Name: imaging the root hair morphology of arabidopsis seedlings in a two-layer microfluidic platform
Uploaded: 2017-08-15T13:00:00.000+00:00
Duration: 9 min 23 s
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>暴露后烘烤硅片 5 分钟在 95 ° c。如果没有发展，回到晶圆片旋转涂布机和自旋上第二层的环氧负性光刻 (80,000 ~76.75%固体 cSt) 在 3000 转，实现第二层高度为 150-200 微米。</li>
		<li>允许硅片，然后才转到 95 ° C 加热 45 分钟休息 5 分钟。后在烤盘上烤，移除晶圆片和允许抗冷却和硬化在室温下另一个 5 分钟在平坦的水平面上。对齐和暴露到第二层掩模为 30 硅片在紫外线接触仪 （剂量大约 300 兆焦耳/厘米2） s。</li>
		<li>执行暴露后烘烤硅片放在 95 ° C，15 分钟的电炉子，然后允许硅片冷却 5 分钟平水平地面上再发展。</li>
		<li>这两种抗蚀剂层并举的将晶片放入塑料盘，淹没在相应的开发人员硅片 （见材料表）。轻轻摇动这道菜，偶尔要洗新鲜的开发人员在晶圆片。</li>
		<li>17 分钟后冲洗与异丙醇 (IPA) 晶片。如果白膜出现，继续循环之间冲洗硅片与开发商和投资促进机构，直到电影消失。干图案的硅片的氮。</li>
	</ol>
	</li>
	<li>聚二甲基硅氧烷软光刻
	<ol>
		<li>公开 30 空气等离子体对硅晶片 s 在等离子清洗上高级设置 （见材料表） 来清洁。Silanize （1 H，1 H，2h，2h-全氟-n-辛基） 三氯硅烷在化学罩下面 2 h 硅烷 (85 ° C) 的闪点的电炉子上晶圆。</li>
		<li>倒到主硅片上的固化剂的聚二甲基硅氧烷 (PDMS) 的比例为 10:1。脱气真空室中混合的 PDMS 防治然后在 70 ° C 烘箱 1 h 或直到完全固化聚二甲基硅氧烷聚合物。</li>
		<li>使用一把手术刀，聚二甲基硅氧烷设备切剥他们从主硅片。使用 1.5 毫米活检冲床创建种子和治疗的入口，冲孔种子进口 45 ° 角，以鼓励根系生长成的主渠道。</li>
		<li>使用透明胶带从聚二甲基硅氧烷设备删除碎片，设备设计端下来放玻璃盖玻片。高压釜组装的设备。</li>
	</ol>
	</li>
</ol>
2.种植设备
<ol>
	<li>拟南芥种子制备
	<ol>
		<li>表面消毒南芥种子 30%商用漂白剂稀释 4 倍用无菌水 7 分钟洗种子去离子水和 0.1%Triton X 洗涤剂溶液微量离心管中。</li>
		<li>分层的种子通过冷冻离心管，一夜之间或一周在 4 ° c。</li>
	</ol>
	</li>
	<li>设备准备
	<ol>
		<li>无菌的设备置于真空脱气室，从气体排除空气渗透 PDMS 和射流渠道来提高易用性的灌装。</li>
		<li>从真空室删除的设备并且立即淹没充满液体 ¼ 强度植物基础培养基 ph 值为 5.7 培养皿中的设备。</li>
		<li>使用吸管，拉液体通过入口填补该设备。请确保没有气泡留在渠道内通过目测。</li>
		<li>将单个设备转移到新干培养皿。倒或移液管的热琼脂周围设备直到琼脂水平几乎与 PDMS 设备的顶部齐平。允许琼脂凝固。 注: 设备现在正准备播种或可以存储在 4 ° C，直到需要。</li>
	</ol>
	</li>
	<li>播种设备内
	<ol>
		<li>在无菌环境中，将一个消毒的种子转移到使用小吸管每个装置的进口。</li>
		<li>盖培养皿用蜡膜 （见材料表） 和光/暗循环生长室或窗台在室温中放置。这样的设备是垂直的重力会鼓励扎根成长通过通道东方培养皿。</li>
	</ol>
	</li>
</ol>
3.处理
<ol>
	<li>实验性治疗
	<ol>
		<li>在幼苗生长发育所需的时间，实验处理幼苗通过添加到每个 8 侧端口通过吸管加入规定的量的治疗。</li>
		<li>重新封装培养皿，回到生长室或窗台。</li>
		<li>继续在所需的时间，以捕获单个时间点或开始时间推移成像成像样品。</li>
	</ol>
	</li>
</ol>
4.光学成像
<ol>
	<li>降低分辨率 (4-20 X) 成像
	<ol>
		<li>整个培养皿中的设备和较低的分辨率 (4-20 X) 明亮的领域或微分干涉 (dic) 成像倒立明场显微镜下幼苗的地方。优化照明条件下，通过调整曝光时间、 灯亮度、 光圈和光的极性澄清感兴趣的形态学特点。开车阶段到所需的根和重点区域的根或 root hair(s) 感兴趣。</li>
		<li>获取单个时间点或时间序列的图像。若要可视化的根毛生长，形象越来越根毛每分钟一次。若要可视化的主根生长，获得一个图像每隔 30 分钟返回培养皿和幼苗生长室或在垂直位置的窗台上成像完成后。</li>
	</ol>
	</li>
	<li>高分辨率 (20-63 X) 的图像
	<ol>
		<li>一旦幼苗已成长为所需的时间，用镊子从琼脂中删除盖玻片和设备。干净的底边盖玻片用乙醇湿实验室组织。</li>
		<li>适用于作为显微镜镜头厂商的建议目的推荐的浸泡媒体。盖玻片下来放在显微镜载物台和提高阶段联系浸入式媒体的目标。通过调整曝光时间、 灯亮度、 光圈和光的极性优化的照明条件。对感兴趣的 root hair(s) 开车送去的所需的区域和重点阶段。 注: DIC 需要可视化胞质，和荧光标记需要可视化细胞器运动。</li>
		<li>为了量化根头发的生长，随着时间的推移，获得一个图像每分钟。若要可视化细胞器运动，尽量减少同时仍然保留一个可识别的荧光信号从细胞器荧光照射时间。曝光时间允许的情况下尽快获得图像的细胞器。对于长延时成像，使用一个活细胞成像阶段孵化器保持环境温度和湿度。 注: 63 X 油目的被推荐成像根头发细胞器。</li>
	</ol>
	</li>
</ol>
5.非光学成像
<ol>
	<li>设备准备
	<ol>
		<li>一旦幼苗已成长为所需的时间，</li></ol></li></ol>从培养皿中取出设备和盖玻片。
		<li>把设备弄颠倒了，轻轻剥盖玻片，根留在 PDMS 通道内。</li>
		<li>继续用于 PDMS 设备作为基材，根在高分辨率原子力或扫描电子显微镜。</li>
	
	
	<li>扫描电镜观察
	<ol>
		<li>存款薄 (~ 20 毫微米) 的根和围聚二甲基硅氧烷使用双枪电子梁蒸发室上的铬层。</li>
		<li>将根和聚二甲基硅氧烷设备转移到扫描电子显微镜室。 注意: 成像条件、即电压、 电流和工作距离将需要进行优化，以实现为一个给定的应用程序所需的分辨率。</li>
	</ol>
	</li>
	<li>原子力显微镜 (AFM)
	<ol>
		<li>在原子力显微镜标本持有人上了装载 PDMS 设备根端。增加对比度相机中的更容易区分根从聚二甲基硅氧烷，将平反射 （如涂金云母） 衬底上的聚二甲基硅氧烷设备放置在原子力显微镜标本架上安装设备之前。</li>
		<li>安全在 PDMS 设备液体井附件和装满水保持在成像过程中水化的根源。</li>
		<li>加载到原子力显微镜标本持有人。调整 z-厚度的聚二甲基硅氧烷设备控制和驱动悬臂到该地区的利益为指导使用照相机从根上。</li>
		<li>对齐与悬臂尖端激光。对于接触模式成像的最佳结果，使用悬臂梁与弹簧常数的 0.01 或 0.03 N m 在扫描过程中的根力很小。</li>
		<li>慢慢地降低扫描机制直到悬臂只是使联系与样品。调整为所需的区域扫描尺寸和选择 1 线/s 与 256 电压点每行扫描速度。获取扫描。</li>
	</ol>
	</li>

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

imaging-root-hair-morphology-arabidopsis-seedlings-two-layer

Research

JoVE Journal

Bioengineering

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

8.5K 次观看.  University of Tennessee. 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。

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

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The 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, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

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, ...

科研

生物工程

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

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

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 ...

发育生物学

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through nanoindentations with an atomic force microscope (AFM) coupled with an optical inverted fluorescence microscope. The method consists of applying controlled forces to the sample while measuring its deformation, allowing quantifying parameters such as the apparent Young's modulus of cell walls at subcellular resolutions. It requires a careful mechanical immobilization of the sample and correct selection of indenters and indentation depths. Although it can be used only in external tissues, this method allows characterizing mechanical changes in plant cell walls during development and enables the correlation of these microscopic changes with the growth of an entire organ.

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

原子力显微镜研究活拟 南芥 根表皮细胞的物理性质

A method is described here to characterize the physical properties of the cell wall of epidermal cells of living Arabidopsis roots through ...

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

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

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 ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

生物学

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

模仿神经血管化初始事件的微流体模型

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

测量时空的Ca<sup&gt; 2 +</sup&gt;在拟南芥信号

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

植物根系的轻质板材荧光显微镜生长在一个凝胶表面

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

影像学在两层微流控平台拟南芥幼苗的根头发形态

摘要

探索更多视频

此视频中的章节

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

长期共焦成像根和下胚轴发育的简单室

原子力显微镜研究活拟南芥根表皮细胞的物理性质

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

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

模仿神经血管化初始事件的微流体模型

从长期来看，高解析度激光共聚焦时间流逝成像

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

测量时空的Ca

植物根系的轻质板材荧光显微镜生长在一个凝胶表面

影像学在两层微流控平台拟南芥幼苗的根头发形态

摘要

探索更多视频

此视频中的章节

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

长期共焦成像根和下胚轴发育的简单室

原子力显微镜研究活拟 南芥 根表皮细胞的物理性质

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

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

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