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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 生物样品的制备 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。 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)。<…
Representative Results
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…
Discussion
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。
<p c…Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Growth medium | |||
| 1000x vimatin stock solution | used to make ACM, composition see Stanislas et al., 2017. Add to ACM after autoclaving, before pouring. | ||
| 1-N-Naphthylphthalamic acid (NPA) | Sigma-Aldrich/Merck | 132-66-1 | add to Arabidopsis medium, 10 μM. Add after autoclaving, before pouring. |
| agar-agar | Sigma-Aldrich/Merck | 9002-18-0 | add to Arabidopsis medium, 1% w/v. |
| agarose | Merck Millipore | 9012-36-6 | used to make solid ACM, 0.8% w/v. |
| Arabidopsis medium | Duchefa Biochimie | DU0742.0025 | For in vitro arabidopsis culture, 11.82g/L. |
| Calcium nitrate tetrahydrate | Sigma-Aldrich/Merck | 13477-34-4 | add to Arabidopsis medium, 2mM. |
| MURASHIGE & SKOOG MEDIUM | Duchefa Biochimie | M0221.0025 | Basal salt mixture, used to make ACM, 2.2g/L. |
| N6-benzyladenine (BAP) | Sigma-Aldrich/Merck | 1214-39-7 | used to make ACM, 555 nM. Add to ACM after autoclaving, before pouring. |
| oryzalin | Sigma-Aldrich/Merck | 19044-88-3 | for oryzalin treatement, 10 μg/mL. |
| plant preservation mixture (PPM) | Plant Cell Technology | used to make ACM, 0.1% v/v. Add to ACM after autoclaving, before pouring. | |
| Potassium hydroxide | Duchefa Biochimie | 1310-58-3 | used to make Arabidopsis medium and ACM, both pH 5.8. |
| sucrose | Duchefa Biochimie | 57-50-1 | used to make ACM, 1% w/v. |
| Tools for AFM | |||
| BioScope Catalyst BioAFM | Bruker | The AFM used for turgor pressure measurement in this protocol. | |
| Nanowizard III + CellHesion | JPK (Bruker) | The AFM used for measuring mechanical properties. | |
| Patafix | UHU | D1620 | |
| Reference elasitic structure | NanoIdea | 2Z00026 | |
| Reprorubber-Thin Pour | Flexbar | 16135 | biocompatible glue. |
| Spherical AFM tips | Nanoandmore | SD-SPHERE-NCH-S-10 | Tips used for measuring mechanical properties. |
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Citazione di questo articolo
Bovio, S., Long, Y., Monéger, F. Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues. J. Vis. Exp. (149), e59674, doi:10.3791/59674 (2019).