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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;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 &#38; Skoog Medium (MS-Medium)</td>
			<td >Duchefa</td>
			<td >M0221</td>
			<td ></td>
		</tr>
		<tr >
			<td >Phytagel&#160;</td>
			<td >Sigma-Aldrich</td>
			<td >P8169</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sample holder 3D print</td>
			<td>i.materialise</td>
			<td ><a href="https://i.materialise.de/shop/item/sampleholder-openspim-zeisslightsheetz1">https://i.materialise.de/shop/item/sampleholder-openspim-zeisslightsheetz1</a></td>
			<td></td>
		</tr>
		<tr >
			<td >Square petri dishes (245x245x25 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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

科研

生物学

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

荧光显微镜筛选和新一代测序:参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

作为寄主植物挥发物的筛选工具electroantennographic生物活性

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

使用高解析度电脑断层可视化的三维结构和功能的植物血管

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

多层安装于斑马鱼的长期光表镜

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...