这项工作是由美国国家科学基金会（奖号IOS-1031416）资助的赠款，并与内森·米勒，洛根·约翰逊和美国威斯康星大学的埃德加·斯伯丁和Brian Bockelman，卡尔Lundstedt和大卫·斯万森的合作已经进行内布拉斯加州的荷兰计算中心大学。

1。图像采集协议注意事项: 这个协议是最有效的两个人进行的，尽管它有可能为1单独工作。工作最好在本实验室的安排是为了一个人来制备板进行扫描，而在扫描仪设置的另一个作品，那么这两个一起放置在平板扫描仪，并启动扫描过程。 同样重要的是要注意，在这个项目上的扫描仪是垂直方向的扫描仪盖子搁在扫描仪的后面。自定义支持是为了保持菜肴在这个垂直位置，并贴于平板表面3M无痕条（ 图2）。附带在这个协议（爱普生V700）使用的扫描仪可移动文件盖内衬一面用黑色毡。文件盖被靠住平板用蹦极绳索来持板就位，并提供图像的对比度（ 图3）。 的足够的分辨率的任何扫描仪可用于图像采集。在爱普生Perfection V700被选中，是因为它的方形轮廓（因此很容易垂直位置），其分辨率高，以及附加的从床上盖既扫描，并使用红外通道。这些额外的选项并没有在这个协议中使用。 一旦板被从生长室中取出，这是必须的协议继续到结束。 板准备 使用了含有10ml透明介质和9的种子在每个板的中间种标准培养皿。 :对于板材的标签，媒体准备和种植过程可以在这里找到<a href="http://www.doane.edu/doane-phytomorph">http://www.doane.edu/doane-phytomorph</a> <ol><l我&gt;提取第一琼脂平板和吸收收集的冷凝在琼脂平板上用的Kimwipe的盖的盖和轮辋。 </li><li>适用的Triton X-100（洗涤剂）来盖了的Kimwipe - 大方。 （需要注意的是的Triton X-100有助于防止结露盖子的建设作为平板扫描。一位慷慨的应用程序（足以创造的盖子表面的膜）将有助于确保该盖在整个扫描仪运行保持透明。） </li><li>用微孔胶带包裹板来固定盖，并且允许用于通风。 </li></ol> 扫描仪设置和图像采集 这个协议假定，超过1扫描仪正在使用，并提供指示，开始从单一的电脑多台扫描仪。 <ol start="4"><li>用于存储从每个扫描仪图像创建文件夹。每个扫描仪将持有两大板块，所以创建文件夹时，记住这一点。一可以选择使用元数据作为文件名，如为每个板块唯一的ID，苗龄，种子大小，和股票种植ID的组成部分。数据收集在含有这些元数据中使用的文件夹名称的示例是"1652-2-SM-9-92-17-1653-2-​​LG-88-79-161"。 </li><li>设置插座计时器指定收集时间（9小时是在这个实验室中使用）。请务必准备设置额外的时间（一个小时左右）。 （请注意，扫描仪必须插在插座上的定时器，以设定的采集时间。虽然VueScan软件允许用户反复采集图像，它不允许用户指明多少图像采集要不了多久来收集图像。） </li><li>打开第一台扫描仪，等待大约10秒的扫描仪要经过初始热身。 </li><li>打开VueScan程序一次。 VueScan版本9.0.20用于本协议（见材料表），但更近的版本，可以少modific使用通报BULLETIN。确保"更多"按钮以显示下面描述的菜单选项被压在用户界面的底部面板上。 </li><li>设置自动重复 :下拉框没有设置预览区域下方的输入标签， 裁切标签下:到最大 （ 图4）。按"预览"。 </li><li>创建一个裁剪框，将通过用鼠标点击并拖动整个的预览图像上感兴趣的区域捕获感兴趣的区域。设置可能会被改变为在作物选项卡中感兴趣的区域。用于裁切框的典型设置为:x偏移量为0.675，y偏移量在1.924，虽然这调整为捕捉育苗面积每扫描仪。使用裁切框的大小是宽7.246在高1.1（ 图5）。 </li><li>移动裁切框，按住Shift键的同时用鼠标拖动。确保裁切框包含了所有要扫描的苗加上可能包含一个标签（ 图5）在任何需要的元数据。 </li><li>根据作物选项卡，设置预览区:以裁剪框，然后按"预览"。 </li><li>转到Output选项卡，并选择正确的文件扫描仪（ 图5）。 </li><li>重复所有扫描仪一台计算机的步骤1.7-1.12。当被问及是否开启VueScan的多个实例，选择"是"选项。 </li><li>通过每个标签，并确认设置是否正确。 （请注意，所有的规格可以改变，以适应个别实验室的需求，包括图像的色彩，分辨率等 。但是，在这个协议中使用的设置，可以直接应用到PArticular扫描一个给定实验的硬件，由于VueScan软件的通用接口。请参阅随附的规格列表，以查看该项目所使用的参数，利用VueScan版本9.0.20）。 </li><li>在输入选项卡中选择连续的自动重复:字段，或者根据需要选择图像之间的时间间隔越长。时间间隔的时间长度保存最后的影像，并开始采集下一个图像后，扫描仪会暂停。在连续模式下，3-4分钟分辨率为4800 DPI来获得。 </li><li>对于连接到一台电脑扫描仪的其余部分重复步骤1.14-1.15。 </li><li>将制备的板与幼苗水平方向的正确扫描仪（不gravistimulate）。 </li><li>暂时放在一个黑色的，感觉对板的背景，使他们不从有机玻璃模板下降。重复所有扫描净入学率。 （ 注:在这个项目中，黑色的毡片被附加到文档涵盖随设备提供的，以防止眩光，并提供对根组织对比中使用的特定背景颜色将取决于被成像组织的颜色）。 </li><li>有一个人转板90°（板逆时针转动在此协议），并立即更换毛毡背景。 </li><li>其他人应该站在电脑，使他们可以马上按下"扫描"按钮。 </li><li>固定的背景，扫描器用松紧绳（ 图3）。有一个人保持背景的地方，而另一个位置的蹦极绳索。 （ 注:立即gravistimulation板的90°（旋转）和毛毡背景放置后，"扫描"应该被按下）。 </li><li>在一个COMPUT为扫描仪的其余部分重复步骤,1.17-1 .21呃。 </li><li>下一组扫描仪（如适用）重复步骤1.6-1.22。 </li><li>不要让扫描仪，直到几个图像被收集，以确保它们被正确保存。 </li><li>它是理想的保持扫描仪在一个地区，将免费为干扰所指定的扫描时间的。这也是审慎考虑在扫描区域内的环境条件，以确保理想的表型反应。 </li><li>当数据收集完成后，按每个VueScan窗口，每个扫描仪重合绿色中止按钮。 </li><li>收出计算机上的所有程序。 </li><li>重新启动计算机，关闭开始另一轮的图像采集前的所有扫描器。 </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+feature+counting+and+measurement+of+roots.">High-throughput feature counting and measurement of roots.</a> Bioinformatics. 27, 1337-1338 (2011).</li></ol>

作者宣称，他们有没有竞争的财务权益。

准确的表型观察是了解生物体中的基因功能的表现是至关重要的。获得表型信息的一种方式是通过高分辨率的图像数据的采集。开发了扫描仪为基础的平台，使收集许多图像（200张/扫描周期）的高分辨率（4,800 dpi）的在数个小时。此外，该平台是很容易适应各种实验室和教室环境中由于VueScan软件运行几千使用通用接口18不同的扫描器的灵活性。 这里介绍的方法填充在高通量的图像捕获的空隙，从大规模的表型设备和自动化系统可实现在单个实验室延伸。目前可用的高通量平台倾向于使用专门的成像硬件，包括摄像机安装在机器人支撑，捕捉的p高分辨率图像rimarily地上植物组织（如中心植物一体化技术和Scanalyzer HTS由LemnaTec）20,21。利用X射线和MRI技术专业成像系统也已开发，以地下组织具有显着的分辨率的图像，因为它们在土壤环境中（如中心厂一体化技术）11,22,23成长。这方面的发展更加专业化的技术一般是在吞吐量的成本，使动态表型的研究比较困难。重要的是，这些高端平台的成本和基础设施的需求使它们大多是不可行的小型实验室执行。 平台也已开发了使用更标准图像捕捉技术和非常适合于动态响应如根响应于重力的刺激进行测量。例如，CCD照相机已被用于捕获个人幼苗光反应和重力在高空间和时间分辨率1,8,12。其他系统已经开发，允许从一个单一的图像（例如RootTipMulti由iPlant合作）17,24多根的根尖方向的测量。在前者的情况下，可以通过相对较低是只有一个苗是由每个摄像机一次成像，而在后一种情况下的吞吐量较高，但通常在分辨率为代价。 本文概述的过程呈现捕捉高分辨率的图像在高吞吐量的设备和软件，都是现成的，比较实惠的平台。利用此设置，1080个人根响应可以每星期被收集在一个单一的实验室装备的6扫描器银行。在收集平均每星期864个人回答15个月，共有41,625苗被扫描的基因组学研究。约15％的个人藏品由于安装错误，netwo失败RK故障或设备故障。另外22％的响应失败，原因是缺乏发芽或根系生长不足引发一个生长反应。最终的数据集包括27,475个人幼苗的反应，从163个重组自交系重力刺激加99近等基因系。数据被收集在一个单一的实验室，使之成为一个非常高通量的方法。即使考虑到用于收购的设备相对便宜，它甚至与大量使用可靠地运作了两年多。 虽然这个协议一直是这个群体的研究目标非常有用，一些限制仍然存在。因为大约50 GB的每一天未压缩的图像数据的吞吐量，很显然，大量的空间是需要的房子的图像，除非有效的压缩方案可以发展。存储问题暂时通过购买外部硬盘驱动器为每台计算机解决。此外，2 10 TB的网络关联存储设备购买。以后，压缩算法被开发中，如上所述，这可以帮助减少高达60％（ 图8）降低数据大小。要注意，在该数据可以被保存到一个网络相关联的存储装置中的速度依赖于网络连接的速度是很重要的。压缩方案也受到限制，由于以防止图像数据丢失的愿望。 特定于扫描仪为基础的成像系统等的限制也正在考虑。例如，在一台扫描仪为基础的方法的幼苗在每次扫描过程中的白色和潜在的红外线范围内暴露于高强度的光。这可能会影响幼苗生长，幼苗虽然仍然可以观察到经历健壮响应重力刺激（ 图7）。未来的改善可能涉及到编程扫描仪这样，只有红外线LED是积极的。在活跃事业发展情况的地区t是创造良好匹配于这些图像数据的分辨率和吞吐量的分析算法。使用这种扫描仪为基础的方法所产生的大数据集已非常适用于强大的工具，用于育苗的图像高通量分型发展。在图7所示的这些图像所采用的压缩算法支持的说法，他们是经得起图像分析应用。此外，所生成的图像可以由先前公布的算法，RootTrace 17,24进行分析，如果它们被收集在较低的分辨率（低于1,200 dpi）的，并且个别苗从图像分析使用前上述压缩算法分割。根系生长数据可以提取图像降低到1,200 dpi的同时刀尖角度数据可以从图像降低到900 dpi的（未发表的观察）中提取。 本文概述的程序安装到在袋鼠的世界自己的利基吨成像，因为它是高通量，高解析度，同时仍然比较实惠。这种方法的一个额外的好处是，它可以很容易地进行定制，以适应特定的研究组的成像需求。

收集高分辨率表型数据的是在研究，旨在了解遗传学的相互作用和环境调解有机体功能1,2有用。是这种性质的研究也天生规模大，使得它另外必要的，用于测量表型在这方面的方法是高通量3,4。在建立表型组学规模的研究方法，吞吐量和分辨率之间进行权衡来发挥作用。方法是在更高的吞吐量也往往在分辨率较低，使之更难以检测遗传或环境5小的影响。可替换地，更多的仔细测量所需表型的方法也往往在吞吐量降低，使得难以广泛调查，遗传和环境的影响。此外，手工方法量化表型，包括视力检查，可以不受变化，由于每个人的差异ception 6。 成像技术可以在获得表型观察7-9提供吞吐量和分辨率之间的有益桥梁。在一般情况下，一个图像是相对容易地捕获，便利的吞吐量，而当在足够高的分辨率拍摄，细微的表现型可检测1,2,7。成像技术往往是修改，以适应感兴趣的系统或过程，一般是可扩展的10-12。正因为如此，成像技术是非常适合的生物体功能的大规模研究的发展。 主根对重力刺激的反应是发生在一个简单形态的器官，一个复杂的生理过程。该反应涉及激活信令通过根器官，传播和其级数是由环境和遗传因素的影响，包括对环境的影响12-14遗传因素决定通路的</suP&gt;。主根对重力刺激的反应自达尔文在研究最少，但还有很多需要学习它是如何工作，特别是在早期信号事件，并在调解响应可塑性12,14,15的因素。获得这一反应的动力学进行详细的了解是在寻找方法来提高苗木的能力，成功地成为在一个特定的环境中16建立了重要的。此外，根的形状，使得它适合用于图像处理的应用程序8,12,17。两者合计，根向地响应是用于高通量成像技术的发展进行有机体功能的基因组层面的研究的目的，一个理想的系统。 在此报告中，高通量，高分辨率方法，利用便宜的，市售的平板扫描仪的根向地响应的图像捕捉呈现。的概述协议， 如图1所示。苗种植在琼脂平板上放置装有定制的有机玻璃板持有者垂直方向的平板式扫描仪。图像采集，每隔几分钟在4,800 dpi的，并保存在本地驱动器或数据服务器上。与每个图象系列关联的元数据存储在数据库和所存储的图像进行处理。该方法使用图像捕捉VueScan软件。 VueScan可用于运行在Windows，Mac或Linux操作系统超过2100不同的扫描仪（见材料表）。 4800 dpi的扫描仪的分辨率用在这个应用程序，以符合使用固定CCD摄像机1,8,12以往的研究取得的分辨率。该VueScan软件以及通用接口的灵活性，它使用它运行，用户可以很容易地采取足够的分辨率几乎所有的扫描仪硬件在本文提出的协议的任何扫描仪。电流通过允许集合每天216个人的反应。该技术的适应性和可扩展适用于机构，从高中到大学的研究。此外，所收集的图像具有足够的质量，用于图像分析的应用程序。

<table border="0" cellpadding="0" cellspacing="0" width="798">
	<tbody>
		<tr>
			<td>Epson Perfection V700 Photo Scanners</td>
			<td>Epson</td>
			<td>B11B178011</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Plexiglas Scanner Template</td>
			<td>-</td>
			<td>-</td>
			<td>Custom made. See Figure 2.</td>
		</tr>
		<tr>
			<td>Smart Strap Bungee Cords</td>
			<td>SmartStraps</td>
			<td>Wal-Mart 1079478</td>
			<td></td>
		</tr>
		<tr>
			<td>Brinks Digital Outdoor Timers</td>
			<td>Brinks</td>
			<td>Wal-Mart 42-1014-2</td>
			<td></td>
		</tr>
		<tr>
			<td>VueScan Software</td>
			<td>Hamrick Software</td>
			<td><a href="http://www.hamrick.com/">http://www.hamrick.com</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Segmentation Software</td>
			<td>Chris Wentworth, Doane College</td>
			<td></td>
			<td>https://sites.google.com/a/doane.edu/compphy-doane/projects/root-gravitropism/image-segmentation</td>
		</tr>
		<tr>
			<td>3M Micropore Tape</td>
			<td>Fisher Scientific</td>
			<td>19-061-655</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Holding racks</td>
			<td>-</td>
			<td>-</td>
			<td>Custom made by gluing two cookie racks together.</td>
		</tr>
	</tbody>
</table>

using flatbed scanners to collect high-resolution time-lapsed images of the arabidopsis root gravitropic response

在生物学的研究工作越来越需要使用方法，使高分辨率数据的大批量采集。实验室可以面对的挑战是这些方法的发展和实现。表型中的感兴趣的过程的观察是研究实验室研究基因功能的一个典型目的和这往往是通过图像捕获来实现。一个具体的过程，是适合于观察用成像方法是已被移位从对准与重力矢量幼苗根的生长纠正。用于测量根向地响应成像平台可以是昂贵的，相对低的吞吐量，和/或劳动强度大。这些问题都已经解决了使用廉价的，但高解析度，平板扫描仪发展高通量图像采集方法。使用这种方法，图像可以在4,800 dpi的捕获每隔几分钟。当前的设置使收集的216单独的R每天esponses。收集到的图像数据是充分的品质进行图像分析的应用。

代表图片 这种方法可以快速生产的高分辨率时间序列拟南芥幼苗生长的影响。扫描仪运行的第一个和最后一个图像示于图7A和7B。图7C和7D示出最佳的结果从半全扫描仪的图像。可以影响图像质量的一些问题示于图7A和7B。这些问题包括变化中发芽，变化幼苗生长轨迹在运行的开始，并凝结在扫描过程中积累。缩合可以在很大程度上增加的Triton X-100的施加到板盖内的量来解决。可能阻碍精确的图像采集等因素是相对于板位置和定位板使得它们倾斜就裁剪框裁剪框的配置不正确。 图像分析中的应用:图像压缩 一旦已经获得扫描仪的图像的时间序列，它必须被安全地存储在一个网络可访问的方式，以方便图像分析。与单个扫描仪的运行相关的图像文件占用了显著量的硬盘空间。在4,800 dpi的收集单一的TIFF文件约为220 MB和一个典型的扫描仪运行200生成的图像文件。因此，大约44 GB的硬盘空间是每次运行需要。以减少与图像分析相关联的存储和网络传输成本，希望减少需要存储图像数据，而在同一时间最大限度地减少数据丢失的空间量。下游分析将涉及识别每个幼苗与实验运行相关的后续的图像文件。因此，分割出从扫描仪图像的单苗可以促进下游分析。因为幼苗远离个的其余部分的分割E图像也可以显著降低存储不必要的背景像素，这种做法也导致了显著减少数据大小。此外，如果下游分析集中根组织上它可能不是必要的，以保持颜色信息，因为根像素是相对窄于它们的颜色空间。计算机图像处理协议和代码由双方分割出单个苗和图像转换为灰度来减少数据量已经研制成功。该方法导致在存储空间需求减少了60％。 用来实现这种数据压缩的工作流程在下面的步骤描述如下: <ol><li>开始在一个文件夹中的时间序列扫描的图像文件。 </li><li>对于每个图像，从RGB转换为灰度（ 图8，上图）。 </li><li>分割的图像分割成左，右两侧。 </li><li>从图像中提取各幼苗到它自己的文件（ 图8）。这是通过施加一个阈值以像素为单位转换成黑色或白色，然后计算每个图像行的总像素强度进行。最高强度的行标识，每个像素被归类为&#39;植物&#39;或基于其邻国的强度"nonplant"。此行中的每个"植物"的中心被发现，从该点一预定大小的裁剪框绘制（ 图8，底部）。 </li><li>创建的图像（左和右）为每个育苗储存个人的时间序列图像文件的单独子文件夹的每一侧单独的文件夹。 </li><li>归档结果文件夹到一个ZIP压缩文件。 </li></ol>了完成这些步骤的代码已经被使用的编程语言Python的20开发的。该算法允许在数据量减少大约60％，并成功识别90％的扫描仪成像的所有个别苗Ë文件迄今进行分析。该代码是免费提供给在GNU通用公共许可证版本3下载（见材料表）。 <img alt="图1" fo:content-width="4in" fo:src="/files/ftp_upload/50878/50878fig1highres.jpg" src="/files/ftp_upload/50878/50878fig1.jpg" width="500px" /> 图1。在扫描过程开始于种子种植（每盘最多九拟南芥种子），并具有数据存储和图像处理结束。 <a href="https://www.jove.com/files/ftp_upload/50878/50878fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="4in" fo:src="/files/ftp_upload/50878/50878fig2highres.jpg" src="/files/ftp_upload/50878/50878fig2.jpg" width="500px" /> 图2。牛逼emplate建设的培养皿支持。的PLE<html> xiglas被切断，使得宽度适合的平板（在本例227毫米）和长度为128毫米。两个圆用88毫米直径的切出的剩余片，使得它们被均匀地沿支承的宽度和长度方向分布的。该支持是贴有3M无痕条平板。 <a href="https://www.jove.com/files/ftp_upload/50878/50878fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/50878/50878fig3highres.jpg" src="/files/ftp_upload/50878/50878fig3.jpg" width="500px" /> 苗已gravistimulated和文档盖板定位图3。扫描仪配置后，这是扫描仪在扫描仪设置和图像采集步骤1.21的配置。"_blank"&gt;点击这里查看大图。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/50878/50878fig4highres.jpg" src="/files/ftp_upload/50878/50878fig4.jpg" width="500px" /> 图4。屏幕截图选择扫描仪设置和图像采集步骤1.8设置。 <a href="https://www.jove.com/files/ftp_upload/50878/50878fig4highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/50878/50878fig5highres.jpg" src="/files/ftp_upload/50878/50878fig5.jpg" width="500px" /> 图5中的步骤1.9 ​​和扫描仪设置和图像采集的1.10屏幕截图VueScan软件。红色框突出显示裁切尺寸，而蓝盒子突出FO具体设置<html> R X和Y偏移量，以便捕捉苗和标签信息使用。要扫描的平板的区域显示为在预览区域中的点线。 <a href="https://www.jove.com/files/ftp_upload/50878/50878fig5highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" fo:content-width="4in" fo:src="/files/ftp_upload/50878/50878fig6highres.jpg" src="/files/ftp_upload/50878/50878fig6.jpg" width="400px" /> 图6。选择的扫描仪设置和图像采集步骤1.12目标文件夹。按@按钮旁边的默认文件夹对话框（红色箭头）允许用户选择相应的目标文件夹。 <a href="https://www.jove.com/files/ftp_upload/50878/50878fig6highres.jpg" target="_blank">点击这里查看大图</a> 。 再7"FO:内容宽度="5英寸"FO:SRC ="/ files/ftp_upload/50878/50878fig7highres.jpg"SRC ="/ files/ftp_upload/50878/50878fig7.jpg"宽度="600px的"/&gt; 图7（AD），上述图像是使用本文介绍的方法所收集的例子。图A，B和C，D分别为第一和最后的图像，分别从单一的扫描周期A，B展示全扫描的区域，而C，D是被扫描区域的裁剪区域，显示出一个单一的板。数不一致可以观察到。图A显示变化的萌发和成长轨迹。 B组（相同的苗形象的 ; 9小时后）可以看出，板可以积累凝结。面板C和 D被认为是由于强劲的增长Ø良好的效果f苗和整个运行的图像质量。 <a href="https://www.jove.com/files/ftp_upload/50878/50878fig7highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图8" fo:content-width="5in" fo:src="/files/ftp_upload/50878/50878fig8highres.jpg" src="/files/ftp_upload/50878/50878fig8.jpg" width="600px" /> 扫描器图像灰度（上图）图8。开发的图像压缩算法转换 。该图像被分为左，右两半和图像的边界被去除（未示出）。每个半个人苗的位置是确定的发现该行拥有最大总像素强度。这些位置被用来定义一个新的裁剪区域，适用于全苗盘上（下图）。 <a href="https://www.jove.com/files/ftp_upload/50878/50878fig8highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response at JoVE.com

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

这个协议描述了用于快速收集拟南芥幼苗用市售的平板式扫描仪响应重力刺激的图像的处理。该方法允许对廉价，高容量的高清晰度图像适合用于下游分析算法捕获。

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 ...

using-flatbed-scanners-to-collect-high-resolution-time-lapsed-images

Research

JoVE Journal

Biology

12.3K 次观看.  Doane College. 这个协议描述了用于快速收集拟南芥幼苗用市售的平板式扫描仪响应重力刺激的图像的处理。该方法允许对廉价，高容量的高清晰度图像适合用于下游分析算法捕获。 

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

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

科研

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

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

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

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

使用高解析度电脑断层可视化的三维结构和功能的植物血管

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

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

侧根诱导系统<em&gt;拟南芥</em&gt;和玉米

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

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 ...

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-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.

鼠标窦房结的高分辨率光学测绘

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

拟南芥种子高通量灭菌的标准化方法

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

The goal of the protocol presented here is to study the transcriptomic response of endosphere-isolated Bacillus mycoides to potato root exudates. This method facilitates the identification of important bacterial genes involved in plant-microbe interactions and is in principle applicable to other endophytes and plants, with minor adjustments.