作者要感谢玉米遗传学合作组织，玉米细胞基因组学项目，Dave Jackson（纽约冷泉港实验室），Anne W. Sylvester（伊利诺伊州芝加哥大学海洋生物实验室），Andrea Gallavoti（罗格斯大学，新泽西州）和Carolyn G. Rasmussen（加州大学河滨分校）提供突变和转基因种群，以及大学先进光学显微镜核心的Robert F. Baker和Alexander Jurkevich。密苏里-哥伦比亚在共聚焦显微镜方面的协助。JMR得到了J. William Fulbright奖学金，Diane P.和Robert E. Sharp基金以及国家科学基金会的植物基因组研究计划（IOS-1546873）的支持。CDTC，EDCDP和RJRR由DOST-SEI S&T本科奖学金支持。DODL由Thomas Steinbugler SJ学术奖学金支持。RJRR 由国际教育——高等教育途径奖学金支持。这项工作得到了马尼拉雅典耀大学科学与工程学院和黎刹图书馆的支持。

改进的玉米叶片原基成像方法

作者没有任何利益冲突需要披露。

我们提出了两种制备玉米叶原基用于细胞成像的方法。第一种方法（协议部分 2）允许测量原基进行横向截面分析，而第二种方法（协议第 3 部分）允许展开和展平原基以进行整体安装分析。这些方法有助于玉米叶原基24中FPs的细胞成像（如图4和图5所示），并为玉米叶片成像的挑战提供了简单的解决方案。协议第 2 节通过在切片前测量原基而不是仅依靠分期参数 9,16 来减少解剖时间并提高采样精度。通过市售纳米胶带，方案第3部分解决了玉米全叶原基成像的长期问题。该协议改进了先前使用透析管31的方法，并且是CT和MRI11,32,33的更便宜的替代方案。然而，当涉及到可视化叶片解剖特征并产生最佳结果时，两种方案都有一些局限性，表2中概述了这些局限性，下面将更详细地讨论。
在方案第2部分中，我们在叶原基的厚横向切片中难以可视化细胞轮廓，并且用细胞壁或质膜结合荧光染料复染未提供令人满意的结果。例如，与质膜FP标志物p Zm PIP2-1：：ZmPIP2-1：CFP39（PIP2-1-CFP;图3A-D）。为了克服这一限制，我们建议使用振动切片机产生更薄的组织切片（~0.1 mm）58，这将允许对细胞轮廓进行生动的明场成像或优化复染方案47,59。
在协议部分3中，主要限制是难以在不撕裂，损坏或气泡的情况下安装叶片，如协议步骤3.2.5-3.2.6（图3E-K）中所述。由于玉米叶片是双边对称的，因此半叶安装而不是全叶安装可能足以进行可视化9。为此，可以在将原基展开到中脉后，用剃须刀片沿纵轴切割原基，只允许安装叶子的一半。协议第3节的另一个限制是叶片的厚度会限制深度成像过程中荧光团信号的光学分辨率。为了解决这个问题，可以采用组织清除技术60。然而，我们发现ClearSee61是一种用于植物组织成像的常用清除试剂，与该方案不兼容，因为它会导致样品和盖玻片从纳米胶带上分离。这个问题的一个潜在解决方案可能是在叶片样品上施加半透膜31，允许它用透明溶液处理，同时被纳米胶带固定到位。这种允许将液体溶液应用于展开的叶子的方法也可用于全安装RNA原位杂交和免疫定位技术，这些技术以前已针对玉米花序发育进行了优化，但不适用于全叶原基62,63。
我们描述了玉米的方案，即使在幼苗阶段，玉米也有大的叶原。其他具有更小叶原基的草种，如水稻、大麦、小麦、Setaria 和 Brachypodium16、23、64、65,66，可能需要使用额外的精密工具来有效地应用这些协议。此外，这些协议不适用于活细胞成像，活细胞成像捕获组织形成和细胞反应的实时动态过程。然而，随着荧光探针，成像技术和计算能力在植物67活细胞成像方面的不断进步，未来的研究可以建立在这些协议的基础上，开发适合草叶原初生物独特特征的活细胞成像策略。

草作物是全球人口的主要食物和生物燃料来源1，改善叶片解剖结构有可能提高其生产力2,3。然而，我们目前对草叶解剖结构如何调节的理解是有限的4，需要对叶原基进行分析，因为叶的许多解剖学和生理学特征是在发育早期预先确定的5,6,7。细胞成像技术，如荧光和共聚焦成像，对于研究草叶解剖结构和细胞性状是必不可少的，但这些技术很难应用于草叶原基，因为它们深深地包裹在叶涡内并滚动。我们通过开发用于玉米叶片原初荧光和共聚焦分析的横向切片和展开的全叶支架的制备方法解决了这个问题，这是一种研究草叶解剖学和发育的模型系统2,8。
玉米叶和所有草叶一样，由带状叶片组成，带鞘缠绕茎和发育芽9,10,11,12,13。叶子从芽顶端分生组织（SAM）以分散模式发育，其中每片新叶子在与前一片叶子相反的位置开始，导致沿垂直轴形成两排叶子（图1A）14。每个叶原基的发育阶段由其相对于SAM的位置确定，最近的原基指定为质体1（P1），随后的原基指定为P2，P3等（图1B，C）2。在发育过程中（图1D），叶原基首先表现为围绕SAM（P1）基部的新月形支撑，然后长成延伸到分生组织（P2）的罩状原基9,10,11。然后，随着尖端向上生长，罩的基缘横向扩展并相互重叠，形成锥形原基（P3-P5）10。然后原基迅速变长，基部的鞘叶边界随着叶子的近轴侧的边缘状突起的形成而变得更加突出（P6/P7）。最后，叶子在稳态生长过程中从漩涡中出来时展开，其中分裂细胞被限制在叶片的小基部区域内，形成沿近端-远端轴（P7 / P8）扩展和分化细胞的梯度15。玉米幼苗的枝顶端包含多个处于不同发育阶段的原基，是研究叶片发育的优良模型8。
对早期叶片发育的准确分析需要分期或使用标准化标准来定义与其他生长或形态参数相关的原始发育的不同阶段。由于叶原基隐藏在草芽中，研究人员通常使用植物的年龄或新生叶子的大小等参数作为叶原基9,16阶段和大小的预测指标。在玉米中，植物的实际年龄由种植或发芽后的天数（DAP/DAG）决定17,18。营养阶段（V期）由最上部的叶子确定，叶子有一个可见的衣领，叶片和鞘之间的远轴侧有一条苍白的线，对应于叶片和耳廓的位置，叶片底部的一对楔形区域（图1A，B）17,19。在20到25 DAG之间，SAM过渡到花序分生组织并停止产生新叶20。玉米叶原的生长速度可以根据环境和植物的基因型而变化。因此，植物年龄和新叶的大小无法准确预测叶子原初的大小;但是，使用这些参数可以帮助预测原始阶段的范围和大小，以便进行实验。
横截面分析是检查玉米和其他草的叶片解剖结构和发育的常用方法，因为它允许在枝条21,22,23的单个截面中对多个质体进行采样。该方法也便于新鲜样品的细胞成像，因为周围的叶子充当支架，在切片和安装期间将叶原基保持在原基的位置24。然而，这种方法的缺点是，当从完整的枝条上切片时，精确定位目标质体和原基内的区域可能具有挑战性。此外，由于叶片生长在塑体上和沿近端-远端轴2,5而变化，因此不精确的采样可能导致对给定部分中原基的发育阶段和区域的错误解释。因此，开发一种精确的横截面采样方法对于确保草叶原初解剖学和发育分析的准确性和可重复性至关重要。
全叶安装分析能够对全器官规模发生的组织和细胞过程进行全面和综合的研究，例如增殖生长25 和静脉模式化 26、27、28。该方法提供了叶子的副皮概述，允许发现不同的过程和模式，否则使用横截面分析很难检测到24,27。与拟南芥不同，拟南芥已经建立了对全叶坐骑进行成像的方法29,30，目前没有标准方法对草丛中展开的全叶坐骑进行成像。先前用于展开分离玉米叶原基的方案涉及不常见的材料，不适合细胞成像31。先进的成像技术，如计算机断层扫描（CT）和磁共振成像（MRI），可以在不分离和展开原始11,32,33的情况下获取3D解剖信息，但它们价格昂贵且需要专用设备。开发一种技术来克服玉米和其他草中叶原基的卷曲和圆锥形形态所施加的限制，将推进对其解剖学和发育性状的研究。
在这里，我们提出了制备玉米叶原基的横向切片和展开的整个安装以进行荧光和共聚焦成像的方法。我们使用这些方法量化静脉数量，并用荧光蛋白（FPs）绘制玉米叶原基中的时空激素分布图24。第一种方法涉及用剥线钳从玉米幼苗上去除老叶的上部（图1E）。通过暴露原基的尖端（P5-P7），可以确定其长度，而无需完全去除周围的较老的叶子，从而实现轻松准确的切片。第二种方法涉及用透明的双面纳米胶带展开和安装全叶原基（P3-P7）（图1F）。这些方法适用于可视化各种FPs24，但需要优化使用荧光染料和清除试剂。此外，我们还概述了在 ImageJ/FIJI 34 中展平 z 堆栈、拼接图像和合并通道的一些过程，这些过程适用于这两种方法生成的图像。这些方法可用于玉米叶片的常规荧光或共聚焦成像，但它们也可以适用于其他模型草种，如水稻、Setaria 和 Brachypodium。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65239/65239fig01.jpg"> 图1：玉米叶原基的组织和形态以及方法概述 。 （A）玉米幼苗示意图。玉米具有二叶叶，新叶子在与前一片叶子相反的位置开始。叶号表示叶子发芽的时间顺序（即第一叶，L1;第二叶，L2;第三叶，L3;等）。每片叶子都有一个远端叶片和一个基鞘，基鞘由与耳廓和耳廓相对应的衣领勾勒出来。最上面的叶子，衣领可见，表示营养阶段。此示例中的幼苗处于 V2 阶段，L2 项圈（箭头）可见。剪刀图标指示中胚叶（me）的位置，必须在那里切割幼苗才能收集。（B）解剖芽的示意图，显示孤立的L1至L4，叶原基L5至L9在（C）中显示为放大图像。质体数表示原基相对于SAM的位置，最年轻的叶原基（P1）最接近SAM，较老的叶原基（P2、P3、P4等）相继远离2。（D）玉米叶原基从P1到P5的形态示意图。（五）玉米叶原基横断面分析方法示意图。（1）用剥线钳修剪老叶。（2）测量原基，切片芽。（3）将切片安装在载玻片上进行成像和处理（4,5）。（F）玉米叶原基全装分析方法示意图概述。（1）去除周围的叶子，提取原基。（2）在纳米带上切割并展开原基。（3）安装样品进行成像和处理（4,5）。缩写：L = 叶子;bl = 刀片;sh = 护套;CO = 项圈;我=中胚叶;V = 植物人;P = 塑体;SAM = 枝顶端分生组织。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65239/65239fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table><tbody><tr><td>Acrylic Gel Clear Double Sided Nano Tape 16.5 ft x 1.2 in, 2 mm thick</td><td>EZlifego Store (Amazon)</td><td>B07YB1ZXG6</td><td>1 roll</td></tr><tr><td>Bellucci Pick Curved micro probe 16.8 cm, 6.6 in</td><td>Bausch &amp;amp; Lomb</td><td>N1692 9</td><td>1 pc</td></tr><tr><td>Clayman guide microprobe Sinskey hook angled shaft, 11.6 cm, 4.6 in</td><td>Storz Opthalmic Instruments</td><td>E0542</td><td>1 pc</td></tr><tr><td>Dental Probe, Bent Needle, 14 cm (5.5 in)</td><td>Ted Pella</td><td>13553</td><td>1 pc</td></tr><tr><td>DOWELL 10-22 AWG Wire Stripper</td><td>Dowell Store (Amazon)</td><td>10-22 AWG</td><td>1 pc</td></tr><tr><td>Feather Double Edge Carbon Steel Blades</td><td>Ted Pella</td><td>121-9</td><td>pkg/10; for fine sectioning</td></tr><tr><td>Frosted End Glass Microscope Slides, 75 mm x 25 mm x 1-1.2 mm</td><td>Ted Pella</td><td>260442</td><td>pkg/144</td></tr><tr><td>GEM Single Edge, Stainless Steel Uncoated Blades</td><td>Ted Pella</td><td>121-1</td><td>box/200; for general cutting/sectioning</td></tr><tr><td>Glycerol</td><td>Thermo Scientific</td><td>PI17904</td><td>1 liter</td></tr><tr><td>ImageJ/FIJI with EDF plugin (version 17.05.2021) and Grid/Collection Stitching plugin</td><td>National Institutes of Health (NIH) USA</td><td>version 2.9.0/1.54s</td><td>The EDF plugin was developed by Alex Prudencio, Jesse Berent, and Daniel Sage for the Biomedical Imaging Group, &amp;Eacute;cole Polytechnique F&amp;eacute;d&amp;eacute;rale de Lausanne (EPFL; http://bigwww.epfl.ch/demo/edf/). The grid/collection stitching software was developed by Stephan Preibisch for the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG).</td></tr><tr><td>Kimwipes Ex-L Small 111.76 mm x 213.36 mm</td><td>Kimtech Science</td><td>34155</td><td>box/280 ply</td></tr><tr><td>Micro Cover Glasses, 22 mm x 22 mm x 0.13 - 0.16 mm thick</td><td>Ted Pella</td><td>260140</td><td>1 ounce</td></tr><tr><td>PU Gel Clear Double Sided Nano Tape 29.5 ft x 1.18 in, 1 mm thick</td><td>Yecaye Store (Amazon)</td><td>L354 W1.18</td><td>2 rolls</td></tr><tr><td>Superslip Cover Glasses, 24 mm x 50 mm x 0.13 - 0.16 mm thick</td><td>Ted Pella</td><td>260166</td><td>1 ounce</td></tr><tr><td>Superslip Cover Glasses, 24 mm x 60 mm x 0.13 - 0.16 mm thick</td><td>Ted Pella</td><td>260168</td><td>1 ounce</td></tr><tr><td>Tempered Glass Cutting Board</td><td>Hacaroa (Amazon)</td><td>B09XMXBT5S</td><td>4 pc</td></tr></tbody></table>

improved methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging

在玉米（Zea mays）和其他草（禾本科）中，叶原基深深地包裹在叶旋内并滚动，因此难以研究早期叶片发育。在这里，我们描述了制备用于荧光和共聚焦成像的玉米叶原基的横向切片和展开的整个安装的方法。第一种方法使用剥线钳去除老叶的上部，露出叶原基的尖端并允许其测量以进行更准确的横向截面采样。第二种方法使用透明的双面纳米胶带展开并安装全叶原基进行成像。我们展示了这两种方法在玉米中可视化和分析荧光蛋白报告基因方面的效用。这些方法为玉米叶原基独特形态带来的挑战提供了解决方案，并将有助于可视化和量化玉米和其他草种的叶片解剖学和发育性状。

玉米叶原基横断面分析 我们使用协议第 2 节来量化静脉数量，并使用 FPs 表征玉米叶原基横向切片中的激素反应模式（图 4）24。为了评估植物激素生长素在叶片生长和叶脉形成中的作用，我们量化了生长素缺乏型玉米突变体消失流苏的叶原叶脉数2 54。在早期塑体中，发育中的脉在玉米叶原基21,22的中间细胞层中表现出明显的细胞化。然而，使用传统的组织学技术22识别和计数静脉可能是劳动密集型和耗时的。因此，为了量化静脉，我们利用玉米生长素外排蛋白标记物p Zm PIN1a：：ZmPIN1a：YFP41（以下简称PIN1a-YFP），它标记发育中的静脉和丙氨酸链（PCS;图 4A）。使用协议部分2，我们能够通过在切片前测量原基来标准化横向切片采样（图4B，C）。我们发现vt2比正常具有更宽的原基和更多的脉络的趋势（图4D，E）24，这与完全展开的叶子55的数据一致，表明vt2的缺陷始于叶片发育的早期。使用协议第2部分，我们还能够系统地检查叶原基中激素反应FP报告基因的表达模式（参见图4F-I中的图像示例）。通过标准化的横截面采样方案，我们绘制了生长素、细胞分裂素（CK）和赤霉酸（GA）响应在不同质体和叶原基区域中的分布，并发现了我们假设对叶片生长和叶脉形成有影响的新响应模式24。因此，这些具有代表性的结果证明了协议第2节在玉米叶原初横截面分析中的实用性。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65239/65239fig04.jpg"> 图4：玉米叶原基横截面分析的代表性结果。 （A-E）用生长素外排蛋白标记PIN1a-YFP量化正常和消失流苏2叶片原基中的脉数和原基宽度。（A）在发育中的静脉和丙链中表达PIN1a-YFP的P5至P7横截面的代表性荧光图像。使用FITC滤光片（495-519nm激发）用落射荧光显微镜对横截面进行成像。静脉数量和原基宽度分别使用FIJI/ImageJ中的多点和手绘线工具进行量化。（B）用剥线钳去除上部叶涡的玉米幼苗，露出第四片叶子（L4）的尖端。括号跨越投影的原基长度，虚线表示中间长度。（C）从P5到P7的不同原基形状的示意图，说明了中长处的脉数和原基宽度（水平虚线）如何根据原基的发育阶段而变化。（D，E）测量正常和vt2的L4中长部分的原基宽度（D）和静脉数（E）。趋势线表示测量值的 10 mm 滚动平均值±平均值的标准误差 （SEM）。（F-I）表达PIN1a-YFP，生长素反应报告基因，DR5-RFP，细胞分裂素反应报告基因，TCS-番茄，赤霉酸响应标记物GAR2-YFP和mDII-Venus（生长素信号输入报告基因DII-Venus的突变版本）组合的叶原基横截面的代表性共聚焦图像。FP通道叠加在每个图像中的明场通道上。比例尺 = 200 μm （A）;10毫米（B）;100微米（F-I）。该图经Robil和McSteen24许可修改和复制。缩写：DAG = 发芽后几天;P = 塑体;VT2 = 消失的流苏2;PCS = 丙链;YFP = 黄色荧光蛋白;FITC = 异硫氰酸荧光素;RFP = 红色荧光蛋白;PIN1a-YFP = p Zm PIN1a：：ZmPIN1a：YFP;DR5-RFP = DR5rev：：mRFPer;TCS-Tomato = TCSv2：：NLS-tdTomato;GAR2-YFP = p Zm GAR2：：ZmGAR2：YFP;mDII-Venus = pZmUbi：mDII：YFP-NLS.<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65239/65239fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
玉米叶原基的全安装分析 我们按照协议第3节可视化和分析玉米叶原基全叶坐骑中的FP表达（图5）。通过使用PIN1a-YFP可视化静脉模式，我们发现在早期质体中，静脉的形成发生在整个原基中，但这一过程在发育后期的近端区域受到限制（图5A）24。作为横截面分析的补充，全叶安装分析揭示了静脉形成过程中激素反应的组织和阶段特异性模式24。一个例子是CK响应报告基因TCSv2：：NLS-tdTomato37（TCS-Tomato）相对于PIN1a-YFP表达的表达模式（图5B）24。通过遵循协议第3节，我们能够对叶原基中的FP表达进行定性和定量分析（图5D-F;未发表的数据）。我们检查了生长素反应报告基因DR5rev：：mRFPer41（DR5-RFP）和GA响应标记物p Zm GAR2：：ZmGAR2：YFP 39（GAR2-YFP）在vt2和矮植物3（d3）的单双突变体（GAR2-YFP）的全叶安装中的表达模式，矮植物3（d3）是GA缺陷突变体56（图5D）。我们还使用p Zm Cyclin-D2B：：ZmCyclin-D2B：YFP42（Cyclin-D2B-YFP）比较了正常和vt2叶原基之间的细胞增殖相对水平，这是细胞周期57中G1 / S转变的标志物（图5E，F）。虽然正常和vt2之间没有显着差异，但Cyclin-D2B-YFP表达与早期质体31的已知细胞增殖曲线一致。我们得出结论，协议第3节是分析玉米叶原基整体坐骑的有效方法，由于其滚动形态而难以成像。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65239/65239fig05.jpg"> 图5：玉米叶原基全安装分析的代表性结果。 （A） 7 个 DAG 玉米幼苗的叶原基的代表性荧光图像，显示发育中的脉络和丙层链，由生长素外排蛋白标记物 PIN1a-YFP 标记。P3-P6原基从基部切除，展开，平整，轴面朝上。插图显示了 P3 和 P4 的特写镜头。在P5和P6中，虚线划定了增殖区的远端，大多数前部链仍在发育和延伸。（乙，丙）代表性共聚焦图像显示PIN1a-YFP和细胞分裂素反应报告基因TCS-Tomato在P5原基近端边缘区域的表达。（D）P4原基的代表性荧光图像，显示生长素反应报告基因DR5-RFP和赤霉酸反应标志物GAR2-YFP在消失的流苏2和矮植株3的正常和单双突变体中的表达。（E）P3和/或P4的代表性共聚焦图像，显示细胞周期蛋白-D2B-YFP的表达，细胞周期中G1 / S跃迁的报告基因。（F） 正常和 vt2 的 P3/P4 叶原基中细胞增殖的相对量，通过使用 ImageJ/FIJI 测量原基面积上细胞周期蛋白-D2B-YFP 信号的积分密度来量化。条形表示平均值测量值±平均值的标准误差。比例尺 = 500 μm （A、D、E）;200微米（B）;100微米（C）。图4A-C经Robil和McSteen24许可修改和复制，而图4D-F是作者未发表的数据。缩写：DAG = 发芽后几天;P = 塑体;VT2 = 消失的流苏2; d3 = 矮植物3; YFP = 黄色荧光蛋白;RFP = 红色荧光蛋白;PIN1a-YFP = p Zm PIN1a：：ZmPIN1a：YFP;TCS-Tomato = TCSv2：：NLS-tdTomato;DR5-RFP = DR5rev：：mRFPer;GAR2-YFP = p Zm GAR2：：ZmGAR2：YFP;Cyclin-D2B-YFP = p Zm Cyclin-D2B：：ZmCyclin-D2B：YFP;ns = 无显著差异;au = 任意单位。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65239/65239fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>
补充文件1：玉米幼苗叶片发育分期实例。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65239/65239_S_File_1_Leaf_staging_maize_seedling_Fall2019%20(1).xlsx" target="_blank">请点击此处下载此文件。</a>
补充图S1：协议中使用的植物样品和材料。 （一，二）玉米幼苗7-8 DAG，使用剥线钳去除上部叶涡。（B） 插图显示了使用曝光的 P6 原基拍摄的特写。（C-G）去除上部螺旋进行横截面分析（C，D）和完全去除周围叶子以进行整体安装分析（E-G）的玉米幼苗芽。（H）一卷聚氨酯凝胶透明双面纳米胶带。（一，一）P6叶原基（I）和P8叶（J）的近端区域展开并用纳米胶带安装在载玻片上。（K）安装在落射荧光显微镜载物台上的带有展开的叶原基的载玻片。缩写：DAG = 发芽后的几天。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/65239/S_FIGURE_1.pdf" target="_blank">请点击此处下载此文件。</a>

Watch this Scientific Journal Video about Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging at JoVE.com

作者聚焦：用于荧光和共聚焦成像的玉米叶片原基横向切片和展开整片的改进方法  

玉米叶原基被深深包裹和卷起，使其难以研究。在这里，我们提出了制备玉米叶原基的横向切片和展开的整个安装以进行荧光和共聚焦成像的方法。

用于荧光和共聚焦成像的玉米叶原基横向切片和展开整片的制备方法的改进方法

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study ...

像玉米这样的草在芽内生长着原叶，因此难以研究。我们通过改进的方案来解决这个问题，用于制备用于荧光和共聚焦成像的玉米叶横向切片和整个支架。第一个方案使用剥线钳切割较老的叶子，允许在切片之前测量原基。第二种方案使用透明的双面纳米胶带展开整个叶原基。这些协议提高了横向切片的准确性，并能够展开叶原基，由于其卷起的圆锥形形态，这很难实现。这些方法将有助于可视化和量化玉米和其他草的叶片解剖学和发育性状。基于这些方法，我们计划开发活细胞成像策略来解决草叶发育中的问题。

improved-methods-for-preparing-transverse-sections-unrolled-whole

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

2.7K 次观看.  Ateneo de Manila University. 像玉米这样的草在芽内生长着原叶，因此难以研究。我们通过改进的方案来解决这个问题，用于制备用于荧光和共聚焦成像的玉米叶横向切片和整个支架。第一个方案使用剥线钳切割较老的叶子，允许在切片之前测量原基。第二种方案使用透明的双面纳米胶带展开整个叶原基。这些协议提高了横向切片的准确性，并能够展开叶原基，由于其卷起的圆锥形形态，这很难实?...

视频: 作者聚焦：用于荧光和共聚焦成像的玉米叶片原基横向切片和展开整片的改进方法  

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

激光显微切割RNA测序实验设计:从玉米叶片发展的经验分析

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

科研

生物化学

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

发育生物学

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

用于在大容量中查找特定细胞目标的串行剖面的多模式分层成像

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

不同植物种的射击顶端共聚焦活图

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

生物学

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 ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

用光片荧光显微镜观察运动轴突导航和小鼠胚轴突树枝状的定量化

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

神经科学

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...