We thank Dr. M. Rosenberg for her advice and help with anatomical sectioning. We also thank D. Eisler for his technical assistance. This research was supported by grants from FP7-PEOPLE-IRG-2008, Binational Agriculture research and Development (BARD; IS-4246-09), and Israel Science Foundation (ISF; 592/13).

1.植物生长<ol><li>表面消毒的种子。 <ol><li>通过湿法或干法消毒的种子（最多30毫克）。作为一个例子，为干热灭菌法，在一个封闭的干燥器在50毫升漂白加入1毫升冰盐酸37％，在玻璃烧杯中，至少4小时和最多过夜。执行在化学罩此步骤。 </li></ol></li><li>传播在0.5倍的Murashige＆Skoog培养基（MS）培养基，0.8％植物琼脂平板上无菌种子。包装用封口膜或多孔带板和铝箔覆盖。 </li><li>存储平板在4℃下1 - 4天推匀发芽。 </li><li>传送板，以适于生长的拟南芥幼苗的生长室。将平板垂直地维持在琼脂培养基的顶端的根生长，并防止渗透。 </li><li>转移苗发芽（DAG）后固定液7天。 </li></ol> 2.固定<ol><li>准备50毫升理查德森搜索解决方案通过在双蒸水1％硼砂（缓冲剂），1％亚甲基蓝和1％天青（组织学染料）等体积的结合Ñ。通过使用前驱动0.22微米的PVDF过滤单元注射器过滤。 </li><li>准备50毫升固定液:1.25％戊二醛在0.05M二甲胂酸钠（缓冲剂）。 注意:这些物质是有毒的，并且必须在化学通风橱中处理。 </li><li>加的理查森溶液100微升（见2.1）的固定溶液（见2.2），得到最终的固定溶液。 </li><li>标记与相应的样品名称的6孔板的每个孔中。 </li><li>放置2 - 3毫升最终固定溶液（见2.3）插入的孔中，从而使幼苗，一旦转移，将在液体中完全覆盖。 </li><li>小心转移，使用镊子，最多10苗每个孔中。使用密封封口膜板。储存在4℃下，在黑暗中，用于至少一个晚上，最多一周。 </li></ol> 3.脱水<ol> &lt;LI&gt;脱水苗。转移他们的新6孔平板的孔之间，含有增加的乙醇浓度，如下所示:10％乙醇15分钟; 30％乙醇15分钟; 50％乙醇15分钟; 70％乙醇15分钟; 85％乙醇15分钟; 95％乙醇15分钟;在4℃，过夜，以最低95％的乙醇。通过他们的子叶，最好用塑料镊子小心地抓住他们处理苗。 </li></ol> 4.渗透<ol><li>准备渗透网上平台。将1袋ヒ套件激活的内容与一个小玻璃瓶50毫升基本树脂液套件。用磁铁20分钟搅拌。浸润培养基可保持在4℃几周。 </li><li>标记与相应的样品名称的新6孔平板的每个孔中，并用2填充 - 3毫升浸润培养基。 </li><li>从95％的乙醇溶液，以使用镊子渗入介质传送苗。确保苗福由介质LLY覆盖。 </li><li>储存在4℃下至少4天，以确保最大的渗透到植物组织。 </li></ol> 5.块准备<ol><li>将小标签，铅笔标明样品名称，嵌入模具的每一个孔里。 </li><li>准备加入1 ml固化剂到15ml渗透介质（见4.1）嵌入介质。不要试图准备一个容量小于15毫升，以避免聚合的问题。 </li><li>填充一半各孔在用200μl包埋介质的模具和与切断模具形状的塔顶薄膜片覆盖，但以一个大小为1毫米的每个侧大。在室温下孵育至少2小时，以允许聚合。不要超过5小时。 </li><li>制备额外批次包埋培养基（见5.2），并保持在冰上，以避免聚合而根尖被布置在模具中。 </li><li>用手术刀切下解剖范围内的每个根尖和非常小心我的位置T:垂直的横截面或横向纵向部分，约2 - 从模具边缘3毫米。与封闭液，还包括与架空电影完全填满模具。注意，一旦它被聚合的封闭溶液将略有收缩。 </li><li>保持在室温下2小时，在最低限度。 </li><li>干块，放置在干燥硅胶的盒子，并在室温离开过夜，在最低限度。 </li></ol> 6切片<ol><li>从准备玻璃棒玻璃刀用刀子机。使用玻璃刀用于组织切片。切玻璃棒成方形，然后切正方形对角线产生两把刀，使用金刚石得分利器。 </li><li>节块分成2 - 3微米宽度切片，用切片机。在载玻片上地方切片，在蒸馏水液滴的顶部，并放置为低热量的热块上的滑动件（50 - 60℃），直到水蒸发。 </li></ol><ol><li>理查德森稀释液（见2.1），以原始溶液的0.2％及使用方法1毫升它覆盖片。放置5秒钟的加热块上，然后用蒸馏水冲洗。 </li><li>干热板上的幻灯片。观察下解剖范围和标记的幻灯片用标记在后面，以指示感兴趣的切片的位置，以便于在显微镜下它们的后面的识别。 </li><li>将100μl封固剂和覆盖的盖玻片。 </li></ol> 8.图像采集<ol><li>放置装有装有适于获得延迟信息和偏振器/干扰光滤光器（ 图1的AD）Polscope系统在光学显微镜下的根部分中的覆盖滑动。 </li><li>选择放大，允许整个样本的清晰的可视化（ 例如 ，40X在这项研究中）和聚焦显微镜E在包含块段，但没有根部分的空场。试着找一个干净的区域，没有瑕疵。使用此区域设置背景。 </li><li>设置成像系统和软件参数:设定范围到17纳米。应用"自动曝光"，并从空场获得背景图像（见图8.2）。一个背景图像被每个实验使用。 </li></ol> 9.纤维素微纤丝取向Polscope分析<ol><li>分析纵向部分。 <ol><li>将含在显微镜下纵段的幻灯片，并获得它的延迟的图像。专注于感兴趣的根部显微镜。提供一个新的文件名。 </li><li>通过"实时Abrio"窗口捕捉abrio图像（迟缓的信息图像）。一定要捕获包括细胞壁，这可以通过沿着电池的周边运行染色纤维素被识别， 如图2C的剖面。</li></ol></li><li>图像分析。 <ol><li>在图像堆栈相关的图像上双击。在"显示设置"选项卡中选择"方向伪彩"。 </li><li>为了估计纤维素微纤丝角，匹配细胞壁的色轮中相应的树荫的。 注意:在图2C的彩色轮反射光沿其指示沿着微纤维的长轴方向上的微纤维躺着的慢轴。纤维素微纤丝的角度是由颜色和单元的长轴指示的角度。例如，在嵌入的伸长细胞显示青色，约90°倾斜的细胞的长轴。 </li><li>为了量化在细胞壁的平均微纤丝角，从软件的工具栏中用"延迟和方位角印记"的工具。在选择工具，点一下就所获得的图像中感兴趣的区域。 注:此WIL升结果在度角（方位角）（ 图2C）的显示。 </li><li>在另一种开放获取偏振光图像分析软件，午餐"波尔分析仪"，从插件菜单插件。在新打开的窗口中，选择"双折射"选项卡，打开并保存背景文件和图像文件。 </li><li>在图像窗口中，选择"规格"。并调整"ROI像素尺寸&#39;（使用5个像素为这项研究）。中的"规格"。窗口中，选择"在结果列表中显示的值"。 </li><li>使用多边形工具，以纪念在图像窗口中感兴趣的区域。选择"方向线"，以获得纤维素微纤维的取向。 注意:这将导致在度角（方位角）的显示。 注意:用两种方法之一计算出的平均角度应然后标准化为沿着滑动根部的定位。为了校正偏差，从沿着滑动整根部的水平定位，量化细胞壁侧翼沿其长轴的小区的平均微纤丝角（即，垂直于该部分和沿着根生长轴， 图2C）。此角度为180°时根部准确沿着滑动地定位。为计算微纤丝角，从180°减去侧翼细胞壁的平均测微纤丝角。所得差添加到细胞壁的平均测微纤丝角，关于滑动平整。 </li></ol></li></ol>结晶纤维素积累10 Polscope分析<ol><li>使用横向（横）截面。注意，结晶纤维素积累只能小区之间进行比较时其纤维素微以类似的角度对准的（如在9确定）。 </li><li>在图像堆栈相关的图像上双击。选择"P"中的"显示设置"选项卡seudo颜色。 </li><li>为了测量延迟。放大-在图像上。选择"地区"，并标记感兴趣的所有区域。移动到"三围"选项卡和"复制到剪贴板"的所有值。 <ol><li>将数据传输到Excel和提取所需的信息（平均延迟的区域）。正常化的数据。 注意:为了考虑切片之间的厚度的差异，表示相对于在滑动一个特定细胞壁边缘的值。正常化，除以不同细胞壁的同一图像中的平均延迟对应于感兴趣的细胞壁的平均延迟。例如，外表皮细胞壁被归一化到在我们的研究中的内皮层细胞壁。 </li></ol></li><li>重复测量每根的至少3个部分，用最少的每个样品3根。 </li></ol>

<ol>
	<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+the+plant+cell+wall.">Growth of the plant cell wall.</a> Nat Rev Mol Cell Biol. 6, 850-861 (2005).</li><li>Wolf, S., Hematy, K., Hofte, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+control+and+cell+wall+signaling+in+plants.">Growth control and cell wall signaling in plants.</a> Annu Rev Plant Biol. 63, 381-407 (2012).</li><li>Mazeau, K., Heux, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Dynamics+Simulations+of+Bulk+Native+Crystalline+and+Amorphous+Structures+of+Cellulose.">Molecular Dynamics Simulations of Bulk Native Crystalline and Amorphous Structures of Cellulose.</a> J. Phys. Chem. B. 107, 2394-2403 (2003).</li><li>Zhao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+cellulose+fiber+structure+by+SEM,+XRD,+NMR+and+acid+hydrolysis.">Studying cellulose fiber structure by SEM, XRD, NMR and acid hydrolysis.</a> Carbohydr Polym. 68, 235-241 (2007).</li><li>Fujita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+microtubules+optimize+cell-wall+crystallinity+to+drive+unidirectional+growth+in+Arabidopsis.">Cortical microtubules optimize cell-wall crystallinity to drive unidirectional growth in Arabidopsis.</a> Plant J. 66, 915-928 (2011).</li><li>Peng, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractional+separation+and+structural+features+of+hemicelluloses+from+sweet+sorghum+leaves.">Fractional separation and structural features of hemicelluloses from sweet sorghum leaves.</a> Bioresources. 7, (2012).</li><li>Xu, S. L., Rahman, A., Baskin, T. I., Kieber, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+leucine-rich+repeat+receptor+kinases+mediate+signaling,+linking+cell+wall+biosynthesis+and+ACC+synthase+in+Arabidopsis.">Two leucine-rich repeat receptor kinases mediate signaling, linking cell wall biosynthesis and ACC synthase in Arabidopsis.</a> Plant Cell. 20, 3065-3079 (2008).</li><li>Anderson, C. T., Carroll, A., Akhmetova, L., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Imaging+of+Cellulose+Reorientation+during+Cell+Wall+Expansion+in+Arabidopsis+Roots.">Real-Time Imaging of Cellulose Reorientation during Cell Wall Expansion in Arabidopsis Roots.</a> Plant Physiol. 152, 787-796 (2010).</li><li>Baskin, T. I., Beemster, G. T., Judy-March, J. E., Marga, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorganization+of+cortical+microtubules+stimulates+tangential+expansion+and+reduces+the+uniformity+of+cellulose+microfibril+alignment+among+cells+in+the+root+of+Arabidopsis.">Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis.</a> Plant Physiol. 135, 2279-2290 (2004).</li><li>Iyer, K. R. K., Neelakantan, P., Radhakrishnan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Birefringence+of+native+cellulosic+fibers.+I.+Untreated+cotton+and+ramie.">Birefringence of native cellulosic fibers. I. Untreated cotton and ramie.</a> J. Polym. Sci. A-2: Polymer Physics. 6, 1747-1758 (1968).</li><li>Abraham, Y., Elbaum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+microfibril+angle+in+secondary+cell+walls+at+subcellular+resolution+by+means+of+polarized+light+microscopy.">Quantification of microfibril angle in secondary cell walls at subcellular resolution by means of polarized light microscopy.</a> New Phytol. 197, 1012-1019 (2013).</li><li>Petricka, J. J., Winter, C. M., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Arabidopsis+root+development.">Control of Arabidopsis root development.</a> Annu Rev Plant Biol. 63, 563-590 (2012).</li><li>Vanstraelen, M., Benkova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+Interactions+in+the+Regulation+of+Plant+Development.">Hormonal Interactions in the Regulation of Plant Development.</a> Annu Rev Cell Dev Biol. , (2012).</li><li>Singh, A. P., Savaldi-Goldstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+control:+brassinosteroid+activity+gets+context.">Growth control: brassinosteroid activity gets context.</a> J Exp Bot. 66, 1123-1132 (2015).</li><li>Fridman, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+growth+is+modulated+by+differential+hormonal+sensitivity+in+neighboring+cells.">Root growth is modulated by differential hormonal sensitivity in neighboring cells.</a> Genes Dev. 28, 912-920 (2014).</li><li>Vragovic, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translatome+analyses+capture+of+opposing+tissue-specific+brassinosteroid+signals+orchestrating+root+meristem+differentiation.">Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation.</a> Proc Natl Acad Sci U S A. 112, 923-928 (2015).</li><li>Hacham, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brassinosteroid+perception+in+the+epidermis+controls+root+meristem+size.">Brassinosteroid perception in the epidermis controls root meristem size.</a> Development. 138, 839-848 (2011).</li><li>Gu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+cellulose+synthase-associated+protein+required+for+cellulose+biosynthesis.">Identification of a cellulose synthase-associated protein required for cellulose biosynthesis.</a> Proc Natl Acad Sci U S A. 107, 12866-12871 (2010).</li><li>Fendrych, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+controlled+by+ANAC033/SOMBRERO+determines+root+cap+organ+size+in+Arabidopsis.">Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis.</a> Curr Biol. 24, 931-940 (2014).</li></ol>

The authors have no conflicts of interest.

在这里，我们提出了一个方法，测定不同组织组成拟南芥根部结晶纤维素的积累，同时保持解剖信息。因此，它提供了对在一个蜂窝分辨率理解在植物生长过程的附加步骤。这种方法也可以应用于其他植物物种和器官的研究。 数点必须应用该方法时，必须考虑。在给定的根段第一，结晶纤维素积累可以组织之间进行比较，从不同基因型的部分之间和处理间，如果他们?...

植物细胞壁是一个动态的结构。生长的细胞是由初级细胞壁，组织其允许细胞扩大包围。细胞停止生长存款更刚性的次生壁，增强植物的机械支撑。两个单元壁是由嵌入在不同的结构（例如 ，半纤维素和果胶），该跨不同发育阶段变化和组织1,2的多糖的基质纤维素微纤维。纤维素被合成为被严格对齐，以形成晶体结构的微纤丝（1,4）-β-D-葡聚糖的链。无定形纤维素是指其中葡聚糖链较少有序的区域。该结晶和无定形结构域之间的比率是认为影响细胞壁的机械特性的一个参数，通过提供机械强度和粘弹性特性，分别为3。几种方法已经开发检测和定量的两种形式的纤维素排列的，其中的X射线衍射和交叉极化/魔角旋转固态核磁共振4。 X射线衍射可以被用来确定样品5中结晶与非晶纤维素域的比例。另一种方法使用的细胞壁含量分馏成酸不溶性和酸可溶材料，分别结晶和无定形纤维素或其它聚合物之间进行区分。在这种方法中，标记的葡萄糖（[14 C]葡萄糖）的掺入来量化纤维素6,7。这些方法需要大量的用于整个器官的分析，在最好的植物材料，因此，是在细胞壁结构组织特异性变异充分敏感。在细胞分辨率纤维素微的可视化可以在用荧光染料8,9-结合实时成像研究来实现，它可以识别在改变纤维素微纤维的取向。然而，这些染料不用于定量，它们不是特定于结晶纤维素和可以与细胞壁8的正常结构干扰。 Polscope是成像技术，其依赖于结晶纤维素分裂光束和延缓的光10的一部分的能力。光相位差是最强的为垂直骗光传播方向的微纤维。对于具有类似取向的微纤维，结晶度越高，越大的光延迟11。因此，polscope是用来研究两个相对水平和纤维素微纤维的取向。 根部呈现线性的增长，在此期间，细胞产生于干细胞小生境，在根的前端，经过一系列的细胞分裂，它们迅速扩大12之前。包括根细胞在单向（各向异性）展开方式，作为决定由小分子信令激素影响细胞壁13的属性。不同的反应激素，在时间和空间，提供了确保平衡的器官生长14的一种手段。因此，细胞壁结构的高分辨率分析可以提供更好的理解，以整个器官生长的细胞类型特异性应答之间的连接所需的重要信息。在这里，我们报告polscope，研究结晶纤维素的组织特异性的积累拟南芥根系在高品质的解剖观察部分的执行情况。这种方法最近发现响应于激素活性15的空间扰动的结晶纤维素的细胞类型特异性蓄积。

<table>
	<tbody>
		<tr>
			<td>Murashige &#38; Skoog (MS)</td>
			<td>Duchefa</td>
			<td>M0221</td>
			<td></td>
		</tr>
		<tr>
			<td>Borax</td>
			<td>LOBA CHEMIE&#160;</td>
			<td>6038</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma</td>
			<td>M-9140</td>
			<td></td>
		</tr>
		<tr>
			<td>Azure</td>
			<td>Sigma</td>
			<td>861065</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Driven 0.22mm PVDF Filter&#160;</td>
			<td>MILLEX-GV</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>EMS</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate</td>
			<td>Sigma</td>
			<td>C-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica kit historesin&#160;</td>
			<td>Leica</td>
			<td>7022 18 500</td>
			<td></td>
		</tr>
		<tr>
			<td>embedding molds</td>
			<td>Agar Scientific</td>
			<td>AGG3530</td>
			<td></td>
		</tr>
		<tr>
			<td>film 100&#181; P.P.C.&#160;</td>
			<td>www.Jolybar.com</td>
			<td></td>
			<td>overhead film</td>
		</tr>
		<tr>
			<td>Knivemaker</td>
			<td>LKB</td>
			<td>7800</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultratome III</td>
			<td>LKB</td>
			<td>8800</td>
			<td>Ultratome</td>
		</tr>
		<tr>
			<td>Shandon immumount&#160;</td>
			<td>Thermo</td>
			<td>9990402</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>light microscope</td>
			<td>Nikon</td>
			<td>Eclipse 80i</td>
			<td></td>
		</tr>
		<tr>
			<td>Abrio imaging system</td>
			<td>CRI</td>
			<td>Abrio imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>Abrio V2.2 software</td>
			<td>CRI</td>
			<td>Abrio V2.2 software</td>
			<td></td>
		</tr>
		<tr>
			<td>Open access polarized light image analysis software &#160;</td>
			<td>OPS</td>
			<td>OpenPolScope</td>
			<td><a href="http://www.openpolscope.org/">http://www.openpolscope.org/</a></td>
		</tr>
	</tbody>
</table>

high resolution quantification of crystalline cellulose accumulation in arabidopsis roots to monitor tissue-specific cell wall modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

我们研究的细胞类型特异性反应油菜素内酯（BRS）的影响，使用拟南芥根作为模型器官15-17。当BR受体BRI1是有针对性的，在BRI1突变体的背景下，对被称为非毛细胞表皮细胞的一个子集（ 图2A，B）中 ，它抑制相邻小区的单向电池膨胀，并完全根生长15。进行揭示这背后的抑制机制Polscope分析。从野生型和从非毛细胞表达BRI1线得到的...

Watch this Scientific Journal Video about High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications at JoVE.com

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

high-resolution-quantification-crystalline-cellulose-accumulation

Research

JoVE Journal

Biology

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

8.1K 次观看.  Technion-Israel Institute of Technology. Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

视频: High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

科研

生物学

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Here we describe a procedure allowing a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple phosphospecific antibodies.

高分辨率法监测IRF3的磷酸化依赖性激活

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

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 ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

结晶纤维素积累高分辨率的量化

摘要

探索更多视频

此视频中的章节

从海兔的神经细胞培养的高分辨率成像生长锥

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

糖基谱研究植物细胞壁聚合物，用微阵列

从长期来看，高解析度激光共聚焦时间流逝成像

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

组织化学染色

高分辨率法监测IRF3的磷酸化依赖性激活

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

高分辨率呼吸测量，以评估线粒体功能的通透和完整细胞

与VirusMapper超分辨率显微镜开源单粒子分析

结晶纤维素积累高分辨率的量化

摘要

探索更多视频

此视频中的章节

从海兔的神经细胞培养的高分辨率成像生长锥

双链DNA定量，使用日立F - 7000荧光分光光度计和PicoGreen染料

糖基谱研究植物细胞壁聚合物，用微阵列

从长期来看，高解析度激光共聚焦时间流逝成像

使用平板扫描仪，以收集拟南芥根向地反应的高分辨率时间失效图片

组织化学染色

高分辨率法监测IRF3的磷酸化依赖性激活

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

高分辨率呼​​吸测量，以评估线粒体功能的通透和完整细胞

与VirusMapper超分辨率显微镜开源单粒子分析

高分辨率呼吸测量，以评估线粒体功能的通透和完整细胞