Name: use of atomic force microscopy to measure mechanical properties and turgor pressure of plant cells and plant tissues
Uploaded: 2019-07-15T15:00:00
Description: Watch this Scientific Journal Video about Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues at JoVE.com

我们要感谢 PLATIM 团队的技术支持,以及 Arezki Boudaoud 和 RDP 实验室的生物物理团队成员,感谢他们的有益讨论。

1.细胞壁机械性能的测量
注:给出了阿拉伯经体发育的陀螺的例子。
<ol><li>生物样品的制备
	<ol>
<li>根据公布的阶段测定,在阶段9至10(约0.5毫米长)收集封闭的花蕾。在双筒望远镜下,使用细钳子,小心地打开芽,检查发育阶段,并收集位于花中心的陀螺仪。</li>
		<li>将陀螺放在双侧胶带上,放在小培养皿盖的中心(直径为 5 厘米)。 <...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Hertzian+Approach+to+Analyzing+Mechanical+Properties+of+Endothelial+Cells+Probed+by+Atomic+Force+Microscopy.">Non-Hertzian Approach to Analyzing Mechanical Properties of Endothelial Cells Probed by Atomic Force Microscopy.</a> Journal of Biomechanical Engineering. 128 (2), 176-184 (2006).</li><li>Beauzamy, L., Louveaux, M., Hamant, O., Boudaoud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically,+the+shoot+apical+meristem+of+Arabidopsis+behaves+like+a+shell+inflated+by+a+pressure+of+about+1MPa.">Mechanically, the shoot apical meristem of Arabidopsis behaves like a shell inflated by a pressure of about 1MPa.</a> Frontiers in Plant science. 6 (1038), 1-10 (2015).</li><li>Majda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanochemical+polarization+of+contiguous+cell+walls+shapes+plant+pavement+cells.">Mechanochemical polarization of contiguous cell walls shapes plant pavement cells.</a> Developmental Cell. 43 (3), 290-304 (2017).</li><li>Torode, T. 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植物形态的出现主要取决于时间和空间内生长的协调速率和方向。植物细胞被包裹在由多糖基质制成的刚性细胞壁中,这些基质将它们粘在一起。因此,细胞膨胀由细胞壁上的 turgor 压力拉扯和细胞壁的刚度与抵抗这种压力之间的平衡控制。为了理解发育背后的机制,能够测量细胞壁的机械特性以及特定器官的不同组织或细胞中的turgor压力是很重要的。如本文所示,AFM 是在此背景下的选择方法。
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原子力显微镜 (AFM) 属于扫描探针显微镜 (SPM) 系列,其中半径通常为几纳米的尖端扫描样品表面。表面的检测不是通过光学或电子方法实现的,而是通过尖端和样品表面之间的相互作用力实现的。因此,该技术不仅限于样品表面的地形特征化(3D分辨率可下降到几纳米),而且还允许测量任何类型的相互作用力,如静电、范德瓦尔或接触力。此外,尖端可用于在生物样品表面施加力并测量由此产生的变形,即所谓的"缩进",以确定其机械特性(例如,Young 的模量、粘弹性特性)。
植物细胞壁的机械特性是在试图理解发育过程1、2、3的基础机制时必须考虑的。事实上,这些特性在发育过程中受到严格控制,特别是因为细胞壁软化是允许细胞生长所必需的。AFM 可用于测量这些属性,并研究它们在器官、组织或发育阶段之间的变化方式。
在本文中,我们描述了我们如何使用AFM测量细胞壁的机械特性和土姆压力。这两种应用在两种不同的 AFM 显微镜上进行了演示,之后将在此详细介绍。

<table border="0" cellpadding="0" cellspacing="0" style="width:700px;" width="700">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Growth medium</td>
			<td style="width:155px;"></td>
			<td style="width:151px;"></td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">1000x vimatin stock solution</td>
			<td style="width:155px;"></td>
			<td style="width:151px;"></td>
			<td>used to make ACM, composition see Stanislas et al., 2017. Add to ACM after autoclaving, before pouring.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">1-N-Naphthylphthalamic acid (NPA)</td>
			<td>Sigma-Aldrich/Merck</td>
			<td style="width:151px;">132-66-1</td>
			<td>add to Arabidopsis medium, 10 &#956;M. Add after autoclaving, before pouring.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">agar-agar</td>
			<td>Sigma-Aldrich/Merck</td>
			<td style="width:151px;">9002-18-0</td>
			<td>add to Arabidopsis medium, 1% w/v.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">agarose</td>
			<td style="width:155px;">Merck Millipore</td>
			<td style="width:151px;">9012-36-6</td>
			<td>used to make solid ACM, 0.8% w/v.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Arabidopsis medium</td>
			<td style="width:155px;">Duchefa Biochimie</td>
			<td style="width:151px;">DU0742.0025</td>
			<td>For in vitro arabidopsis culture, 11.82g/L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Calcium nitrate tetrahydrate</td>
			<td>Sigma-Aldrich/Merck</td>
			<td style="width:151px;">13477-34-4</td>
			<td>add to Arabidopsis medium, 2mM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">MURASHIGE &#38; SKOOG MEDIUM</td>
			<td style="width:155px;">Duchefa Biochimie</td>
			<td style="width:151px;">M0221.0025</td>
			<td>Basal salt mixture, used to make ACM, 2.2g/L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">N6-benzyladenine (BAP)</td>
			<td>Sigma-Aldrich/Merck</td>
			<td style="width:151px;">1214-39-7</td>
			<td>used to make ACM, 555 nM. Add to ACM after autoclaving, before pouring.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">oryzalin</td>
			<td>Sigma-Aldrich/Merck</td>
			<td style="width:151px;">19044-88-3</td>
			<td>for oryzalin treatement, 10 &#956;g/mL.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">plant preservation mixture (PPM)</td>
			<td style="width:155px;">Plant Cell Technology</td>
			<td style="width:151px;"></td>
			<td>used to make ACM, 0.1% v/v. Add to ACM after autoclaving, before pouring.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Potassium hydroxide</td>
			<td style="width:155px;">Duchefa Biochimie</td>
			<td style="width:151px;">1310-58-3</td>
			<td>used to make Arabidopsis medium and ACM, both pH 5.8.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">sucrose</td>
			<td style="width:155px;">Duchefa Biochimie</td>
			<td style="width:151px;">57-50-1</td>
			<td>used to make ACM, 1% w/v.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tools for AFM</td>
			<td style="width:155px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">BioScope Catalyst BioAFM</td>
			<td style="width:155px;">Bruker</td>
			<td style="width:151px;"></td>
			<td>The AFM used for turgor pressure measurement in this protocol.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Nanowizard III + CellHesion</td>
			<td style="width:155px;">JPK (Bruker)</td>
			<td style="width:151px;"></td>
			<td>The AFM used for measuring mechanical properties.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Patafix</td>
			<td style="width:155px;">UHU</td>
			<td style="width:151px;">D1620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Reference elasitic structure</td>
			<td style="width:155px;">NanoIdea</td>
			<td style="width:151px;">2Z00026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Reprorubber-Thin Pour</td>
			<td style="width:155px;">Flexbar</td>
			<td style="width:151px;">16135</td>
			<td>biocompatible glue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Spherical AFM tips</td>
			<td style="width:155px;">Nanoandmore</td>
			<td style="width:151px;">SD-SPHERE-NCH-S-10</td>
			<td>Tips used for measuring mechanical properties.</td>
		</tr>
	</tbody>
</table>

use of atomic force microscopy to measure mechanical properties and turgor pressure of plant cells and plant tissues

我们在这里介绍使用原子力显微镜来缩进植物组织并恢复其机械性能。在缩进模式下使用两种不同的显微镜,展示了如何测量弹性模量,并用它来评估细胞壁的机械性能。此外,我们还解释了如何评估 turgor 压力。原子力显微镜的主要优点是它是非侵入性的,相对快速(5~20分钟),几乎任何类型的活植物组织表面平坦,无需治疗即可进行分析。分辨率可能非常好,具体取决于尖端大小和单位面积的测量次数。此方法的一个限制是,它只提供直接访问表面细胞层。

图 1A和图 1B显示了一个屏幕截图,其中说明了协议步骤 1.3.4 到 1.3.6 的结果,用于查找获取 QI 映射的感兴趣区域。值得一提的是,选择感兴趣的区域是为了不倾斜表面(即尽可能平坦)。事实上,正如Routier等人所注意到的,如果缩进轴不垂直于表面,那么所测得的杨的模量就被低估了。此效果在图 1</strong...

Watch this Scientific Journal Video about Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues at JoVE.com

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

在这里,我们介绍原子力显微镜(AFM),作为细胞和组织纳米和微缩进工具。该仪器允许同时采集样品的三维表面地形及其机械特性,包括细胞壁杨的模量以及 turgor 压力。

利用原子力显微镜测量植物细胞和植物组织的机械特性和Turgor压力

We present here the use of atomic force microscopy to indent plant tissues and recover its mechanical properties. Using two different microscopes in ...

植物细胞的机械特性在理解发育基础的机制时必须考虑到。原子力显微镜可用于测量这些特性，并遵循它们变化的方式，在器官、组织之间、所有发育阶段。这种技术的主要优点是，它不侵入，它相对快速，它不需要治疗，所以可以直接应用于活样品。对于新到此过程的人，样品的 cis 部分非常关键，因此请确保您真正对样品进行适当的机械固定。这个程序的可视化演示，对于更好地了解样品固定、寄养的质量控制和测量设置的复杂性至关重要。演示这个程序的将是西蒙妮·博维奥，一个工程师和玉辰龙，一个博士后在我的实验室。首先，将一块双面胶带放入直径五厘米的培养皿的中心。将从花蕾分离的基因样本添加到胶带中。快速加入水到盘中，直到样品完全覆盖，以避免脱水。接下来，将样品放在 AFM 阶段并移动头部，以便其位于样品上。在软件中校准悬臂后，确保系统处于 QI 模式，并接近样本的设定点力为 15 纳米牛顿。接下来，将 Z 长度设置为 4 微米，将扫描区域设置为 80 x 80 微米平方，像素数设置为 40 x 40。然后转到高级映像设置面板，将模式设置为恒定速度。此外，设置将进入轨道速度扩展到 200 微米/秒，采样速率扩展到 25 公斤赫兹。设置参数后，将提示移动到样本上感兴趣的区域。验证要扫描的区域是否无碎屑，定位尽可能平坦的区域，然后接合，然后开始扫描并使用快速低力扫描来检查样品是否移动。找到感兴趣的区域后，选择区域为 40 x 40 到 60 x x（60 微米平方），然后将像素数增加至每微米 2 像素。接下来，将设定点增加至五百纳米牛顿，获得一百至两百纳米的缩进。将 Z 长度减小至 2 微米，将扩展进入区域速度降低至每百微米，然后将采样速率提高至 50 千赫，然后开始扫描样品。完成后，将输出同时保存为图像和数据文件。打开数据处理软件并加载数据文件。单击使用此地图进行批处理按钮，以便在地图的所有曲线上使用相同的参数，然后转到加载预定义的进程并选择赫兹拟合。接下来，转到可切换的基准线操作，并设置减去偏移加倾斜，X 分钟介于 40% 到 60% 之间。在垂直尖端位置选项卡上，如果您喜欢处理原始数据，请选择平滑紧固。要选择合适的拟合模型，请转到弹性拟合选项卡，然后根据预期的附着力强度选择其中一个选项。如果不存在或存在弱粘附，则使用接近曲线，并首选使用赫兹 isnader 模型。在粘附力更强的情况下，使用德根·米利亚·多尔托普罗夫或DMD的模型，并处理迟钝曲线。现在根据标称尖端形状设置尖端几何参数。这个实验中使用的尖端是球形尖端，半径为四百纳米。接下来，将泊松比率设置为 0.5，然后选择移位曲线。通过单击主窗口的图标，并重复相同的参数设置，添加第二个弹性拟合例程。最后，在 X min 中指定所需的缩进，然后保留并应用于所有缩进，以迭代地图所有曲线上的上述步骤。保存结果以在点 tsv 文件中获取图像。要测量快速溶液变化，请将样品放在培养皿中，并握住一小块粘合剂粘胶。使用生物相容性胶水快速密封粘胶和样品基座之间的间隙。等待胶水凝固，然后将样品淹没在液体顶点培养基中，含有0.1%植物保鲜混合物。校准系统后，打开采集软件，然后首先进入检查参数窗口。将弹簧常数设置为悬臂制造的弹簧常数或确定的弹簧常数。接下来，将尖端半径设置为四百纳米，将样品波松比设置为 0.5，将样本线设置为一百二十八，以确保快速采集，扫描速率为 0.2 赫兹，扫描大小为 1 微米。然后转到坡道窗口，将斜坡大小设置为 5 微米，将三角阈值设置为最大值，将样本数设置为四千六百零八。设置所有参数后，请手动小心地接近样品。当探头相对靠近样品表面时，单击方法。接触后，逐渐增加扫描大小并修改扫描速率，直到达到所需的平衡而不损坏样品或尖端。如果测量区域未达到所需，请重新定位扫描。满意时，单击按钮、点和射，以启动点和拍摄窗口。指定保存目录和文件名。然后单击下一次扫描的渐变以启动录制。扫描完成后，软件界面将重定向到斜坡窗口。单击扫描的图像以指定要缩进的位置。每个单元格在浆果中心附近至少选择三个缩进点，将缩进设置为每侧重复三次，然后单击渐变并捕获。在分析软件中，打开点 MCA 文件，这显示了扫描图像上每个力曲线的位置。然后打开一个力曲线进行分析。单击基准线校正按钮并拖动力曲线上的蓝色虚线，直到扩展源基准线开始和扩展源基准线停止，或分别以 0% 和 80%。然后单击执行。接下来单击箱体汽车过滤按钮，然后单击执行以平滑力曲线。然后单击缩进按钮。在输入窗口中，将活动曲线设置为扩展，将第五种方法设置为线性化模型，将最大力拟合边界设置为 99%，将最小力拟合边界设置为 75%，将拟合模型设置为刚度。分析批次中的力曲线。为此，请单击"运行历史记录"按钮，指定报表目录，并添加需要相同处理的其他力曲线。完成后，单击"运行"。当 K 的值是批处理时，单击历史记录并转到五个缩进以返回到缩进窗口。一旦存在，最大力拟合边界更改为 10%，最小力拟合边界更改为 1%。然后将拟合模型设置为赫兹安。接下来，打开相应的文件以显示不同的扫描通道。在高通道窗口中，单击剖面按钮。这将允许测量涡轮压力演绎所需的样品表面曲率。然后跨一个单元格的长轴绘制一条线，将虚线边界移动到单元格边缘并记录半径值。最后在文本协议中遵循，计算每个细胞的均值幼光模、弹簧常数、E、K 和 turgor 压力。左侧的图像是年轻人模数的地图，通过分析整个缩进，从用户定义的力设定点，而右图，显示了第一百纳米缩进的分析结果。在这里，这两个地图看起来非常相似，但是，缩进深度的变化在某些情况下会导致更好地突出显示样本异质性，这可能有助于确定位置或提供有关内部结构行为的信息。对于这些地图上的每个点，都有一个基础力曲线。此处所示的曲线有两个值得一提的效果。首先，如果接近部分的力曲线结束在五百纳米牛顿。下坡运动继续。表示尖端施加的最终力高于预期。第二件事要注意的是椭圆突出显示的缩回曲线上的波。这种挥动可以是样品在尖端作用下移动或振动的指示器，可能需要切换到不同的固定方法。使用本文中记录的过程，可以从 AFM 扫描和缩进中检索除单元壁厚外的所有磁油压力扣减关键参数。红十字位置的深压痕力曲线描述了细胞壁幼光的模量和曲线不同系的样品的明显刚度。样品固定至关重要。在测量过程中，注意样品不稳定性的迹象。仔细选择具有平面的面积，以确保垂直缩进。按照此过程，可以监控机械性能随着时间的推移和不同的条件。人们还可以将机械测量与共和图像叠加在相关研究中。这项技术为生物力学与生理学发展和其他生物过程联系起来铺平了道路。

Research

JoVE Journal

Developmental Biology

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

地磁场（GMF）和植物进化：调查逆转基因食品对影响<em&gt;拟南芥</em&gt;开发与基因表达

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) ...

科研

发育生物学

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

长期共焦成像根和下胚轴发育的简单室

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

拟南芥花器官和角果的全装清和染色

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

不同植物种的射击顶端共聚焦活图

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

Development of Targeting Induced Local Lesions IN Genomes TILLING Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

通过乙基甲烷硫素突变在小谷物作物中靶向诱导局部病变(TILLING)种群的发展

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

细胞细胞成像的微管细胞和微机械操作的 阿拉比多普西斯拍摄 阿皮克·梅里斯特姆

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...