Name: light sheet fluorescence microscopy of plant roots growing on the surface of a gel
Uploaded: 2017-01-18T13:00:00.000+00:00
Duration: 6 min 41 s
Description: Watch this Scientific Journal Video about Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel at JoVE.com

我们感谢马加什Fendrych为批判性阅读/查看和斯蒂芬Stadlbauer的音频设备。感谢米巴机械车间在IST奥地利为他们的OpenSPIM贡献。导致这些结果的研究已根据REA赠款协议N°[291734]和欧洲研究委员会（ERC项目2011年获得了资金从人计划（玛丽·居里行动）欧盟第七框架计划（FP7 / 2007-2013）的-StG-20101109-PSDP）。

1.拟南芥培养成像之前<ol><li>通过加入2.15克的MS培养基，10g的蔗糖，0.97克的MES（2-（N-吗啉代）乙磺酸）和1升DDH 2 O的（双蒸水制备半MS培养基（半强度Murashige和Skoog培养基） ）成1升瓶中。用KOH调整pH值至5.8。 </li><li> 15克/ L，结冷胶添加到1/2 MS培养基中并高压灭菌它在121℃下20分钟。 </li><li>倾30毫升的热介质的成正方形皮氏培养皿（245点¯x245×25毫米），厚度为约2mm创建凝胶的层。让菜冷却到室温，以允许介质固化。 </li><li>放灭菌拟南芥的种子到含有1mL的无菌H 2 O使用玻璃移液管或1000微升吸管尖拿起种子和播种它们到凝胶的表面上的1.5毫升反应管。放置的种子分开分别约10mm。密封用胶带板。 </li><li>将培养板在4 24小时°C（分层）。 </li><li>培育在生长培养箱该板， 例如在22℃在一个16/8ħ日/夜周期，120-140微摩尔/平方米/秒的光的6天量。长达10天可用于老厂。 </li></ol> 2.植物样品制备方法注:样品架既可以三维印刷或使用图2C中所描绘的尺寸在机器车间手工制作。 3D模型文件在补充材料（Supplemental_File _-_ 3D_Sample_Holder.stl）提供。见链接在材料清单中订购的3D打印。 <ol><li>添加5克琼脂糖低熔入含50毫升半MS培养基的50ml瓶子和高压灭菌它在121℃下20分钟。 </li><li>分装在1.5毫升的反应管的1％低熔点琼脂糖溶液。储存在4°C（可用于至少两个月）。 </li><li>熔体1 %%低熔点琼脂糖的等分试样在80℃，并让它冷却到33°C。 </li><li>清洗在超声单元的样品架。消毒用70％乙醇的样品架，并用无菌水冲洗。 </li><li>削减使用手术刀周围的植物凝胶。 </li><li>提起用平铲块，小心地将它使用的是第二刮刀样品架。 </li><li>用100微升吸管粘合在样品支架上的凝胶用1％琼脂糖（在33℃）。 </li><li>胶与使用10微升吸管1％琼脂糖凝胶上的植物。使用立体显微镜来验证该叶没有覆盖凝胶。不凝胶上直接感兴趣的区域进行定位。 </li><li>为了防止晒出工厂，不间断地工作。将样品架到1000微升枪头只要有可能。使用吸管尖的帽子，小心地将它在那里的工厂位于样品支架的一端。 </li><li>把样品架在枪头盒，如果需要准备更多的植物。植物可以是直销TLY成像或在生长培养箱放回。 </li></ol> 3.设置显微镜注:LED照明系统是一个定制的灯泡。构建的LED环所需的技术细节可以在图3和材料清单中找到。请参阅板设计的补充材料文件（Supplemental_File _-_ LED_Ring_Board.brd）。 <ol><li>螺钉或胶（ 例如双面胶带）上OpenSPIM X / Y / Z /θ级臂的下侧的LED环形。 </li><li> LED的环比可调电源（0-30 V，最大2A）连接。 </li><li>调整电压为所需的光强度（拟南芥，120-140微摩尔/米2 /秒， 图3D）。 </li><li>消毒用70％乙醇的样品室，并用无菌水冲洗。 </li><li>清洁汽油与和镜头清洁纸物镜。消毒用70％乙醇的物镜。 </li><li>连接perfusi上管，以在样品室中的单向排列。把含有新鲜½MS培养基和另一个空瓶子1升瓶子的蠕动泵灌注旁边。该瓶用一个灌注管样品室的下入口平台连接。样品室的上部出口连接的空瓶回收站使用其他输液管所使用的媒介。 </li><li>流量设置为1毫升/分钟的速度。 注意:要不会溢出的样品室，它具有比流入较高流出是很重要的。要么加大排气速度，或使用一个管具有较大内径的流出。 </li><li>切断的黑色塑料箔成3mm小方块，用70％乙醇洗，并让他们将它们放置在样品室中的水的表面上之前，干燥。 </li><li>除去从样品保持器的吸管尖和在样品室中插入样品架。如果新生产的样品架不适合舞台手臂，用细砂纸张使它更薄或万一使用O形环（∅6毫米）它太薄。 </li><li>要创建盖子，切黑铝箔分成两个50×25mm的碎片。使切每一块的一侧上的中间一个5毫米。折叠以创建一个三角形凹槽（ 图1D）。通过将盖体上朝向样本保持器的三角形凹口的样品室的顶部关闭与两个盖的样品室中。确保植物叶片不在盖子色光和从照明系统接收光。 </li><li>发现使用的x / y / z和旋转台在所述视场定位一个新兴侧根的感兴趣区域。 </li><li>录制前，让系统平衡至少15分钟。 </li><li>安装图像采集。 <ol><li>置217的图像的堆叠3微米的z间隔（650微米），并设置用15分钟摄像间隔的时间流逝为17小时的总时间。 注:详细的文档上如何操作OpenSPIM软件可以上找到（http://openspim.org/Acquisition#Acquiring_a_Stack）。 </li></ol></li><li>开始录制。 </li></ol>

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The authors have nothing to disclose.

光表荧光显微镜具有很大的优势，以低光毒性和超快采集速度，其可以被用来捕捉大量具有高时空分辨率的同时保持样品中的生理状态相结合。光片显微镜的分辨率可以相比，共聚焦显微镜9。然而，光散射和吸收沿激发和发射路径中发生单独和总体图像质量可以是不透明的组织内显著相比降低到表面。为了避免这种复杂人们可以使用的可能性来旋转沿垂直轴的样品和观察从不同的方向相同的体积。但是，这并不总是有利的， 例如侧根根和成像从后面在低图像质量的结果的一侧出现没有获得更多的信息。然而，旋转可以被主要用于定位SAMP乐以最好的方式。物镜的经典横向布置允许样品安装的新方法。植物受益于垂直位置。相比于其它的安装方法，例如嵌入凝胶24,25的内侧的根这里提出，该安装方法"的凝胶的表面上的"具有几个优点。 1）根系统是在与液体介质直接接触。样品室被连接到灌注系统，该系统提供了连续的新鲜培养基。它也可以用于快速交换样品室的整个体积应用不同的介质或药物。 2）之前，样品制备植物生长，因为它们是用来在实验室中生长。植物可以在荧光显微镜下被选择和仅所需的植物需要准备。 3）植物从培养皿到样品保持器，而不被触摸传送。从而植物可以进一步在其上在生长incubato生长在相同的凝胶开发r和机械应力减小到最低限度。 4）在试样上的视图是通畅和光学像差最小化，因为在样品和检测目标之间的空间完全填充有培养基并用不同折射率没有其它材料。 为了进行长期成像，植物照明系统是必要的，以确保植物的光合活性。在大多数实验室植物生长上的透明凝胶， 即根暴露于光。这可能会导致他们的环境不同的反应，诱导其生物化学和发展变化26,27。为了减少根系统上的光的量，黑色的塑料箔被用于覆盖水面以及由黑色铝箔的盖所覆盖的样品室中。光可到达植物的叶通过在盖的中心孔。在这种设置中，在背景灯没有增加观察，suggestin克杂散光从红色和蓝色发光二极管的量显著由GFP过滤器和遮光的方法降低。这使得保持图像采集过程中开了灯不增加相机的背景噪声。 样品架被设计用于三维印刷。但是，材料的选择是关键，因为被测试的几个塑料均不是100％的稳定，从而导致样品的漂移。因此，建议改用树脂或通过研磨一个聚乙烯（PEP）杆构建样品架。当使用光片显微镜设置用双面照明系统的样品夹持器可能会根据旋转角度的光片材之一干扰。从板块舀植物中减少机械应力，用锅铲的平角。该工厂能迅速干透，为第一次体验气流。尽量避免任何空气草案（快速移动，空调流量），WORK尽可能样品架，并不间断地滑向1000微升枪头。显微镜内，这是至关重要的不浸在液体中的全厂和保持叶片干燥。 该技术是理想的成像侧根形成的早期阶段。当执行成熟的根尖长期成像我们必须牢记，拟南芥根与100-300微米/小时迅速增加出来的视场。一个非常有用的今后的实施可能是一个自动跟踪算法，这将使以下根尖生长在延长的时间段。在采集过程控制环境条件，例如光与介质的营养组合物中的能力允许调查植物如何适应变化。根是在与液体介质，其可以用于应用药物化学激活基因表达的直接接触，例如使用地塞米松诱导的28或β型雌adiol诱导系统29。然而，需要时间来交换样品室的整个体积洗出的药物。设置可以通过减少样品室的体积以加速介质交换来改善。然而，该技术具有很大的潜力。安装过程中，标准化的生长条件，并使用光表镜温柔的图像采集相结合，使植物的生长发育具有高分辨率的长期研究在生理水平。这将有助于研究人员能够探索植物发育的基本机制。

其中一个在认识植物生长发育的关键问题是单个细胞器官分化和生长过程中的行为方式。理想情况下，细胞的活动，如基因表达模式和细胞内蛋白质的定位，可以在组织更大范围的光线中。这个目标带来的技术挑战，需要具有高的空间整个器官的观察以及在一段延长的时期，这可能会导致光毒性作用的时间分辨率。由于植物快速适应环境的变化，不断增长的条件必须严格控制。为了做到长期成像，而不与植物的生理状态的干扰，三件事情都必须确保，1）生长条件在样品室中，2）稳定样品放置过长时间，以及3）的摄像低的光强度，以避免光损伤和非生理条件。 生理成长conditi组件在显微镜试样腔室是用于长期实验的关键。有一些描述成像生长室的共焦显微镜1可用的协议- 3。然而，共焦显微镜引入高的光强度的植物，这可能会导致应激反应，通常抑制4的生长。此外，大多数常规显微镜使样品，因为他们试图重新定位自己和生长朝向重力的载体，其是不是最佳的植物仅水平定位。在过去的十年里，光片显微镜已成为一个强大的工具，捕捉到较大的标本在细胞分辨率发展的时间段数天5 - 9。光表镜允许垂直放置标本和植物的研究越来越多地用于研究根系发育10 - 21，最近由伯特审查T和Maizel 22。许多提到的研究10,13的- 18,21进行了优化，并在恩斯特HK施特尔策的使用样本的一种特殊的方式在实验室进行的安装件，其特征通过凝胶17的表面上生长的根。在这些研究中，使用定制的显微镜，其中所述植物是从底部保持。与此相反，大多数广泛提供光片的显微镜保持从顶部样品。因此，这个特定的制备方法不能容易地应用。这里介绍的方法提供了适用于OpenSPIM 23，用于施加和提高选择性平面照明显微镜（SPIM）开放式接入平台行之有效的上表面安装方法的协议。 此协议的总体目标是使拟南芥根系长期成像在OpenSPIM光表镜。这是通过在S直立生长的植物来实现在液体介质中的根的凝胶urface而叶子留在空气中。为了确保植物的光合活性，定制的照明系统照射的叶子，但不根（ 图1）。

<table><tbody><tr><td>Agarose, low melting</td><td>VWR</td><td>AFFY3282125GM</td><td></td></tr><tr><td>Black aluminum foil</td><td>Thorlabs</td><td>BKF12</td><td></td></tr><tr><td>Black plastic foil</td><td>Carl Roth</td><td>HT83.2</td><td></td></tr><tr><td>LED blue (453 nm)</td><td>OSRAM</td><td>LD&amp;nbsp;CN5M-1R1S-35-1</td><td></td></tr><tr><td>LED red (625 nm)</td><td>OSRAM</td><td>LR T66F-ABBB-1-1</td><td></td></tr><tr><td>LED board - PCB design software</td><td>Cadsoft Eagle</td><td></td><td></td></tr><tr><td>MES monohydrate</td><td>Duchefa</td><td>M1503.0100</td><td></td></tr><tr><td>Micropore Surgical Tape</td><td>3M</td><td>1530-1</td><td></td></tr><tr><td>Murashige &amp;amp; Skoog Medium (MS-Medium)</td><td>Duchefa</td><td>M0221</td><td></td></tr><tr><td>Phytagel</td><td>Sigma-Aldrich</td><td>P8169</td><td></td></tr><tr><td>Sample holder 3D print</td><td>i.materialise</td><td>&lt;a href=&quot;https://i.materialise.de/shop/item/sampleholder-openspim-zeisslightsheetz1&quot;&gt;https://i.materialise.de/shop/item/sampleholder-openspim-zeisslightsheetz1&lt;/a&gt;</td><td></td></tr><tr><td>Square Petri dishes (245 x 245 x 25 mm)</td><td>VWR</td><td>734-2179</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097-1KG</td><td></td></tr></tbody></table>

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.

这种样品制备方法允许显微镜样品室内部的植物的培养，同时观察具有光片显微镜（ 图1）的根系统。植物生长凝胶（½含有1.5％吉兰糖胶的MS培养基）的一个层的表面上安装在一专门设计的样本保持器（ 图2）。样品架是用透明树脂作为材料的三维印刷。一个制造版本的突出尺寸如图2C所示。根部浸没在液体（半MS培养基），其连续地灌注系统刷新。叶子留在空气和130微摩尔/平方米/秒，从被布置在所述植物（ 图1A，B和图3A-C）以上的环蓝色和红色LED来的光强度连续地照亮。该LED环形在我们的机器车间制造，我们提供了如何在图3和材料清单构建LED环技术细节。的光强度可连续调节范围从30-250微摩尔/米2 /秒（ 图3D）。根系统是由一个黑色的塑料薄片覆盖在水面的小片（ 图1）的阴影。从照明是由检测透镜收集的任何杂散光由GFP过滤器（ 图3E）过滤。 用这种设置，越来越拟南芥侧根的时间推移，记录用于使用20X / 0.5透镜（ 图4）17小时。侧根有它的中柱鞘细胞层，这是位于主根内很深的渊源。为了证明，即使在组织的内侧更深的成像能力对于延长的时间段，一个更高的放大倍率（40X / 0.75）被用于捕获横向ロ形成OT从第一级原基直至38小时（ 图5）的时间周期内出现了初生根。数据集的示例性二维切片示于图。 5B。该记录允许我们遵循侧根形成的3D模型（ 图5A）的动力学与蜂窝分辨率。 <img alt="图1" src="/files/ftp_upload/55044/55044fig1.jpg" /> 图1: 培养OpenSPIM样品室里面的条件。 A）成像室的素描。该工厂的凝胶的表面上直立生长，安装在定制的样本保持器（也参见图2）。根部生长在液体介质中，这是连续地灌注系统交换。在植物叶子生长在空气和由红色和蓝色发光二极管（也参见图3）的照明。根系统处于阴影机智<html>一个黑色的塑料薄膜覆盖在水面H小片。由黑色铝箔的两片的盖进一步降低了水表面以下的光的量和在样品室保持湿度。右侧的放大的面板突出的植物浸在液体介质凝胶的块的表面上生长。琼脂糖一滴挂载根凝胶上。虚线框表示通过显微镜观察到感兴趣的区域。 B）的成像室（无盖的照片）。数字（1） - （10）中A和B代表:（1）:X / Y / Z /θ级带LED环，（2）:样品架，（3）:盖，（4）: 拟南芥 ，（5）:灌注系统，（7）:检测物镜，（8）:液体介质，（9）:样品室，（10）:照明物镜黑色塑料箔，（6）的片材。 C）的盖是由黑色铝箔的两件。<html>目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55044/55044fig2.jpg" /> 图 2: 样品架。 A）3D模型。 3D模型文件在补充材料提供。透明丙烯酸类塑料，（4）和（5）:树脂，（6）:透明树脂（3） - B）中使用不同的材料（1）的三维打印的照片。 C）的样品架的技术图纸，数字代表毫米。 四 ）与植物制造的样品架的照片安装。虚线面积可通过显微镜观察到。 <a href="http://ecsource.jove.com/files/ftp_upload/55044/55044fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html>上传/ 55044 / 55044fig3.jpg"/&gt; 图3: 植物照明设置。 A）中的灯的示意性电路图。的发光二极管对可以用于定向闪电接通/关逐个。 LED:发光二极管，R:电阻，T:晶体管，JP:针头。印刷电路板）:B）中的照明灯的最终设计用在PCB软件（PCB绘制。我们提供补充材料的电路板设计文件。董事会随后制造和我们学院的采矿公司机加工车间装配。 C）的LED环形的照片接通。四对红色和蓝色LED的布置在环。 D）的电压范围可以3.5 V和14.0 V.抗性之间调整被用来达到光速的范围从30-250微摩尔/米2量/秒（R1-8:220欧姆，R9-12:1,220欧姆）。 E）的灯中，GFP和YFP的发射光谱。<html> TP://ecsource.jove.com/files/ftp_upload/55044/55044fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55044/55044fig4.jpg" /> 图4: 拟南芥 的时间流逝记录 拟南芥 侧根。 5日龄幼苗表达的膜标记（pUBQ10 :: YFP-PIP1; 4）和一个核记者（pGATA23 :: NLS-GUS-GFP）的特异性标记柱鞘该发展成侧根细胞。 217图像（3微米Z间距）的堆栈被抓获了使用20X / 0.5镜头17小时记录每15分钟。 A）四个时间点的69显示在最大强度投影。 B）的六总分217一个时间点的Z堆叠的单个切片被示出。在A和B比例尺代表100微米。负载/ 55044 / 55044fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/55044/55044fig5.jpg" /> 图5: 拟南芥 的时间流逝记录 拟南芥 侧根。 6日龄幼苗表达的膜标记（pUBQ10 :: YFP-PIP1; 4）和一个核记者（pGATA23 :: NLS-GUS-GFP）的特异性标记柱鞘该发展成侧根细胞。 200幅图像（1.5微米Z间距）的堆栈被抓获了使用40X / 0.75镜头38 H记录每15分钟。 A）的四个时间点的3D渲染，在网格中的数字代表通过主根的中心平面以下，B）的单个片。比例尺代表50微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55044/55044fig5large.jpg" target="_blank">请</a>点击此处查看该图的放大版本。 Supplemental_File _-_ 3D_Sample_Holder.stl。设置在3D模型文件。 <a href="http://ecsource.jove.com/files/ftp_upload/55044/Supplemental_File-3D_Sample_Holder.stl" target="_blank">请点击这里下载此文件。</a> 补充文件LED环Board.brd。提供的电路板设计文件。 <a href="http://ecsource.jove.com/files/ftp_upload/55044/Supplemental_File-LED_Ring_Board.brd" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel at JoVE.com

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

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 ...

light-sheet-fluorescence-microscopy-plant-roots-growing-on-surface

Research

JoVE Journal

Biology

14.6K 次观看.  Institute of Science and Technology Austria. 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.

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

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

科研

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, ...

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

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

生物化学

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic nutrient (e.g., P, Fe, Mn) and contaminant (e.g., As, Cd, Pb) solute species in the soil adjacent to plant roots (the &#8216;rhizosphere&#8217;) at sub-millimeter (~100 &#181;m) spatial resolution. The method combines sink-based solute sampling by the diffusive gradients in thin films (DGT) technique with spatially resolved chemical analysis by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The DGT technique is based on thin hydrogels with homogeneously distributed analyte-selective binding phases. The variety of available binding phases allows for the preparation of different DGT gel types following simple gel fabrication procedures. For DGT gel deployment in the rhizosphere, plants are grown in flat, transparent growth containers (rhizotrons), which enable minimal invasive access to a soil-grown root system. After a pre-growth period, DGT gels are applied to selected regions of interest for in situ solute sampling in the rhizosphere. Afterwards, DGT gels are retrieved and prepared for subsequent chemical analysis of the bound solutes using LA-ICP-MS line-scan imaging. Application of internal normalization using 13C and external calibration using matrix-matched gel standards further allows for the quantification of the 2D solute fluxes. This method is unique in its capability to generate quantitative, sub-mm scale 2D images of multi-element solute fluxes in soil-plant environments, exceeding the achievable spatial resolution of other methods for measuring solute gradients in the rhizosphere substantially. We present the application and evaluation of the method for imaging multiple cationic and anionic solute species in the rhizosphere of terrestrial plants and highlight the possibility of combining this method with complementary solute imaging techniques.

This protocol presents a workflow for sub-mm 2D visualization of multiple labile inorganic nutrient and contaminant solute species using diffusive gradients in thin films (DGT) combined with mass spectrometry imaging. Solute sampling and high-resolution chemical analysis are described in detail for quantitative mapping of solutes in the rhizosphere of terrestrial plants.

土壤中实验室、无机植物养分和污染物的二维可视化和量化

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic ...

环境科学

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

生物学

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 ...

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 ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...