我们特别感谢伊夫Couder许多有益的讨论。我们感谢阿提夫Asnacios的悬臂和讨论的校准。我们感谢丽莎·威利斯，艾略特迈耶罗维茨，和奥利弗Hamant批判性的阅读。这项工作被资助了一部分由人类前沿科学计划授出RGP0062/2005-C;在法新社国立德拉RECHERCHE项目&#39;&#39; Growpec，&#39;&#39;和&#39;&#39;&#39;&#39; Mechastem。

1，准备玻璃幻灯片的安装示例<ol><li>准备嵌入琼脂糖介质：10％甘露糖醇（在水中）0.7％低熔点琼脂糖。 </li><li>使用强金属工具（ 如钻头尖，酸橙），蚀刻出一0.5×0.5厘米面积在显微镜载玻片上的中心位置。或者相反，粘上一小块玻璃薄片（约20×200微米），以使用爱牢达胶水的载玻片上。注：此变粗糙的表面，以便于琼脂糖的附着力，以确保琼脂糖介质棒或修复的幻灯片。该滑动可重复使用。 </li><li>放置约20微升嵌入琼脂糖介质到玻璃载片的蚀刻区域的液滴。这使琼脂糖的薄膜。 </li><li>储存在潮湿箱中的载玻片，并等待琼脂糖介质固化。 </li></ol> 2，解剖和安装分生组织样品<ol><li> Microdissect分生组织的共聚焦想象力吴：从干拉下来了超精细镊子取出花芽一个接一个。重要的是要消除所有的花蕾最多时不显示（ 即 P1原基14）萼片的味蕾是很重要的。 </li><li>用刀片或镊子的尖端，切分生组织离干。切口应平行于分生组织表面的将要被使用的原子力显微镜测得的区域。得到的顶点应该是300微米，600微米高，以适应针尖下的。 </li><li>推顶到在步骤1.1.4制备琼脂糖介质的膜。选择在载玻片/琼脂糖的位置并轻推，这样的分生组织，既是直接与载玻片接触，并从琼脂糖伸出。关键是要遵循从它的茎切了几秒钟之内定位在琼脂糖的分生组织。注意：这是为了防止从分生组织的干燥。在这个阶段，分生组织将站在一个CRACķ因为琼脂糖时施加压力破碎。 </li><li>准备几个分生组织这种方式为批处理成像，只要它们都具有相同的高度并彼此上滑动定位小于500微米。保持分生组织准备在潮湿的盒子，同时进一步解剖标本。 注意：它限制了变化，从校准到来。理想的情况是2个样本的每个条件的可能做好准备，并随机测量。到目前为止，有一批成像对测量（应力诱导反应）没有观察到的效果。这可能是由于应激信号传导分子中的琼脂和/或所述安装的溶液稀释。备选的应激反应是一样的，不管对样品的制备量。 </li><li>之前移动到下一个步骤，确保分生组织和花芽之间的边界是干的。如果没有，用滤纸轻轻去除多余水分。这应防止在接下来的阶段，该熔融琼脂糖洪水样品由于毛细作用的力量。 </li><li>用20微升移液器，补充足够的安装融化琼脂糖填写步骤1.2.3创建裂纹。和以包围各分生组织。琼脂糖应达到除去花芽（原基）的伤痕的程度。为了实现这一目标，它可能有必要在裂纹的每一端加琼脂糖。 </li><li>当琼脂糖被添加，它会迅速涌入裂缝;使用吸管，吸脱熔安装琼脂糖，以确保分生组织是幻灯片上的最高点的一部分。这解决了分生组织，使其不能成像过程中移动。 </li></ol> 3，安装根或胚轴样品<ol><li>对于根和下胚轴，没有剥离是必要的。在琼脂糖上制备的玻璃载片的薄膜，使一个槽是直接在上面的玻璃蚀刻或沿着一个玻璃薄片。放置在该槽中根或胚轴确保它是与接触玻璃沿其整个长度。 </li><li>之前移动到下一个步骤，确保样品是干在其表面。如果没有，用滤纸轻轻去除多余的水。 </li><li>加入融化的样品周围琼脂糖作为安装步骤1.2.6。 </li></ol> 4，原子力显微镜制备及灵敏度校准（一JPK Nanowizard AFM） <ol><li>安装在原子力显微镜的悬臂。悬臂的安装应与圆形压头。半径将决定在x，y和z的分辨率和压痕深度在哪个精确的测量可被存档。 <ol><li>采取细观纳米级测量在表皮表面，使用50纳米半径圆压头;采取尺度测量，使用一个0.5微米的半径圆压头;采取微尺度测量（横跨2-3个细胞），使用2.5微米半径的圆压头。 </li></ol></li><li>定位激光在悬臂的尖端。激光对准中间使用镜像的俘虏的。 </li><li>放置干净的玻璃的原子力显微镜下。 注意：以下设置变换的原子力显微镜受体的激光位置的信号转换成悬臂的变形，并通过评估悬臂的弹簧常数，把它翻译成一种力量。 </li><li>与1 V的目标部队“设定点”的​​方法</li><li>选择“力谱”。由最大的“相对集中点”设置为2 V做一个压痕，在“扩展速度”到40微米/秒，而“Z长”到5微米。 </li><li>从“校准管理”菜单中，选择适合使用“选择合适的范围”按钮灵敏度的远期曲线的线性部分。 “相对集中点”应该从“伏”转化为“米”。 </li><li>从“校准管理”菜单，选择“弹性系数”来评价的ELasticity利用热波动的悬臂。按“运行”，以获得悬臂的频谱密度图。找到具有最低频率的共振峰。使用“选择合适的范围”按钮适应它。 注1：关于热波动调整的更多细节，请阅读库克等人15。 注2：这是优​​选使用的直接方法使用参考悬臂（或材料与已知的刚度），以评估在悬臂的弹性。这里使用一个是好心阿提夫Asnacios 16捐赠。 </li><li>加入200μl的悬臂尖端下10％甘露醇溶液。重新对准激光来使用镜子的捕捉体的中间。 </li><li>重新校准灵敏度：从“校准管理”菜单中，单击“未知”按钮;重复步骤2.3至2.5。 </li></ol> 5，数据采集：表观杨氏模量制图<ol&gt; <li>定位装样品的原子力显微镜下，并添加10％的甘露醇溶液中的500微升小滴在样品上。的甘露醇溶液plasmolyzes分钟内的单元格。 </li><li>用20 NN目标力（“设定点”）的方式（“设定点”不应该超过3 V） </li><li>缩进为200 NN“相对设定点”，一个“延伸速度”40微米/秒，而为5μm的“z长度”。 </li><li>调整“相对设定点”，以获得100nm的缩进处安装悬臂或0.5微米的为1微米/ 5微米安装悬臂200。 </li><li>在“力映射”模式，选择一个区域进行扫描：对分生组织，70微米×70微米，分辨率为64×64（“像素”）;对于下胚轴和根，100微米×100微米与64×64的分辨率。 </li><li>按下扫描启动实验。保存输出。 </LI&gt; </ol> 6，数据分析：表观杨氏模量的计算<ol><li>在数据分析软件打开数据文件。 </li><li>选择“批量处理”，以同样的分析适用于单个地图中的所有曲线。 </li><li>选择“正确的高度为悬臂的弯曲”，所以提取压痕深度。 </li><li>选择“杨氏模量的拟合函数”，将假定该力正比于压痕的面积。将“笔尖形状”，以抛物线假设压痕形状是抛物线。 </li><li>表明尖的半径。 </li><li>优先选择的“回退”曲线的分析，因为它是地形较不敏感比“扩展”曲线。然而，如果有探针的显著粘固在“回退”，用“扩展”曲线，这是不敏感的粘附。 </li><li>集泊松比为0.5。按分析和保存输出。 </li></ol>

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作者什么都没有透露。

在植物中，改变机械性能起到引导生长和形态发生了重大作用。到目前为止，已经在解开控制植物生长的遗传和化学网络很大的进步，但我们对这些网络如何促进并受到改变机械性能知识是最基本的。这个方法应该使我们能够填补这一空白，所以它应该是浓厚的兴趣，科学家研究植物生长或形态发生的任何方面的。现在我们总结了该方法的挑战和局限，以及未来展望。 在协?...

在植物生长受周围的生物体的每一个细胞的刚性细胞壁的协调扩张来实现。越来越多的证据表明它是通过细胞壁化学修饰的植物局部控制这种膨胀。膨胀被认为是主要受应变的细胞壁，引起细胞的高膨压上;此应变响应膨压是由细胞壁1的机械性能约束。鲜为人知的是，这些机械性能和他们在开发过程中如何变化。而且很少有人知道如何将这些机械性能的控制和反馈是否有助于改变细胞壁化学在显然是跨组织协调的方式。如果我们要了解发育过程中植物细胞壁的化学和机械变化，最终如何将这些微观相互作用支配植物之间的连接的宏观增长，一个是可以监控的细胞壁中，在细胞或组织规模发展器官的机械性能的方法是必需的。 这里的原子力显微镜（AFM）所描述的方法，它是基于微米或纳米级组织的压缩或压痕，开发了精确测量的细胞壁中的亚细胞分辨率同时显影机构和跨组织的整个区域的机械性能。其他的方法有两种，分辨率太低或太高：引伸计仅能够在毫米尺度2-4，其规模是例如过大而无法 ​​测量中的早期事件，以测量整个组织的平均机械性能器官;该microindenter可以进行测量以在纳米尺度的亚细胞的分辨率，但它被限制在测定分离的细胞，而不是细胞或器官5-7的基团。用原子力显微镜的要求三维组织，细胞，亚细胞分辨率可达到8-10。最近几个协议已被开发专门用于测量植物组织中，可能也可用于11，12的结构。 我们将在这里介绍了如何通过表观杨氏模13的测量评估组织的弹性。 杨氏模量通常是用来描述材料的刚度。在小变形而变形的材料所需要的力是正比于压痕的面积。杨氏模量是该系数。在一个连续的均匀的材料的情况下相同的系数将被考虑的缩进类型（大小和形状）的测定，但将与所述测量的速度发生变化。在植物组织中的复杂结构的情况下，我们观察到，到目前为止，该力正比于该变形容许的确定比例系数，我们命名为“表观杨氏模量”。在从连续媒体中的植物相比，这明显的杨氏模量是压痕的大小敏感。它不对应于一个纯细胞壁的年轻模量。它最能说明细胞壁组织的脚手架的弹性。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AFM</td>
			<td style="width:348px;">JPK</td>
			<td style="width:119px;">NanoWizard</td>
			<td style="width:167px;">All the 3-generation are abele to do the work withe the same preferment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AFM stage</td>
			<td style="width:348px;">JPK</td>
			<td style="width:119px;">CellHesion</td>
			<td>Required for sample withe low topography (les then 11&#181;m between the lowest and the highest point in the aria of force scanning).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AFM optics</td>
			<td style="width:348px;">JPK</td>
			<td style="width:119px;">Top View Optics&#160;</td>
			<td>Very important in order to position the sample. Cold be replaces by long range a binocular or microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo Microscopes&#160;</td>
			<td style="width:348px;">Leica</td>
			<td style="width:119px;">M125</td>
			<td>Any type of stereo microscopes could do.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:348px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">150nm mounted cantilever&#160;</td>
			<td style="width:348px;">nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel Switzerland</td>
			<td style="width:119px;">R150-NCL-10</td>
			<td>To measure only the cell wall at the surface of the epidermis use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1&#181;m mounted cantilever&#160;</td>
			<td style="width:348px;">nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel Switzerland</td>
			<td>SD-Sphere-NCH-S-10&#160;</td>
			<td>to measure the mechanics of the cell wall orthogonal to the surface of the epidermis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tipless cantiliver</td>
			<td style="width:348px;">nanosensors Rue Jaquet-Droz 1Case Postale 216 CH-2002 Neuchatel Switzerland</td>
			<td>TL-NCH-20&#160;</td>
			<td>to measure the local mechanics of the tissue (2-3 cell wide) use a 5&#181;m mounted cantilever. We attached a 5&#181;m borasilicate bead to a tipless cantiliver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5&#181;m silicon microspheres</td>
			<td style="width:348px;">Corpuscular</td>
			<td style="width:119px;">C-SIO-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aradilte&#160;</td>
			<td style="width:348px;">Bartik S.A. 77170 Coubet France</td>
			<td style="width:119px;"></td>
			<td>Aradilte for fixing the bead to the tip les cantiliver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:348px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;low melting Agarows</td>
			<td style="width:348px;">Fishersci Fair Lawn , new jersey 07410</td>
			<td style="width:119px;">BP160-100</td>
			<td>34-45 Gelation Temperature</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">D-Mannitol</td>
			<td style="width:348px;">Sigma-Aldrich, 3050 Spruce Street, St Louis Mo 63103 USA)</td>
			<td style="width:119px;">M4125-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:348px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">2&#160; Stainless Steel No. 5 Tweezers</td>
			<td style="width:348px;">Ideal-Tek 6828 Balerna Switzerland&#160;</td>
			<td align="right">951199</td>
			<td></td>
		</tr>
	</tbody>
</table>

afm-based mapping of the elastic properties of cell walls: at tissue, cellular, and subcellular resolutions

我们描述了一种最近开发的方法来测量植物组织的表面的机械特性使用原子力显微镜（AFM）的微/纳米压痕，要JPK原子力显微镜。具体来说，在这个协议中，我们测量视杨氏模量的细胞壁在跨区域的亚细胞分辨率可达100 微米 ×100微米花分生组织，下胚轴和根。这需要精心准备的样品，正确选用微压头和压痕深度。只占，测量在甘露醇的高度浓缩的溶液以质壁分离的细胞中，从而除去细胞膨压的贡献进行细胞壁的属性。 相对于其他现存的技术，通过使用不同的压头和压痕深度，该方法允许同时多尺度测量<em&gt;即在细胞内的决议和在数百细胞包括组织的。这意味着，现在有可能在空间上，时间上的特征所发生的细胞壁的机械性质在开发过程中，使这些变化与生长和分化相关联的变化。这是一个关键的步骤，了解协调的微观细胞的变化如何带来宏观形态发生的事件。 然而，一些局限性依然存在：该方法只能用于在相当小的样品（大约100μm直径），并且只在外部组织;该方法是将组织形貌敏感;它测量的组织复杂的机械特性只有某些方面。该技术正在迅速发展，很可能大多数的这些限制将在不久的将来得到解决。

在图1中，我们提出花分生组织（ 图1A和1B），年轻人和老年人的下胚轴（ 图1C-F）和根分生组织（ 图1G和1H）典型的年轻模量地图。在所有实验中压头是半球形的，但它的半径不同，使不同空间分辨率可以达到图1C和1D显示了中观纳米压头（50纳米半径）与介观纳米压痕（50 nm的深度）的典型结果。...

Watch this Scientific Journal Video about AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions at JoVE.com

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

afm-based-mapping-elastic-properties-cell-walls-at-tissue-cellular

Research

JoVE Journal

Biology

12.8K 次观看.  Université Paris Diderot. We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

视频: AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

双层弹性性能的监测变化的单分子方法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

综合植物细胞壁成分分析（木质纤维素生物质）第二部分：碳水化合物

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

培养HL-1心房肌细胞单层的电压和钙双通道光映射

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

在出生后的小鼠听泡解剖：中耳骨骼和组织学分析的分离

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic properties. Density is a cell type identifying property, directly related to its metabolic rate, differentiation, and activation status. Magnetic levitation allows a one-step approach to successfully separate, image and characterize circulating blood cells, and to detect anemia, sickle cell disease, and circulating tumor cells based on density and magnetic properties. This approach is also amenable to detecting soluble antigens present in a solution by using sets of low- and high-density beads coated with capture and detection antibodies, respectively. If the antigen is present in solution, it will bridge the two sets&#160;of beads, generating a new bead-bead complex, which will levitate in between the rows of antibody-coated beads. Increased concentration of the target antigen in solution will generate a larger number of bead-bead complexes when compared to lower concentrations of antigen, thus allowing for quantitative measurements of the target antigen. Magnetic levitation is advantageous to other methods due to its decreased sample preparation time and lack of dependance on classical readout methods. The image generated is easily captured and analyzed using a standard microscope or mobile device, such as a smartphone or a tablet.

This paper describes a magnetic levitation-based method that can specifically detect the presence of antigens, either soluble or membrane-bound, by quantifying changes in the levitation height of capture beads with fixed densities.

使用磁悬浮定量细胞密度和抗原特性

The described method was developed based on the principles of magnetic levitation, which separates cells and particles based on their density and magnetic ...