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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作者没有透露。

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 ...

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

8.4K 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

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

展示新型引力光谱仪的使用拉伸和测量纤维蛋白

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

科研

生物工程

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

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 ...

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, ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

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

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

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

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 for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

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

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

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 ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

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

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

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

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

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 ...