Name: imaging the root hair morphology of arabidopsis seedlings in a two-layer microfluidic platform
Uploaded: 2017-08-15T13:00:00
Description: Watch this Scientific Journal Video about Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform at JoVE.com

这份手稿创作的 UT-巴特尔，LLC 根据合同号德-AC05-00OR22725 与美国能源部。美国政府保留和发布服务器，通过接受出版，文章承认，美国政府保留非独占、 缴足股款、 不可撤销的、 世界范围内的许可发布或复制已发布的表单的这份手稿，或允许其他人这样做，为美国政府的目的。能源署将提供对这些结果根据能源部公共访问计划 (http://energy.gov/downloads/doe-public-access-plan) 由联邦政府赞助研究公共访问。
这项工作被支持部分的基因组科学计划、 美国能源部、 办公室的科学、 生物和环境研究，作为植物微生物接口科学重点领域 (http://pmi.ornl.gov) 的一部分。制作的微流控平台进行了在中心的纳米加工研究实验室的纳米材料科学，这是一个美国能源部科学用户设施办公室。JAA 支持由 NSF 研究生研究奖学金 DGE-1452154

1.两层平台制作
<ol>
	<li>多层大师的制备
	<ol>
		<li>旋涂环氧负性光刻 (1,250 ~63.45%固体 cSt) 根据制造商的规格 (45 2，000rpm s) 到 4 英寸直径硅片，以获得所需的 20 微米的第一个设计层高度。</li>
		<li>软烤涂层的抗晶片为 4 分钟，95 ° c。允许硅片冷却 5 分钟暴露于 UV 光线为 15 硅片 s (~ 150 兆焦耳/厘米2在 365 nm) 通过在 UV 光掩膜接触定位仪来确定底层的几何形状。</li>
		<li>暴露后?...

<ol>
	<li>Pritchard, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soil+organisms+and+global+climate+change.">Soil organisms and global climate change.</a> Plant Pathol. 60 (1), 82-99 (2011).</li><li>Norby, R. J., Ledford, J., Reilly, C. D., Miller, N. E., O'Neill, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine-root+production+dominates+response+of+a+deciduous+forest+to+atmospheric+CO2+enrichment.">Fine-root production dominates response of a deciduous forest to atmospheric CO2 enrichment.</a> Proc. Natll. Acad. Sci. USA. 101 (26), 9689-9693 (2004).</li><li>McCormack, M. 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=Redefining+fine+roots+improves+understanding+of+below-ground+contributions+to+terrestrial+biosphere+processes.">Redefining fine roots improves understanding of below-ground contributions to terrestrial biosphere processes.</a> New Phytol. 207 (3), 505-518 (2015).</li><li>Mangano, S., Juarez, S. P. D., Estevez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+regulation+of+polar-growth+in+plant+cells.">ROS regulation of polar-growth in plant cells.</a> Plant Physiol. 171 (3), 1593-1605 (2016).</li><li>Ketelaar, T., Emons, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Actin+Cytoskeleton+in+Root+Hairs:+A+Cell+Elongation+Device.">The Actin Cytoskeleton in Root Hairs: A Cell Elongation Device.</a> Root Hairs. , 211-232 (2009).</li><li>Grossmann, G., 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+RootChip:+an+integrated+microfluidic+chip+for+plant+science.">The RootChip: an integrated microfluidic chip for plant science.</a> Plant Cell. 23 (12), 4234-4240 (2011).</li><li>Grossmann, G., 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=Time-lapse+fluorescence+imaging+of+Arabidopsis+root+growth+with+rapid+manipulation+of+the+root+environment+using+the+RootChip.">Time-lapse fluorescence imaging of Arabidopsis root growth with rapid manipulation of the root environment using the RootChip.</a> J. Vis. Exp. (65), (2012).</li><li>Jiang, H., Xu, Z., Aluru, M. R., Dong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+chip+for+high-throughput+phenotyping+of+Arabidopsis.">Plant chip for high-throughput phenotyping of Arabidopsis.</a> Lab Chip. 14 (7), 1281 (2014).</li><li>Busch, W., 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=A+microfluidic+device+and+computational+platform+for+high-throughput+live+imaging+of+gene+expression.">A microfluidic device and computational platform for high-throughput live imaging of gene expression.</a> Nat. Methods. 9 (11), (2012).</li><li>Meier, M., Lucchettta, E., Ismagilov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Stimulation+of+the+Arabidopsis+thaliana+Root+using+Multi-Laminar+Flow+on+a+Microfluidic+Chip.">Chemical Stimulation of the Arabidopsis thaliana Root using Multi-Laminar Flow on a Microfluidic Chip.</a> Lab Chip. 10 (16), 2147-2153 (2010).</li><li>Ozoe, K., Hida, H., Kanno, I., Higashiyama, T., Notaguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+characterization+method+of+plant+root+adaptability+to+soil+environments.">Early characterization method of plant root adaptability to soil environments.</a> Proc. of 28th IEEE Interntl. Conf. Micro. Electro Mech. Syst. , (2015).</li><li>Sanati Nezhad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+platforms+for+plant+cells+studies.">Microfluidic platforms for plant cells studies.</a> Lab on a chip. , 3262-3274 (2014).</li><li>Parashar, A., Pandey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-in-chip:+Microfluidic+system+for+studying+root+growth+and+pathogenic+interactions+in+Arabidopsis.">Plant-in-chip: Microfluidic system for studying root growth and pathogenic interactions in Arabidopsis.</a> App. Phys. Lett. 98 (26), 2009-2012 (2011).</li><li>Rigas, 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=Root+gravitropism+and+root+hair+development+constitute+coupled+developmental+responses+regulated+by+auxin+homeostasis+in+the+Arabidopsis+root+apex.">Root gravitropism and root hair development constitute coupled developmental responses regulated by auxin homeostasis in the Arabidopsis root apex.</a> New Phytolol. 197 (4), 1130-1141 (2013).</li><li>Bengough, A. G., McKenzie, B. M., Hallett, P. D., Valentine, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+elongation,+water+stress,+and+mechanical+impedance:+A+review+of+limiting+stresses+and+beneficial+root+tip+traits.">Root elongation, water stress, and mechanical impedance: A review of limiting stresses and beneficial root tip traits.</a> J. Exp. Bot. 62 (1), 59-68 (2011).</li><li>Sia, S. K., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+fabricated+in+poly(dimethylsiloxane)+for+biological+studies.">Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies.</a> Electrophor. 24 (21), 3563-3576 (2003).</li><li>Millet, L. J., Stewart, M. E., Sweedler, J. V., Nuzzo, R. G., Gillette, M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+culturing+primary+mammalian+neurons+at+low+densities.">Microfluidic devices for culturing primary mammalian neurons at low densities.</a> Lab chip. 7 (8), 987-994 (2007).</li><li>Nelson, B. K., Cai, X., Nebenf&#252;hr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicolored+set+of+in+vivo+organelle+markers+for+co-localization+studies+in+Arabidopsis+and+other+plants.">A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants.</a> Plant J. 51 (6), 1126-1136 (2007).</li><li>Talbot, M. J., White, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+and+cell+outline+imaging+in+plant+tissues+using+the+backscattered+electron+detector+in+a+variable+pressure+scanning+electron+microscope.">Cell surface and cell outline imaging in plant tissues using the backscattered electron detector in a variable pressure scanning electron microscope.</a> Plant Methods. 9 (1), 40 (2013).</li></ol>

用于创建一个片上植物平台本文中描述的方法是独特的两层设计的范围根毛到单一的成像平面和平台可能解构和作为基材用于高分辨率非光学成像.使用高分辨率的非光学成像可以提供有价值的信息不能从单独的光学成像获得的植物组织。例如，原子力显微镜成像可以提供力测量计算根组织的弹性，在开发过程中或特定的化学或生物治疗后。同样，扫描电镜成像可以提供高分辨率细节表面形貌的根...

细根功能增加水和养分采集植物，探索新的土壤空间和增加总根表面面积。这些细根功能的营业额在起主要作用刺激地下食品链1和细根在某些植物物种数目是预期到双下大气二氧化碳浓度升高2。细根一般定义为那些小于 2 毫米直径，虽然新增倡导表征其功能3细根。像许多细根，根毛提供的摄取和吸收功能，但占用更小的空间，直径相当于微米。由于其体积小、 根毛难以图像原位，往往忽略了作为整体的根建筑领域规模实验和模型的一部分。
Ex terra根头发研究，如从琼脂平板上生长的幼苗，科学界提供了有价值的信息，对细胞生长和运输4，5。虽然琼脂板允许根系统成像无损和真正的时间，他们不提供另外的实验性治疗，如营养、 植物激素或细菌高环境控制。促进高分辨率成像，同时还提供动态环境控制新兴解决方案已经过植物研究的微流控平台的出现。这些平台已启用的非破坏性生长和可视化为高吞吐量分型6，7，8，9，孤立的化学处理的几个植物物种。10、 力测量11，12和微生物13的加法。微流控平台设计集中在单一的开放空间流体层根可能传播，允许根毛时有时无光学聚焦在增长或治疗过程中的使用。
在这里，我们目前的程序制订一个两层微流控平台使用照片和软光刻方法，在以前的片上植物设计基础上了围到主根相同的成像平面的幼苗根毛。这使我们能够跟踪根头发发展在真正的时间，在高分辨率下，和在整个实验治疗过程。我们培养的方法允许拟南芥幼苗萌发出种子在平台内而又有教养的水合和无菌的环境中不需要使用注射器泵设备一周。一旦延时成像实验已经结束，在这里提出了一种平台可以打开而不会干扰细根功能的位置。这允许使用其他高分辨率成像方法。在这里我们提供代表结果量化和可视化的根毛的形态结构，在这个平台上由光学、 扫描电子显微镜 (SEM) 和原子力显微技术 (AFM)。

<table width="810">
	<tbody>
		<tr height="72">
			<td height="72" style="width:209px;height:72px;">Silicon Wafer</td>
			<td style="width:191px;">WRS Materials</td>
			<td style="width:283px;">100mm diameter, 500-550um thickness, Prime, 10-20 resistivity, N/Phos&#60;100&#62;</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="width:209px;height:32px;">Quintel Contact Aligner</td>
			<td style="width:191px;">Neutronix Quintel Corp</td>
			<td style="width:283px;">NXQ 7500 Mask Aligner</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="width:209px;height:32px;">Fluorescent Microscope</td>
			<td style="width:191px;">Nikon</td>
			<td style="width:283px;">Eclipse Ti-U</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="width:209px;height:30px;">laboratory tissue</td>
			<td style="width:191px;">Kimberly Clark</td>
			<td>Kimwipe KIMTECH SCIENCE Brand, 34155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Negative Photoresist Epoxy</td>
			<td style="width:191px;">Microchem</td>
			<td style="width:283px;">SU-8 2000s series</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Photoresist developer</td>
			<td style="width:191px;">Microchem</td>
			<td style="width:283px;">Su-8 developer</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:209px;height:42px;">trichloro(1H,1H,2H,2H-perfluoro-octyl)silane</td>
			<td style="width:191px;">Sigma Aldrich</td>
			<td></td>
			<td>use in chemical hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Air Plasma Cleaner</td>
			<td style="width:191px;">Harrick Plasma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">PDMS</td>
			<td style="width:191px;">Dow Corning</td>
			<td>Sylgard 184 Silicone elastomer base</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">PDMS curing agent</td>
			<td style="width:191px;">Dow Corning</td>
			<td>Sylgard 184 Silicone elastomer curing agent</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Dessicator</td>
			<td style="width:191px;">Bel-Art</td>
			<td>F42010-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Scalpel</td>
			<td style="width:191px;">X-acto knife</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Biopsy Punch</td>
			<td style="width:191px;">Ted Pella</td>
			<td>15110-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Adhesive tape</td>
			<td style="width:191px;">Staples</td>
			<td>Invisible Tape</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Microfuge tube</td>
			<td style="width:191px;">Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Triton X</td>
			<td style="width:191px;">J.T.Baker XI98-07</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Bleach</td>
			<td style="width:191px;">Chlorox</td>
			<td>concentrated</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:209px;height:42px;">Plant-Based Media</td>
			<td style="width:191px;">Phyto Technology Laboratories</td>
			<td>M524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Agar</td>
			<td style="width:191px;">Teknova</td>
			<td>A7777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Wax film</td>
			<td style="width:191px;">Parafilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">microscope</td>
			<td style="width:191px;">Olympus</td>
			<td>IX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Atomic Force Microscope</td>
			<td style="width:191px;">Keysight Technologies</td>
			<td>5500 PicoPlus AFM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Petri dish</td>
			<td style="width:191px;">VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:209px;height:21px;">Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>7400</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:209px;height:42px;">Dual Gun Electron Beam Evaporator</td>
			<td>Thermionics</td>
			<td>Custom Dual Electron Gun Evaporation System</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging the root hair morphology of arabidopsis seedlings in a two-layer microfluidic platform

根毛增加根表面积为更好的水吸收及养分吸收的植物。因为他们是体积小而且经常掩映在它们生存的自然环境，根毛的形态结构和功能很难研究和从植物研究经常被排除在外。近年来，微流控平台提供的方式，而不会干扰到成像系统的传输过程中的根可视化根系统在高分辨率。微流控平台在这里生成以前的片上植物研究把相结合提出两层设备限制根毛相同的光学平面的拟南芥主要根源。这种设计使量化的根毛细胞和细胞器水平，也可以防止漂流在另外的实验性治疗过程中的 z 轴。我们描述如何在包含水合的环境中，而无需射流泵，而同时保持苗定环境存储设备。光学成像实验后，该设备可能拆卸和作为基材用于原子力或同时保持细根结构完整扫描电镜观察。

此处所述的两层 PDMS 微流控设备有 200 微米高渠道为主要的拟南芥根和 20 μ m 的高分庭，以限制侧向生长根毛 (图 1A)。这种设计可能用于类似根直径植物拟南芥和可以随时修改，以适应不同尺寸.的物种该设计采用用于植物的入口以及任何所需的化学或生物治疗的 8 侧入口。与媒体细化设备和浇筑设备周围的琼脂保持根环境水水的?...

Watch this Scientific Journal Video about Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform at JoVE.com

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

影像学在两层微流控平台拟南芥幼苗的根头发形态

这篇文章演示了如何以文化拟南芥幼苗在一个局限的主根和根毛向单一光学平面的两层微流控平台。这个平台可以用于实时光学成像的细根形态以及对于高分辨率成像，通过其他的手段。

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

Research

JoVE Journal

Bioengineering

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

High-throughput Protein Expression Generator Using a Microfluidic Platform

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

科研

生物工程

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

时间推移荧光成像拟南芥根系生长的根环境的快速操作使用RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...