Name: development of microfluidic devices to study the elongation capability of tip-growing plant cells in extremely small spaces
Uploaded: 2018-05-22T12:30:00
Description: Watch this Scientific Journal Video about Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces at JoVE.com

我们感谢 h. Tsutsui 和 d. 栗原为我们提供了转基因植物, 包括T fournieriRPS5Ap::H2B-tdTomato线和南芥 UBQ10pro::H2B-mClover线分别。这项工作得到了名古屋大学变革性生物分子研究所和日本先进植物科学网的支持。这项工作的财政支助是由日本科技署 (ERATO 项目赠款) 提供的。JPMJER1004 为金斗勋), 为创新领域的科学研究提供资助 (编号JP16H06465 和 JP16H06464 为金斗勋), 和日本促进科学协会 (jsp) 资助为挑战性的探索性研究 (批准26600061为纽约和授予 nos 25650075 和15K14542 为林宏)。

1. 制备 Microdevice 研究生长的花粉管和苔藓 Protonemata
注: 我们使用了无掩模光刻仪器在硅片上制备了硅烷模具。本手稿中省略了有关系统操作的详细信息。使用光掩模的标准光刻技术9 也可用于创建本手稿中所描述的注塑模具。
<ol><li>将11克的预聚合物塑料混合物 (弹性体底座: 固化剂的比例为 10:1) 倒入每4英寸的模具中。</li>
	<li>将步骤1.1 中准备的模具放在?...

<ol>
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Cell. 25 (3), 317-323 (2013).</li><li>Rensing, S., 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=The+Physcomitrella+genome+reveals+evolutionary+insights+into+the+conquest+of+land+by+plants.">The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants.</a> Science. 319, 64-69 (2008).</li><li>Yanagisawa, N., Sugimoto, N., Arata, H., Higashiyama, T., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capability+of+tip-growing+plant+cells+to+penetrate+into+extremely+narrow+gaps.">Capability of tip-growing plant cells to penetrate into extremely narrow gaps.</a> Sci. Rep. 7 (1), 1403 (2017).</li><li>Dolan, L., 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=Clonal+relationships+and+cell+patterning+in+the+root+epidermis+of+Arabidopsis.">Clonal relationships and cell patterning in the root epidermis of Arabidopsis.</a> Development. 120, 2465-2474 (1994).</li><li>Denais, C. 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=Nuclear+envelope+rupture+and+repair+during+cancer+cell+migration.">Nuclear envelope rupture and repair during cancer cell migration.</a> Science. 352 (6283), 353-358 (2016).</li></ol>

为了获得上述结果, 必须遵循议定书中的几个关键步骤。首先, 在粘合之前, 必须在足够的时间内用等离子处理该层和玻璃底碟表面。否则, 当尖端生长的细胞穿过 microgaps 时, 该层可能会在本地与玻璃表面分离。根头发和苔藓 protonemata 协议的另一个关键步骤是 microdevice 的灭菌。通常, 根毛和苔藓 protonemata 细胞需要培养在 microdevice 几个星期。在不杀菌设备的情况下, 微生物可能会蓬勃发展, 这可能会...

花粉粒在柱头上发芽后, 每粒谷物都会产生一个花粉管, 将精子细胞带到卵细胞和胚珠中的中心细胞进行双重受精。花粉管拉长的风格, 并最终达到胚珠通过感知多个指导线索沿其方式1。在伸长过程中, 花粉管遇到一系列物理障碍;传送的轨道是充满细胞, 和花粉管必须进入分钟珠孔端打开胚珠达到他们的目标 (图1A) 2.因此, 花粉管必须具有穿透物理障碍物的能力, 同时能耐受周围的压力。根毛发是另一种尖端生长的植物细胞, 它必须经受住环境中的物理障碍, 以填充土壤颗粒 (图 1B) 的形式。
研究了花粉管的各种力学特性, 包括细胞顶端区域的膨压力和刚度, 可以用初始质壁分离方法3、4和细胞力显微镜来测量.CFM)5,6分别。然而, 这些方法本身并没有揭示花粉管是否能够通过其生长路径上的物理屏障拉长。一种替代技术, 允许花粉管伸长率被监测体内是两个光子显微镜7。然而, 用这种方法很难观察到胚珠组织内的单个花粉管的形态学变化。此外, 土壤中的根毛发生长可以用 X 射线计算机断层扫描 (CT) 和磁共振成像 (MRI)8来可视化, 尽管分辨率较低。在这里, 我们提出了一个方法, 可用于获取高分辨率图像的细胞的变形过程, 在常规显微镜。
这里描述的方法的总体目标是可视化尖端生长的植物细胞的伸长能力, 包括花粉管, 根毛和苔藓 protonemata, 在极小的空间。本文介绍的聚甲基 (microdevices) 在光学上具有透明和透气的特点, 可以在显微镜下培养活细胞, 观察其生长行为。通过使用模具的软光刻技术9也可以创建微 ~ 纳米尺度空间。这些特性使我们能够研究在物理密闭环境中尖端生长的植物细胞的伸长能力。
在这项工作中, 我们在微流控装置上构造了1µm 宽间隙 (4 µm), 并研究了花粉管穿透这些人工障碍物的能力, 这些障碍远小于圆柱形花粉管直径 (约8µm)。这个实验平台使我们能够可视化花粉管对 microgaps 的反应, 捕捉响应的时间推移图像, 跟踪细胞的变形过程。我们还开发了 microdevices, 可用于研究根毛和苔藓 protonemata 的穿透能力。已报告了若干 microdevices, 使植物根10、11、12、13和 moss protonemata14在高分辨率下实现可视化。在我们的设备, 一系列根毛发生长通道垂直连接到一个根生长室, 和个别根毛 (约7µm 直径) 被引导到射流通道与1µm 宽的差距。我们还在含有 microgaps 的 microdevice 中培养苔藓 protonemata (直径约20µm), 以检查它们对这些物理屏障的反应。本文提出的微流控方法使我们能够探索各种尖端生长的植物细胞在极小的空间中拉长的能力, 目前尚无任何其他方法可供研究。

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<tbody>
		<tr height="18">
			<td height="18" style="height:19px;width:216px;">PDMS</td>
			<td style="width:165px;">Dow Corning Co.</td>
			<td style="width:119px;">Sylgard184</td>
			<td style="width:427px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Murashige &#38; Skoog Medium</td>
			<td style="width:165px;">Wako Pure Chemical</td>
			<td style="width:119px;">392-00591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MES</td>
			<td style="width:165px;">Dojindo</td>
			<td style="width:119px;">345-01625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:165px;">Wako Pure Chemical</td>
			<td style="width:119px;">196-00015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mm glass-bottom dish</td>
			<td style="width:165px;">Matsunami Glass</td>
			<td style="width:119px;">D210402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm glass-bottom dish</td>
			<td style="width:165px;">Iwaki&#160;</td>
			<td style="width:119px;">3971-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:165px;">Feather</td>
			<td style="width:119px;">No.11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">biopsy punches</td>
			<td style="width:165px;">Harris</td>
			<td style="width:119px;"></td>
			<td>Uni-Core</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel loading tips</td>
			<td style="width:165px;">Bio-Bik</td>
			<td style="width:119px;">124-R-204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted Microscope</td>
			<td style="width:165px;">Olympus</td>
			<td style="width:119px;">IX83</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:216px;">CSU-W1</td>
			<td style="width:165px;">Yokogawa Electric</td>
			<td style="width:119px;"></td>
			<td>No Catalog number is avairable for this customized microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MetaMorph imaging software</td>
			<td style="width:165px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of microfluidic devices to study the elongation capability of tip-growing plant cells in extremely small spaces

在体内, 尖端生长的植物细胞需要克服一系列物理障碍;然而, 研究人员缺乏在这种限制性条件下可视化细胞行为的方法。为了解决这一问题, 我们开发了生长室, 用于尖端生长的植物细胞, 其中含有一系列狭窄的, 微型制造的缺口 (1 µm) 在一个多甲基 (基板) 基质。这种透明材料允许用户在 microgap 穿透过程中通过时间推移成像监测单个细胞中的尖端伸长率。利用这个实验平台, 我们观察了花粉管的形态学变化, 因为它们穿透了 microgap。在这个过程中, 我们捕获了一个荧光标记的营养细胞核和精子细胞的形状的动态变化。此外, 我们展示了根毛和苔藓 protonemata 的能力, 以穿透1µm 的差距。此体外平台可用于研究单个单元格对物理受限空间的响应方式, 并可提供对尖端生长机制的洞察力。

如图 1所示, 尖端生长的植物细胞在其生长路径体内中遇到一系列物理屏障。本研究提出的微流控体外细胞培养平台使三种植物细胞 (花粉管、根毛和苔藓 protonemata) 的尖端生长过程能够通过1µm 人工间隙进行检查 (图 3,图 4,图 5)。对于花粉管的研究, 用活细胞成像监测花?...

Watch this Scientific Journal Video about Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces at JoVE.com

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

我们描述了一种方法来调查尖端生长的植物细胞, 包括花粉管, 根毛和苔藓 protonemata, 以拉长通过极窄的差距 (约1µm) 在微流控装置。

微流控装置在极小空间中研究尖端生长植物细胞的伸长能力

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

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Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...