平将增加，表明纳米移液器由于感测样品表面而被撤回。由于AFM标准上的安装不均匀，感兴趣区域的最高点可能是未知的。因此，将移液器缩回的振幅设置为至少200至500纳米。注意不要超过Z压电致动器运动的上限，以低分辨率开始成像。一旦确定了样品在成像区域的最高点，请降低跳幅并沿Z轴缩回移液器约200微米，以防止在将样品移动到新的X Y位置之前与样品发生任何不希望的碰撞。找到感兴趣区域后，开始以更高的分辨率成像。对于听觉毛细胞成像，使用牙线或柔性玻璃移液器将刚分离的Corti器官牢固地固定到腔室中。使用双面胶带将腔室牢固地固定到X Y压电级，并如图所示将新的纳米移液器加载到支架上。检查纳米移液器电阻后，使用膜片钳微型机械手将纳米移液器定位在毛细胞区域，同时在倒置显微镜中观察Corti X植物的器官。在示波器上记录实时电流和Z定位信号，以检查系统是否稳定，设定点为0.5%6或更低，并确定最佳设定点并如图所示接近样品。使用至少6至8微米的跳跃幅度对样品进行低分辨率成像。如果纳米移液器需要移动到新的X Y位置，请将移液器缩回约500纳米以避免与组织内的任何高特征发生碰撞，并重复低分辨率HPICM成像，直到毛细胞所在的感兴趣区域。然后在15分钟或更短的时间内以更高的分辨率对感兴趣区域进行成像。HPICM方案可用于可视化具有复杂地形的任何活细胞，例如活大鼠听觉毛细胞束。尽管与扫描电子显微镜图像相比，HPICM图像显示出较低的X Y分辨率，但HPICM图像可以成功解析不同的立体纤毛行。立体纤毛尖端的形状，甚至是连接相邻立体纤毛的小五纳米链接。鉴于HPICM成像的非接触性质，可以对同一毛细胞束进行连续延时成像数小时，而不会损害束粘性。请注意，在设定点非常低的情况下，系统可能会将电流的微小波动解释为遇到电池表面，从而导致图像中的白点噪声。同样，较大的跳跃幅度可能会增加移液器的横向共振，也会导致产生噪声像素。相反，如果跳跃幅度太小，或者设定值太高，纳米移液器可能会与样品碰撞，导致成像伪影或毛束损坏。尝试此过程时要记住的最重要的事情是在处理活细胞时，每个毛细胞束花费的时间少于15分钟。在获得发束的图像后，可以尝试从立体纤毛表面的特定位置获取单通道记录，以回答有关机械加速器通道属性的问题。

Research

JoVE Journal

Neuroscience

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

科研

神经科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

神经元的密集核心囊泡的超分辨率成像

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

高分辨率<em&gt;在体内</em&gt;使用3T磁共振成像手动分割协议为人类海马子字段

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Optogenetic Stimulation of the Auditory Nerve

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...