作者希望感谢小号Castorani，AJ旅游Eustis，甘贝塔GA，CM Manuck，Z Nasafi，和A泽丹。这项工作是由美国农业部农业研究服务局的研究现状信息系统的资金（研究项目。5306-21220-004-00先进的光源是支持的，科学办公室主任，办公室基础美国能源部能源科学，合同号DE-AC02-05CH11231）;和NIFA特色作物研究计划，拨款，以AJM。

协议详细说明，下面是专门为工作的先进光源8.3.2光束线。的适应可能需要工作在其他的同步加速器设施。使用这些设施需要适当的安全和辐射培训。 1。样品制备活的植物<ol><li>在〜10厘米直径盆种植植物，并确保主阀杆（或植物的部分，以进行扫描）为中心尽可能和取向在锅中垂直。的物理尺寸的的HRCT仪器双雄在先进光源的限制，活的植物〜1米的高度。因此，成像是最好的活植物苗/小花盆中种植的树苗。根据实验，不同土壤类型，可用于控制土壤水分含量（ 例如在干旱实验），可以灵活的芽植物（ 如葡萄树）长芽仔细图cked到丙烯酸类管下面描述（见图1和图2）。 </li><li>现场盆栽安装在一个特制的硬质铝壶持有人。顶板的高度可以调节，以适应多种锅高度。板的顶部被设计以配合的顶部和植物的土壤表面，从两个部分的板的中心突出。锅支架的目的是，以确保牢固地保持在植物的茎的地方，以尽量减少振动或样本运动。在扫描过程中最小化样本运动是必不可少的。 </li><li>一旦安装在支架中，测量茎水势或叶蒸腾使用Scholander风格的压力室或剪辑叶气孔计，分别确定植物的生理状态的扫描之前。 </li><li>接管该工厂和铝厂支架顶部的薄壁丙烯酸圆筒放置并固定它代替粘土腻子稳定样品（ 图2）。上部的枝叶的任何振动或运动将被发送的下阀杆，并导致植物组织的扫描区域内的移动，最终导致图像失真。缸是用来包含植物的叶子，并防止从摩擦件等在笼的设备，在扫描期间的振动会导致植物叶片。应当使用附加的保鲜膜，擦手纸，和磁带，为进一步减少的植物部分（参见与图4中的示例运动有关的问题）的振动和运动。为了减少它的X-射线的吸收（在一个给定的曝光时间，可以降低图像质量的），包含汽缸应具有尽可能薄的壁，同时保持足够的刚性，以执行其功能。 </li><li>自定义锅支架附加空气轴承阶段和锁定（螺钉）之间的X-射线源和成像传感器和照相机设备入到位。 Positi在阀杆上尽可能和中心垂直磁夹头底座上，以确保样品停留在旋转期间的视场。 </li></ol> 2。样品制备新鲜，离体植物组织<ol><li>新鲜的植物材料，通常茎或叶柄，可以扫描后立即清除从活体植物。如果实验的意图是形象化的木质部的整个网络，水内的船只必须抽真空，并用空气取代。要做到这一点，装入样品在Scholander风格的压力室，约5分钟内，在低压力（&lt;0.05 MPa）的压缩空气或氮气通过样品推。物种撤离容器网络所需的时间将有所不同。如果其目的是评估的程度栓塞形成的新鲜植物组织，然后使用新鲜的刀片从植物和消费样本，使水切割下。 </li><li>用样品在一层封口膜公关在扫描期间的事件干燥。 </li><li>样品安装在钻头卡盘固定到空气轴承阶段被拧入的金属板。中心和定位垂直如上所述，为了确保样本的样本保持在视场。 </li></ol> 3。样品制备干伍迪组织<ol><li>为了获得最佳的组织样本的可视化和图像对比度，这是必要的，慢慢脱水整个木本组织样本。剪切样品约6厘米的长度。选择针对性的扫描区域中尽可能地直的样品，并有一个直径≤1厘米。 </li><li>木本组织样本放置到干燥烘箱中在低温下，慢慢地干燥，而不会造成任何开裂或分裂的组织样本。这种过程可能会有所不同，物种之间和组织。对于木质茎，在一个40℃的烘箱中12小时通常是足够的，以提供优异的对比度，而不引起显著茶NGES在阀杆的物理结构（参见图3中表现出的问题的，快速干燥）。 </li><li>在某些情况下，理想的是有一个受托标记内的样本，使得随后的解剖和用扫描电子显微镜的可视化可以被定向HRCT图像中的特定点。要做到这一点，贴上的金属或玻璃珠或线的外侧的干用石蜡膜。另一种方法是使用有机硅树脂（ 例如 RTV-141，蓝星有机硅，东不伦瑞克，新泽西州），可以被注入到一个单一的木质部导管（见实施例在布罗德森等人 2010）。一旦硬化，有机硅树脂与样品中是清晰可见的，很容易区别于其他的空气填充的船只。使用此标记精确定位的样本的特定区域。 </li><li>样品安装在钻头卡盘和中心如上所述。 </li></ol> 4。样品制备叶Tissue为二维（2D）影像学<ol><li>叶在近实时的可视化容器的内容，叶可进行扫描，以产生一个二维的X线片，牙科X射线。安装在两片薄的丙烯酸塑料叶片的边缘，并确保与剪辑。然后将其附加到一个保持器后的系统和位置旁边的成像系统和X-射线源的光学面包板上安装的样品。 </li></ol> 5。 8.3.2双雄扫描的样本。 <ol><li>决定的放大倍率，将工作最适合你的应用程序。 ALS光束线8.3.2有能力扫描镜头，放大倍率为2倍，5倍，10倍。这些结果在图像的像素大小为4.5，2.25，和0.9微米，分别。根据放大倍率，样品必须适当大小，作为视场随倍率的增加而减小。查看详情选择摄像机和镜头和所得到的图像在表1中的参数。</li></ol><table border="1"><tbody><tr><td></td><td colspan="2"> PCO.4000（4008x2672） </td><td colspan="2"> PCO.Edge（2560x2160）（OPTIQUE彼得） </td></tr><tr><td> 镜片 </td><td> 像素（微米） </td><td> 视场（毫米） </td><td> 像素（微米） </td><td> 视场（毫米） </td></tr><tr><td> 10倍</td><td> 0.9 </td><td> 3.6 </td><td> 0.65（0.69） </td><td> 1.7（1.7） </td></tr><tr><td> 5倍（4X） </td><td> 1.8 </td><td> 7.2 </td><td> 1.3（1.72） </td><td> 3.3（4.4） </td></tr><tr><td> 2倍</td><td> 4.5 </td><td> 18 </td><td> 3.25（3.44） </td><td> 8.3（8.8） </td></tr><tr><td> 1个</td><td> 9 </td><td> 36 </td><td> 6.5（ - ） </td><td> 16.6（ - ） </td></tr></tbody></table> 表1。提供在ALS 8.3.2的相机和镜头。 <ol start="2"><li>设置的X-射线能量至15千电子伏。这已被证明对于大多数植物的应用程序提供出色的图像对比（见布罗德森等，2010，2011，2012A，B）。曝光时间一般都依赖于样品的厚度和密度的（并因此使用的放大倍率）介于100和1000毫秒。更长的曝光时间（只要作为检测器像素是不饱和的），通常会导致更高的信号噪声比，但在成本增加的扫描时间。 </li><li>选择一个角度增量为您的应用程序是适当的。样品被旋转180°，在扫描过程中，和旋转期间拍摄的图像的数量可以有一个显着的大小的数据集，扫描间隔的长度，和最终的图像质量的影响，但一般有在质量上的收益递减。典型的扫描进行增量为0.25°，得到721幅图像扫描。降低incremen吨至0.125°的结果更好的图像可视化精致的细节，但产量1,440的图像和更大的数据集（一个典型的地区的利益，这意味着10-30 GB的数据与5 GB）。然而，信号噪声比往往改善，值得增加扫描时间和数据大小。可以是在扫描过程中不太可能变形/收缩的干茎进行较长的时间间隔（更小的角度增量）而不损害。当成像活的植物，其中的生物过程（ 如栓塞修复）在短的时间尺度上发生，选择较短的扫描时间间隔优选限制潜在的破坏性影响，x射线辐射，在此组织，虽然这来自于一个潜在的损失的图像质量。使用连续的层析成像期间，设置样品连续旋转图像被捕获，而可以实现更短的扫描时间间隔。 </li><li>每次扫描时，“亮场”和“暗场图像必须correcte四。明场图像是没有在光束中的样本。这些通常被扫描之前和之后的样品通过水平翻译样品收集。黑暗的字段收集关闭这个测得的信号的量的相机中显示没有X-射线的X-射线快门。 </li></ol> 6。数据处理<ol><li> “原始”2D。TIF图像，从采集计算机导出到文件服务器上，传输到数据处理计算机。如果计算机具有足够的RAM，可以将数据复制到一个所谓的“RAM驱动器”（部分出现的RAM在计算机上的硬盘驱动器）。通过这种方式，软件没有进入高速旋转的硬盘驱动器，这是比较慢相比，固态驱动器或闪存。此步骤显着地减少了所需的时间量来处理数据集。 </li><li>的图像必须被转换为一个百分比传输规模。光束线8.3.2有一个自定义的BAckground正常化插件，可以下载并使用免费提供的套装软件ImageJ的或斐济（ <a href="http://fiji.sc/" target="_blank">http://fiji.sc/</a> ）的。从图像中减去暗计数和标准化的样本图像的明亮的领域产生的图像显示传输％。归一化图像加载的八达通的软件包（ <a href="http://www.inct.be/en/software/octopus" target="_blank">http://www.inct.be/en/software/octopus</a> ），和“重建”的3D数据集的2D原料。TIF文件，使用指定的处理步骤（归一化图像，环形切除创造，正弦图，平行光束重建）。然后，这个过程产生了一系列的。TIF横向（横截面）图像组成的“像素”（体积象素单元），每个与一个x，y，z坐标和强度值表示的X-射线的线性吸收系数。 </li></ol> 7。可视化<ol><li> Visuali平仄堆栈的图像，在各种软件包中的一个。免费软件（ 例如 Drishti， <a href="http://anusf.anu.edu.au/Vizlab/drishti/index.shtml" target="_blank">http://anusf.anu.edu.au/Vizlab/drishti/index.shtml</a> ）可以用于可视化卷或个别或堆栈的图像（ 例如 ImageJ的或FIJI）。其他的套装软件，可用于三维可视化。我们的研究小组使用的Avizo软件包（ <a href="http://www.vsg3d.com/avizo/overview" target="_blank">http://www.vsg3d.com/avizo/overview</a> ），但其他如阿米拉（ <a href="http://www.amira.com/ " target="_blank">http://www.amira.com/</a> ）和VGStudioMax（ <a href="http://www.volumegraphics.com/" target="_blank">http://www。 volumegraphics.com /</a> ）也是常用的。 </li><li>数据集加载到系统内存中，并显示在虚拟的横向，纵向或径向切片方向的样品。由于3D属性的数据集，虚拟切片通过SAMPLe可以旋转，以配合该地区的利益，显着改善了传统的串行光镜（详细的例子，看电影1-3）在任何平面上。 </li><li>以可视化的样品，根据需要，在3D中，“段”的示例中使用各种半自动化的和手动的例程Avizo从周围组织中分离容器的流明或其他结构。分割是指定义感兴趣的对象之间的边界，因此，分离或分割成单独的区域。在三维可视化软件进行渲染卷。做到这一点的一种方法是直接体绘制，卷中的，其中每个点被假定为发射和吸收光的发射和吸收的量和颜色可以被定义在一个给定的方向上是使用“色彩映射表”，和所得到的投影显示在屏幕上。或者，代表分段边界线框或三维网格表面被构造成显示的3D模型吨他的兴趣结构。多边形元素组成的三维网格，和元素的总数将同时影响结构再现保真度和相关联的数据文件的大小（ 即更多的元素，导致更高的保真度，但较大的文件大小）。各种内的可视化软件提供的图像处理模块的控制容积再现输出，以及控制图像的亮度，对比度，透明度，降低噪音等 </li></ol> 8。量化<ol><li>一旦分割已经完成，它是可能的量化目标植物的结构或功能上的变化，体积，长度，宽度，存在或不存在，例如水，空气， 等 ，布罗德森等人 （2010）使用Avizo软件量化内部葡萄注墨船只的水滴的体积变化。植物进行扫描，在四到八个小时，每30分钟的时间LAPSE序列的容器补充。每个扫描重建，并加载到Avizo，单个液滴随着时间的推移，它们的体积增加。 </li></ol>

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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifuge+technique+consistently+overestimates+vulnerability+to+water-stress+induced+cavitation+in+grapevines+as+confirmed+with+high+resolution+computed+tomography.">Centrifuge technique consistently overestimates vulnerability to water-stress induced cavitation in grapevines as confirmed with high resolution computed tomography.</a> New Phytologist. , (2012).</li><li>Lee, K., Avondo, J., 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=Visualizing+plant+development+and+gene+expression+in+three+dimensions+using+optical+projection+tomography.">Visualizing plant development and gene expression in three dimensions using optical projection tomography.</a> Plant Cell. 18, 2145-2156 (2006).</li><li>Truernit, E., Bauby, 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=High-resolution+whole-mount+imaging+of+three-dimensional+tissue+organization+and+gene+expression+enables+the+study+of+phloem+development+and+structure+in+Arabidopsis.">High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis.</a> Plant Cell. 20, 1494-1503 (2008).</li><li>Jahnke, S., Menzel, M. I., 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=Combined+MRI-PET+dissects+dynamic+changes+in+plant+structures+and+functions.">Combined MRI-PET dissects dynamic changes in plant structures and functions.</a> The Plant Journal. 59, 634-644 (2009).</li><li>Iyer-Pascuzzi, A. S., Symonova, O., 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=Imaging+and+analysis+platform+for+automatic+phenotyping+and+trait+ranking+of+plant+root+systems.">Imaging and analysis platform for automatic phenotyping and trait ranking of plant root systems.</a> , (2010).</li></ol>

我们什么都没有透露。

Synchotron HRCT植物生物学家提供一个强大的，非破坏性的工具，探索植物脉管系统的内部运作，在令人难以置信的细节。这项技术已被用来为确定以前描述过的葡萄树木质部的解剖结构的差 ​​异改变木质部网络的连接，不同的葡萄品种（布罗德森等。2012B，出版中） -这种连接可以极大地改变血管病原体和栓子传播的能力破坏性整个木质部网络。第一个成功的扫描活的植物还透露判罚尺度?...

水被输送，从植物的根称为木质部 - 一个网络相互连接的管道，纤维，和生活，代谢活跃的细胞中的血管组织的叶子。必须保持植物的木质部运输功能，提供养分和水分的叶子进行光合作用，生长，并最终生存。水上运输木质部导管可以中断时，的木质部网络受到病原微生物。响应于此类感染植物经常产生凝胶，树胶和侵填体作为一种手段来隔离病原体传播（ 例如 McElrone等人，2008年，2010年）。干旱胁迫也可以限制水的木质部运输。由于植物失去水在长时间的干旱，紧张建立在木质部。在张力下的水是亚（ 即在一个特定阈值的紧张局势变得足够大，空化水木质部导管中的列）。气蚀发生后，可以形成一个气泡（栓塞），并填写在CONDUIT，有效地阻止水的运动（1989年泰里和Sperry），一个在深海潜水员减压病（ 即 “弯曲”）类似的现象。 尽管关于这一主题的历史和当代文学（泰里齐默尔曼，2002年，霍尔布鲁克等人 ，2005）证明了一个巨大的身体的最佳植物功能木质部水运输的重要性，但仍有木质部网络方面，仍然是难以捉摸的。最近的几个研究小组已开始利用高分辨率X射线计算机微断层扫描（HRCT），以评估更精细的细节木材解剖和血管组织（ 如梅奥等人，2010年，2008年曼纳斯等。2010年布罗德森等人2010年，2011，2012A，B，前田和三宅一生，2009年，草原等，2004）。 HRCT是一种非破坏性的用于可视化在内部的固态物体的功能和它们的3-D结构性质，得到的数字信息的技术。 HRCT不同于传统的医疗CAT扫描在其解决在一微米的大小为小的细节的能力，即使对于高密度的对象。同步HRCT技术的最新进展，提高了图像的分辨率和信号的信噪比足以使植物船只网络和intervessel的连接可以可视化，分配的三维坐标，并远销水工模型模拟。布罗德森等人 （2011）最近从木质部网络的数据，自动地提取在高得多的分辨率相比，有可能与传统的解剖方法（ 即串行切片用切片机与一个Fortran模型相结合，所产生的同步加速器HRCT三维重建先进的这种技术和光镜， 如图像采集与齐默尔曼1971年）。这项工作也被用来优化液压木质部系统模型和确定的独特的特征的交通（ 即反向流在一些已经ssels期间的峰值蒸腾）（李等人 ，在审查）。 同步HRCT现在可以使用可视化的木质部的功能，气蚀的敏感性，以及一个植物修复能力，栓塞导管。未能重新建立栓塞管道中流动，降低了液压能力，限制光合作用，导致植物死亡，在极端的情况下（麦克道威尔等人，2008）。调水通过连接相邻的功能管道的坑，周围堵塞和不断增长的新的木质部，以取代失去的液压能力，的植物可以应付栓子。有些植物具有的能力，修复断裂的水柱，但几十年来，紧张木质部这个过程中的细节仍不清楚。布罗德森等人 （2010年）的可视化和定量加气过程中现场葡萄HRCT。成功的容器加气是依赖于活细胞周围的水涌入的XYLEM管道，随着时间的推移，其中个别的水滴扩大，充满血管，被迫解散的残存气体。不同的植物修复受损的木质部和控制机制这些维修的能力，目前正在调查中。 ALS的设施8.3.2光束线的说明在劳伦斯伯克利国家实验室（伯克利分校CA USA）的先进光源硬X射线微断层扫描光束线8.3.2在我们的工作迄今已进行。植物样品被放置在引线内衬笼位于20米从X-射线源，由一个6特斯拉超导弯曲磁体偶极子的高级光源电子储存环操作在一个关键的能量为11.5千电子伏内产生的。在图1中所示的示意性的端站。的x-射线进入笼的光束大小的40倍〜4.6毫米，并通过样品，其安装在一个电动旋转阶段​​。 “发送的X-射线照射上的晶体的闪烁体（两个常用的材料是LuAG或CdWO 4）X-射线转换ccd的图像采集通过透镜到被中继的可见光。摄像头，闪烁和光学元件都包含在一个不透光的方块的轨道上，允许样品到闪烁相位衬度成像距离进行优化。 所有的样品被安装在直径10厘米的旋转台，而这又是安装在水平和垂直的翻译阶段的样品定位。生活在一个植物性样品，与根系统安装在一个定制的盆栽植物保持架和丙烯酸类管中所载的叶子，在图2中可以看出。典型的曝光时间范围可以从0.1-1秒用10-18千电子伏，扫描持续时间范围从5-40分钟，根据一个特定样本的最优化设置。对于高采样（典型植物木质部网络的），数据扫描平铺通过与样品在不同的高度，这是控制自动重复的测量，允许无缝连续切片沿〜10厘米的最大采样高度。最大样品宽度在4.5微米的分辨率是成像时〜1厘米的样品在垂直方向几乎是完美的。使用下面列出的协议完成数据的生成和处理。由于空气和水之间的差异在X射线衰减，优异的图像的对比度可以在植物中获得对比度医疗CT系统的典型的解决方案，而无需使用。空气填充的血管腔是很容易区分，从周围的充满水的水合的植物组织中。

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using high resolution computed tomography to visualize the three dimensional structure and function of plant vasculature

高分辨率X射线计算机断层扫描（HRCT）是一种非破坏性的诊断成像技术，具有亚微米分辨率的能力，现在被用于评价在三维（3D）的结构和功能的植物木质部网络（ 例如 Brodersen 等2010，2011，2012A，B）。 HRCT成像是根据医疗CT系统相同的原则，但高强度同步加速器X-射线源的查询结果在较高的空间分辨率，并减少图像的采集时间。在这里，我们展示了如何基于同步加速器的详细HRCT（光源 - 劳伦斯伯克利国家实验室的高级伯克利，CA，USA）进行结合Avizo软件（VSG公司，伯灵顿，MA，USA）被用来探索植物木质部切除组织和活的植物。这种新的成像工具，允许用户将超越传统的静态的2D光或电子显微镜研究样本，在任何平面上使用虚拟串口的部分。无限多的在任何方向C片可以对同一样品，实际上是不可能的一个特点，就是使用传统的显微镜方法。 结果表明，HRCT可以适用于草本和木本植物物种，以及各种植物器官（ 即叶，叶柄，茎，树干，树根）。数字是在这里展示了一系列有代表性的植物血管解剖和类型从的HRCT资料集，包括扫描海岸红杉（ 红杉 ），核桃（ 胡桃属），橡木（ 栎属 ），枫提取的细节（ 宏碁属）的树苗，向日葵（ 油葵 ），葡萄（ 葡萄种），蕨类（ 蕨和狗脊黑斑病的 ）。切除和干燥的样品，木本物种是最简单的扫描，通常产生最佳的影像。然而，最近的改进（ 即更快速的扫描和样品稳定），它POSSIBLE使用可视化技术，绿色组织（ 如叶柄）和活的植物。有时收缩的水合绿色的植物组织，会导致图像模糊和方法，以避免这些问题进行了阐述。这些最新进展与HRCT提供有前途的新的见解植物的血管功能。

HRCT扫描已经Synchotron已成功实施各种各样的植物组织和种光束线8.3.2（ 图5），并以前所未有的分辨率在3D植物的木质部的结构和功能，提供了新的见解。三维重建（如在图6-8中所示;影1-3）所提供的可视化和勘探能力允许精确测定木质部网络上都切除样本的结构的位置和取向，以及在活体植物。 在某些情况下，样本移动或无意的振动引起的扭曲在最终...

Watch this Scientific Journal Video about Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature at JoVE.com

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

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

高分辨率X射线计算机断层扫描（HRCT）是一种非破坏性的诊断成像技术，可以被用来研究在3D中的结构和功能的植物脉管。我们演示如何HRCT促进木质部网络的探索范围广泛的植物组织和种类。

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 ...

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Biology

Plant Biology

Plant Structure, Growth, and Nutrition

SCIENTISTS IN ACTION

20.9K 次观看.  U.S. Department of Agriculture. 高分辨率X射线计算机断层扫描（HRCT）是一种非破坏性的诊断成像技术，可以被用来研究在3D中的结构和功能的植物脉管。我们演示如何HRCT促进木质部网络的探索范围广泛的植物组织和种类。

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

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

科研

生物学

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

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.

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 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 ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

在天然化合物存在下测定完整β细胞线粒体功能的高分辨率呼吸

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

单个细胞分辨率三维成像的有机体

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an International Space Station (ISS) mission. However, the underlying mechanisms of these spaceflight-induced changes are currently unknown. The purpose of the present study was to determine the impact of the spaceflight environment on ocular structures by evaluating the thickness of the mouse retina, the retinal pigment epithelium (RPE), the choroid and the sclera layer using micro-CT imaging. Ten-week-old C57BL/6 male mice were housed aboard the ISS for a 35-day mission and then returned to Earth alive for tissue analysis. For comparison, ground control (GC) mice on Earth were maintained in identical environmental conditions and hardware. Ocular tissue samples were collected for micro-CT analysis within 38(&#177;4) hours after splashdown. The images of the cross-section of the retina, the RPE, the choroid, and the sclera layer of the fixed eye was recorded in an axial and sagittal view using a micro-CT imaging acquisition method. The micro-CT analysis showed that the cross-section areas of the retina, RPE, and choroid layer thickness were changed in spaceflight samples compared to GC, with spaceflight samples showing significantly thinner cross-sections and layers compared to controls. The findings from this study indicate that micro-CT evaluation is a sensitive and reliable method to characterize ocular structure changes. These results are expected to improve the understanding of the impact of environmental stress on global ocular structures.

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method

We present a protocol using high-resolution micro-computed tomography imaging to determine whether spaceflight induced damage on ocular structures. The protocol shows the micro-CT-derived measurement of ex vivo rodent ocular structures. We demonstrate the ability to assess ocular morphological changes following spaceflight using a non-destructive tridimensional technique to evaluate ocular damage.

使用微计算断层扫描（微CT）成像方法对太空飞行后全球眼结构进行评估

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an ...

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron spin echo, diffraction, and inelastic scattering. Unlike neutron scattering techniques that obtain information in reciprocal space, attenuation-based neutron imaging measures a signal in real space that is resolved on the order of tens of micrometers. The principle of neutron imaging follows the Beer-Lambert law and is based on the measurement of the bulk neutron attenuation through a sample. Greater attenuation is exhibited by some light elements (most notably, hydrogen), which are major components of biological samples. Contrast agents such as deuterium, gadolinium, or lithium compounds can be used to enhance contrast in a similar fashion as it is done in medical imaging, including techniques such as optical imaging, magnetic resonance imaging, X-ray, and positron emission tomography. For biological systems, neutron radiography and computed tomography have increasingly been used to investigate the complexity of the underground plant root network, its interaction with soils, and the dynamics of water flux in situ. Moreover, efforts to understand contrast details in animal samples, such as soft tissues and bones, have been explored. This manuscript focuses on the advances in neutron bioimaging such as sample preparation, instrumentation, data acquisition strategy, and data analysis using the High Flux Isotope Reactor CG-1D neutron imaging beamline. The aforementioned capabilities will be illustrated using a selection of examples in plant physiology&#160;(herbaceous plant/root/soil system)&#160;and biomedical applications&#160;(rat femur and mouse lung).

Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory&#039;s High Flux Isotope Reactor

This manuscript describes a protocol for neutron radiography and computed tomography of biological samples using a High Flux Isotope Reactor (HFIR) CG-1D beamline to measure a metal implant in a rat femur, a mouse lung, and an herbaceous plant root/soil system.

橡树岭国家实验室高通量同位素反应堆的中子射线照相和生物系统计算机断层扫描

Neutrons have historically been used for a broad range of biological applications employing techniques such as small-angle neutron scattering, neutron ...

Construction of a Realistic, Whole-Body, Three-Dimensional Equine Skeletal Model using Computed Tomography Data

Therapies based upon whole-body biomechanical assessments are successful for injury prevention and rehabilitation in human athletes. Similar approaches have rarely been used to study equine athletic injury. Degenerative osteoarthritis caused by mechanical stress can originate from chronic postural dysfunction, which, because the primary dysfunction is often distant from the site of tissue injury, is best identified through modeling whole-body biomechanics. To characterize whole-body equine kinematics, a realistic skeletal model of a horse was created from equine computed tomography (CT) data that can be used for functional anatomical and biomechanical modeling. Equine CT data were reconstructed into individual three-dimensional (3D) data sets (i.e., bones) using 3D visualization software and assembled into a complete 3D skeletal model. The model was then rigged and animated using 3D animation and modeling software. The resulting 3D skeletal model can be used to characterize equine postures associated with degenerative tissue changes as well as to identify postures that reduce mechanical stress at the sites of tissue injury. In addition, when animated into 4D, the model can be used to demonstrate unhealthy and healthy skeletal movements and can be used to develop preventative and rehabilitative individualized therapies for horses with degenerative lamenesses. Although the model will soon be available for download, it is currently in a format that requires access to the 3D animation and modeling software, which has quite a learning curve for new users. This protocol will guide users in (1) developing such a model for any organism of interest and (2) using this specific equine model for their own research questions.

The purpose of this protocol is to describe the method of creation of a realistic, whole-body, skeletal model of a horse that can be used for functional anatomical and biomechanical modeling to characterize whole-body mechanics.

使用计算机断层扫描数据构建逼真的全身三维马骨骼模型

Therapies based upon whole-body biomechanical assessments are successful for injury prevention and rehabilitation in human athletes. Similar approaches ...