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 中准备的模具放在真空室中20分钟。</li>
	<li>在非对流式烤箱中固化65摄氏度90分钟后, 将其从模具上剥离, 然后使用活检拳将进入孔的穿孔进入射流通道。 注: 对于花粉管装置, 必须调整孔的大小以反映雌蕊的直径。因此, 这一价值将因物种而异。在这个实验中, 我们打了一个1毫米孔的雌蕊入口和1.5 毫米孔的液体介质油藏。对于青苔 protonemata 装置, 一个4毫米孔被用于样品水库。</li>
	<li>五十年代, 将该层和玻璃底盘 (直径 3.5-5 厘米) 暴露在空气等离子体中。</li>
	<li>在非对流式烤箱中按下玻纤层进入玻璃底盘, 加热65摄氏度30分钟, 完全封闭微流控网络。</li>
</ol>2. Microdevice 根毛的制备
<ol><li>重复步骤 1.1-1.3 使用模具, 为根和根头发 microdevices 准备两个注塑层。 注: 我们在根 microdevice 中使用了一个2毫米的液体介质储层孔。</li>
	<li>在五十年代将两个聚硅烷层暴露在空气等离子体中。</li>
	<li>使用定制的桌面光刻, 在显微镜下组装两层。 注意: 这些微的层间必须面对面。在组装之前, 请确保两个图层上的对齐标记匹配。</li>
	<li>在非对流式烤箱中加热65摄氏度30分钟, 完全封闭微流控网络。</li>
	<li>从所构造的 microdevice 上取下盖板, 将设备放在直径为5厘米的玻璃底部盘上。</li>
</ol>3. 花粉管的体外细胞培养培养基的制备 (蓝猪耳耳)
<ol><li>准备修改后的 Nitsch 介质, 如前所述15和蒸压釜的介质在121°c 为20分钟。 注意: 修饰 Nitsch 的介质的组成是 NH4没有3 (80 毫克/升), 是一个3 (125 毫克/升), Ca (无3)2‧ 4H2O (500 毫克/升), MgSO4‧ 7H2O (125 毫克/升), KH2PO4 (125 毫克/升), MnSO4‧ 4H2O (3 毫克/升), ZnSO4‧ 7H2O (0.5 毫克/升), H3BO3 (10 毫克/升), 丘索4‧ 5H2o (0.025 毫克/升), Na2MoO4‧ 2H2O (0.025 毫克/升),蔗糖 (5万毫克/升), 酪蛋白 (500 毫克/升)。所制备的培养基至少可贮存在4°c 4 月。</li>
	<li>用蒸压去离子水制备 26% (w/v) 聚乙二醇, 用0.3 µm 孔过滤器过滤溶液。 注: 所制备的培养基可储存在4摄氏度, 1 月。</li>
	<li>将步骤3.1 和3.2 中准备的试剂混合在 1:1 (v/v) 的比率中。 注: 此介质应为每次使用而准备新鲜。</li>
</ol>4. 制备根毛的体外细胞培养培养基 (拟南芥)
<ol><li>准备一个解决方案, 0.215% (w/v) Murashige & Skoog 培养基, 0.05% (瓦特/v) MES, 1% (瓦特/v) 蔗糖和 1% (w/v) 琼脂在去离子水。蒸压釜溶液在121°c 为20分钟。</li>
</ol>5. 制备苔藓 Protonemata 的体外细胞培养培养基 (Physcomitrella 专利)
<ol><li>在培养皿中, 在 BCDAT 中等的16中预孵化苔藓 protonemata。 注: BCDAT 介质的组成为 1 mM MgSO4, 10 mM 熟悉3, 45 µM FeSO4, 1.8 毫米的.2PO4 (pH 值调整为6.5 与 KOH), 微量元素解决方案 (0.22 µM 丘索4, 0.19 µM ZnSO4, 10 µM H3BO3、0.1 µM Na2MoO4、2µM MnCl2、0.23 µM CoCl2、0.17 µM 基)、1 mm CaCl2和5毫米二铵 (+) 酒石酸。</li>
	<li>在 microdevice 的 BCDATG 培养基中培养苔藓。 注: BCDATG 培养基为 BCDAT 培养基, 含 0.5% (瓦特/v) 葡萄糖。所制备的培养基可贮存在4°c 1 月。</li>
</ol>6.体外培养 Microdevice 中的T. 耳花粉管
<ol><li>将花粉管 microdevice 在真空室中, 再加20分钟。</li>
	<li>将 microdevice 从真空室中取出, 并使用一个非常精细的微将生长培养基引入雌蕊入口。用同样的介质把剩余的水井填满。等待几分钟, 直到所有的微都是由微内产生的真空填充介质。</li>
	<li>在玻璃底部的盘子里放一些湿纸巾, 以保持盘子里的湿度。</li>
	<li>用解剖针将花粉粒从野生型耳' 蓝色和白色 ' 的花朵转移到其柱头上。 注意: 我们还进行了这项实验的转基因T. 耳' 皇冠紫罗兰 ' 花 ( RPS5Ap::H2B-tdTomato线17), 与花粉管含有荧光标记精子细胞和营养细胞核。</li>
	<li>使用刀片切割授粉方式 (1 厘米长)。</li>
	<li>将剪切样式插入设备的入口, 如图 2所示。</li>
	<li>把盘子盖上, 用胶带封住。</li>
	<li>在黑暗中将设备放置在28摄氏度的孵化器中, 5-6 小时。</li>
</ol>7.体外培养 Microdevice 中的南芥根毛
注: 步骤 7.1-7.9 (除7.3 和 7.5) 应在层流罩中进行。
<ol><li>通过浸泡在无菌液体中 (5% (v/v) 家用漂白剂和 0.02% (v/v) 海卫 X-100), 将种子从南芥哥伦比亚 (Col-0) 和转基因线UBQ10pro::H2B-mClover (含有荧光核标记) 中消毒5分钟。</li>
	<li>用蒸压水彻底冲洗灭菌过的种子。</li>
	<li>把漂洗过的种子在水中存放2天, 在4摄氏度的黑暗中。</li>
	<li>在紫外线照射下 microdevice 消毒。</li>
	<li>将 microdevice 放在真空室中, 再加20分钟。</li>
	<li>使用微将生长介质引入设备的井中。等待几分钟, 直到真空填满所有的微与介质。</li>
	<li>将蒸压湿纸巾放在玻璃底部的盘子里, 以保持盘子里的湿度。</li>
	<li>将被灭菌的种子转移到设备的入口。</li>
	<li>把盘子盖上, 用胶带封住。</li>
	<li>在连续白光下, 将设备垂直放置在22摄氏度的孵化器中。</li>
	<li>检查根头发生长后 4-10 天的孵化。使用倒置显微镜获得明亮的场和荧光图像。</li>
</ol>8.体外在微流控装置中培养P. 专利(苔藓) Protonemata
注: 步骤 8.2-8.6 (除 8.3) 应在层流罩中执行。
<ol><li>Preculture P. 专利应变 Gransden 200418在 BCDAT 培养基上, 用玻璃纸在培养皿中覆盖一周, 连续白光在25摄氏度。</li>
	<li>在 microdevice 的紫外线照射下, 在层流罩中消毒。</li>
	<li>将 microdevice 放在真空室中, 再加20分钟。</li>
	<li>用微将生长介质引入井中。等待几分钟, 直到真空填满所有的微与介质。</li>
	<li>将蒸压水添加到 microdevice 周围的盘子中, 以保持盘子里的湿度。</li>
	<li>将一小块苔藓 protonemata 组织移植到 microdevice 的入口, 并在连续白光下25摄氏度的培养皿中组织。</li>
	<li>检查 2-3 周的孵化后苔藓 protonemata 生长。使用显微镜获得明亮的场图像。</li>
</ol>9.耳花粉管生长的时间推移成像
<ol><li>将 microdevice 放在装有图像采集硬件 (e. g) 和软件的倒置荧光显微镜上。找到 microgap 的位置。 注: 对于图像采集软件, 我们使用了商业上可用的产品 (材料表)。开放源码显微镜软件, 如µManager 也可以在线 (https://micro-manager.org/wiki/Micro-Manager)。我们建议在显微镜上安装显微镜冷凝器, 以获得更高分辨率的图像。</li>
	<li>每十年代使用显微镜图像采集软件捕捉明亮的现场图像。</li>
	<li>观察RPS5Ap::H2B-tdTomato线花粉管中的荧光标记的精子细胞和营养细胞核, 用 561 nm 激光照射试样, 并使用带通光过滤器 (578/105 nm)。 注: 虽然花粉管生长的时间推移图像经常被捕获, 但成像并没有影响生长。但是, 总是建议尽量减少激光强度、曝光时间和时间间隔, 以尽量减少荧光的毒性和漂白。</li>
	<li>调整图像的亮度和对比度, 并使用 ImageJ 软件 (https://imagej.nih.gov/ij/) 准备视频文件。</li>
</ol>

<ol>
	<li>Higashiyama, T., Takeuchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mechanism+and+Key+Molecules+Involved+in+Pollen+Tube+Guidance.">The Mechanism and Key Molecules Involved in Pollen Tube Guidance.</a> Annu. Rev. Plant. Biol. 66, 393-413 (2015).</li><li>Vogler, H., Shamsudhin, N., Nelson, B. J., Grossniklaus, U., Obermeyer, G., Feij&#243;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+cytomechanical+forces+on+growing+pollen+tubes.">Measuring cytomechanical forces on growing pollen tubes.</a> Pollen Tip Growth. , 65-85 (2017).</li><li>Kehr, J., Wagner, C., Willmitzer, L., Fisahn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+modified+carbon+allocation+on+turgor,+osmolality,+sugar+and+potassium+content+and+membrane+potential+in+the+epidermis+of+transgenic+potato+(Solanum+tuberosum+L.)+plants.">Effect of modified carbon allocation on turgor, osmolality, sugar and potassium content and membrane potential in the epidermis of transgenic potato (Solanum tuberosum L.) plants.</a> J. Exp. Bot. 50 (334), 565-571 (1999).</li><li>Benkert, R., Obermeyer, G., Bentrup, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+turgor+pressure+of+growing+lily+pollen+tubes.">The turgor pressure of growing lily pollen tubes.</a> Protoplasma. 198, 1-8 (1997).</li><li>Vogler, H., 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+pollen+tube:+a+soft+shell+with+a+hard+core.">The pollen tube: a soft shell with a hard core.</a> Plant J. 73 (4), 617-627 (2013).</li><li>Shamsudhin, N., 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=Massively+parallelized+pollen+tube+guidance+and+mechanical+measurements+on+a+Lab-on-a-Chip+platform.">Massively parallelized pollen tube guidance and mechanical measurements on a Lab-on-a-Chip platform.</a> PloS one. 11 (12), e0168138 (2016).</li><li>Cheung, A. Y., Boavida, L. C., Aggarwal, M., Wu, H. M., Feij&#243;, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pollen+tube+journey+in+the+pistil+and+imaging+the+in+vivo+process+by+two-photon+microscopy.">The pollen tube journey in the pistil and imaging the in vivo process by two-photon microscopy.</a> J Exp Bot. 61 (7), 1907-1915 (2010).</li><li>Metzner, R., 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=Direct+comparison+of+MRI+and+X-ray+CT+technologies+for+3D+imaging+of+root+systems+in+soil:+Potential+and+challenges+for+root+trait+quantification.">Direct comparison of MRI and X-ray CT technologies for 3D imaging of root systems in soil: Potential and challenges for root trait quantification.</a> Plant Methods. 11 (1), 1-11 (2015).</li><li>Xia, Y., 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=Soft+Lithography.">Soft Lithography.</a> Angew Chemie Int Ed. 37 (5), 550-575 (1998).</li><li>Massalha, H., Korenblum, E., Malitsky, S., Shapiro, O. H., Aharoni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+root-bacteria+interactions+in+a+microfluidics+setup.">Live imaging of root-bacteria interactions in a microfluidics setup.</a> P Natl Acad Sci. 114 (17), 4549-4554 (2017).</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>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> Nature Methods. 9, 1101-1106 (2012).</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> Appl. Phys. Lett. 98, 263703 (2011).</li><li>Bascom, C. S., Wu, S. -. Z., Nelson, K., Oakey, J., Bezanilla, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Growth+of+Moss.">Long-Term Growth of Moss.</a> in Microfluidic Devices Enables Subcellular Studies in Development. Plant Physiol. 172 (1), 28-37 (2016).</li><li>Higashiyama, T., Kuroiwa, H., Kawano, S., Kuroiwa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+in+vitro+of+the+pollen+tube+to+the+naked+embryo+sac+of+Torenia+fournieri.">Guidance in vitro of the pollen tube to the naked embryo sac of Torenia fournieri.</a> Plant Cell. 10, 2019-2032 (1998).</li><li>Nishiyama, T., Hiwatashi, Y., Sakakibara, K., Kato, M., Hasebe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tagged+mutagenesis+and+gene-trap+in+the+moss,+Physcomitrella+patens+by+shuttle+mutagenesis.">Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis.</a> DNA Res. 7, 9-17 (2000).</li><li>Maruyama, D., 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=Independent+Control+by+Each+Female+Gamete+Prevents+the+Attraction+of+Multiple+Pollen+Tubes.">Independent Control by Each Female Gamete Prevents the Attraction of Multiple Pollen Tubes.</a> Dev. 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>

作者声明他们没有竞争的财政利益。

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, 这可能会影响这些尖端生长细胞的生长。由于实验是在半天内完成的, 花粉管装置的灭菌并不那么重要。我们经常使用的花粉管设备没有绝育, 并没有观察到任何不良影响, 在实验中。
微通道设计应根据感兴趣的细胞进行修改。在我们的工作中, 用于耳根头发实验的通道设计不同于用于 T.. ..........专利protonemata 实验。需要不同的设备, 因为样品有不同的尺寸;例如, 根毛比花粉管和苔藓 protonemata 短得多。因此, 我们设计了根发装置, 使 microgap 区域与主根通道相邻。除了渠道设计, 根发装置的制作程序也不同于用于花粉管或苔藓 protonemata 的程序。对于花粉管和苔藓 protonemata 的研究, 我们使用了一个 microdevice 包含一个单一的一层, 与微密封的玻璃底部菜。然而, 用于研究根毛的设备包括两层: 顶层带有微通道 (深度: 200 µm), 主要根被密封在底层, 包含微 (深度: 4 µm) 的根毛。用于制造硅模具的标准光刻技术仅限于5:1 左右的纵横比。因此, 要创建1µm 宽的间隙, 通道高度将限制在大约5µm。在技术上, 精确地对齐一个深通道 (e. g, 200 µm), 在1µm 宽的硅模具上的缺口上, 这也是一个技术性的挑战。因此, 我们为主根和根毛准备了单独的聚硅烷层, 并将其密封在一起。
虽然该协议成功地用于可视化花粉管的尖端生长过程通过 microgaps (补充影片1和 2), 在 microgap 地区根头发和苔藓 protonemata 伸长的时间推移成像将更挑战。在用于培养 microdevice 根毛的使用中, microgaps 所在的侧通道的高度 (4 µm) 比根生长室 (200 µm) 浅得多, 因此大多数根毛无法进入侧通道. 即使根毛确实找到了侧通道入口, 也很难预测它们何时进入。为了捕获 microgap 的根毛发生长的时间推移图像, 我们建议使用一台装有自动舞台的显微镜, 它可以被编程为图像采集的多个位置。当这种仪器无法使用时, 我们建议搜索已经进入侧面通道但尚未越过 microgaps 的根毛发。自A的增长速率以来. 拟南芥的根毛比T. 耳花粉管 (通常为 22-23 µm/分钟19) 要慢得多 (通常为 1-2.5 µm/分20), 可能会捕获这些根毛的时间推移图像。即将穿越 microgaps。另一方面, 苔藓 protonemata 观察需要一个时间推移系统, 可以专门用于这项研究, 因为长期的观察周期是由于缓慢的增长速度。
这里提出的微流控方法是第一种方法, 使我们能够研究尖端生长的植物细胞的能力拉长通过非常狭窄的空间。这些 microdevices 可能有助于筛选化验, 研究与细胞壁生物合成相关的基因功能和尖端生长细胞的完整性, 通过他们的渗透能力。此外, Denais et al。21最近演示了一种核包络断裂和修复机制, 通过在微流控装置中制备的密闭空间挤压癌细胞, 可以研究在花粉管中是否存在类似的机制, 使用我们的装置。这个新开发的实验平台可以用来研究单个细胞如何响应物理约束空间, 从而增强我们对尖端生长机制的理解。

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><tbody><tr><td>PDMS</td><td>Dow Corning Co.</td><td>Sylgard184</td><td></td></tr><tr><td>Murashige &amp;amp; Skoog Medium</td><td>Wako Pure Chemical</td><td>392-00591</td><td></td></tr><tr><td>MES</td><td>Dojindo</td><td>345-01625</td><td></td></tr><tr><td>Sucrose</td><td>Wako Pure Chemical</td><td>196-00015</td><td></td></tr><tr><td>50 mm glass-bottom dish</td><td>Matsunami Glass</td><td>D210402</td><td></td></tr><tr><td>35 mm glass-bottom dish</td><td>Iwaki&amp;nbsp;</td><td>3971-035</td><td></td></tr><tr><td>Surgical blade</td><td>Feather</td><td>No.11</td><td></td></tr><tr><td>biopsy punches</td><td>Harris</td><td></td><td>Uni-Core</td></tr><tr><td>Gel loading tips</td><td>Bio-Bik</td><td>124-R-204</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus</td><td>IX83</td><td></td></tr><tr><td>CSU-W1</td><td>Yokogawa Electric</td><td></td><td>No Catalog number is avairable for this customized microscope</td></tr><tr><td>MetaMorph imaging software</td><td>Molecular Devices</td><td></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粉管顶端区域的形态学变化, 以及植物细胞核和精子细胞, 以应对遇到一个非常小的空间 (补充电影1和 2)。
虽然大多数 microgaps 是通过使用此处描述的方法成功地制造出来的, 但我们注意到其中一些已完全关闭 (图 6)。由于在硅模具上创建的1µm 宽通道是易碎的, 重复使用这种模具可能会损坏缺口, 导致在基片层上的缝隙堵塞。因此, 在进行实验之前, 检查 microgaps 是否完好无损是很重要的。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57262/57262fig1.jpg"> 图 1: 将尖端生长的植物细胞渗透到物理约束空间中.(A)花粉管通过几个物理屏障延伸到胚珠上。障碍包括传播的形式和珠孔周围的层珠被组织。(B)根毛穿透致密土壤。在(A)和(B)中插入将显示盒装区域的放大。<a href="/files/ftp_upload/57262/57262fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57262/57262fig2.jpg"> 图 2: 用于研究耳花粉管、microdevices 根毛和 P. 专利protonemata 的特性。在微的示意图中, 一个样本放置在星号标记 (*) 所指示的位置。概述了微通道的设计、每种设备的照片、通道结构和细胞培养周期。此数字已通过柳泽et al的权限进行了修改。201719。缩放条, 20 µm.<a href="/files/ftp_upload/57262/57262fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57262/57262fig3.jpg"> 图 3:耳花粉管伸长率通过1µm 间隙.(A)花粉管通过1µm 宽和4µm 高间隙。利用带旋转圆盘共焦系统的倒置显微镜, 捕捉到了延时亮场图像。缩放条, 20 µm. (B)荧光标记植物细胞核和精子细胞在 microgap RPS5Ap::H2B-tdTomato线的花粉管中的时间推移图像。缩放栏, 20 µm. 图(A)和(B)已通过柳泽et al的权限进行了修改。201719。<a href="/files/ftp_upload/57262/57262fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57262/57262fig4.jpg"> 图4。南芥根发伸长率通过1µm 隙.(A) microdevice 中的根和根毛发生长。microgaps (宽度为1µm, 高度为4µm) 位于根生长通道的左侧。右侧的微不包含 microgaps, 可以用作控件。缩放条, 100 µm. (B)亮字段, (C)荧光, (D)合并了通过 microgaps 的根毛的图像。根毛发中的原子核在UBQ10pro::H2B-mClover行中荧光标记。每根头发的尖端由箭头指示。刻度条, 30 µm。这些数字已通过柳泽et的权限进行了修改。201719。<a href="/files/ftp_upload/57262/57262fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57262/57262fig5.jpg"> 图5。专利通过1µm 间隙 protonemata 伸长率.苔藓 protonemata 细胞穿过1µm 间隙 (高度为4µm), 由于 protonemata 的膨压力而加宽。刻度条, 20 µm。该图形已通过柳泽et al的权限进行了修改。201719。<a href="/files/ftp_upload/57262/57262fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57262/57262fig6.jpg"> 图6。已关闭 microgaps1µm 间隙区域的 SEM 图像。右边的 microgap 完全封闭了。<a href="/files/ftp_upload/57262/57262fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
辅助影片1。花粉管生长通过 1 µm 隙.时间推移明亮的现场图像是每十年代拍摄的, 使用倒置显微镜配备了旋转圆盘共焦显微镜.缩放栏, 20 µm.<a href="/files/ftp_upload/57262/movie1.avi">请单击此处下载此文件.</a>
辅助影片2。营养细胞核和精子细胞穿透 1 µm 间隙.利用反转荧光显微镜, 每年二十年代捕获到时间过亮场和荧光图像。缩放栏, 20 µm.<a href="/files/ftp_upload/57262/movie2_3fps.avi">请单击此处下载此文件.</a>

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 ...

development-microfluidic-devices-to-study-elongation-capability-tip

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.3K 次观看.  Nagoya University. 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。

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

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

科研

生物学

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

一种微流体装置研究多个不同的菌株

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

生物工程

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, ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

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

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

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

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 their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

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

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

影像学在两层微流控平台拟南芥幼苗的根头发形态

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 ...

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 ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

用于在细胞水平上探测真菌 - 微生物相互作用的微流体工具

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...