Name: micron-scale phenotyping techniques of maize vascular bundles based on x-ray microcomputed tomography
Uploaded: 2018-10-09T15:00:00
Description: Watch this Scientific Journal Video about Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography at JoVE.com

这项研究得到了中国国家自然科学基金 (31671577)、北京农林科学院科技创新专项建设资助项目 (KJCX20180423) 的支持, 研究中国开发项目 (2016YFD0300605-01), 北京自然科学基金 (5174033), 北京博士后研究基金会 (2016 ZZ-66), 北京农林科学院 (KJCX20170404) (JNKYT201604)。

1. 样品准备协议
<ol><li>对于取样, 从新鲜的玉米植株收集茎、叶和根, 并将它们分成三类样本组 (每组四个复制)。然后, 用手术刀片将其切成小段: (1) 切下中间茎节间的段, 长度为 1-1.5 厘米;(2) 用主脉沿垂直方向切开叶片最大宽度的一段, 长度为 0.5-3 厘米;(3) 将冠根的一段长度切开0.5 厘米。</li>
	<li>对于联邦航空局的固定, 浸泡在 faa 解决方案 (90:5: 5 伏/v/v, 70% ethanol:100 打印张数%acetic 酸) 的样品段至...

<ol>
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随着 CT 技术在生物医学和材料科学领域的成功应用, 该技术已逐步引入植物学和农业领域, 促进了植物生命科学的研究, 成为一种有前途的技术工具。.二十世纪九十年代代末, CT 技术首次用于研究植物根系的形态结构和发育。在过去的十年中, 同步辐射已经成为植物生物学家的一种强大的、无损的工具, 已经成功地用于鉴定葡萄血管系统的组织结构8、拟南芥叶片的组织结构<...

玉米血管系统贯穿整个植物, 从根部到茎叶, 形成了输送水、矿物质养分和有机物质的关键运输途径1。血管系统的另一个重要功能是为玉米植株提供机械支持。例如, 根和茎中血管束的形态、数量和分布与2、3玉米植株的抗倒伏性密切相关。目前, 对血管束解剖结构的研究主要是利用显微和超微结构技术来显示茎、叶或根的某一部分的解剖结构, 然后测量和计数这些结构的通过人工调查的兴趣。毫无疑问, 大规模 microimages 中各种显微结构的手工测量是一项非常繁琐、效率低下的工作, 由于其主观性和不一致4, 严重限制了 microphenotypic 特性的精度, 5。
玉米无二次生长, 细胞含量主要由初级生分生中的水组成。未经任何预处理, 可以用微 CT 装置直接扫描玉米组织的新鲜样品;然而, 扫描结果可能是差和粗糙。主要原因如下: (1) 植物组织的衰减密度低, 导致图像中原子数和高噪声的对比度低;(2) 在正常的扫描环境中, 新鲜植物材料容易脱水和收缩, 如杜6所报告。上述问题已成为玉米、小麦、水稻等单子叶植物 microphenotyping 技术发展和应用的主要制约因素。在这里, 我们介绍了 "样品准备协议", 以预处理玉米茎, 叶和根的样品。该协议避免了在 CT 扫描过程中植物材料的脱水和变形;因此, 用 nondeformation 增加植物样品的保存时间是有益的。此外, 基于固体碘的染色步骤也提高了植物材料的对比度;从而大大提高了显微 CT 成像质量。此外, 我们还开发了名为 VesselParser 的图像处理软件来处理玉米茎叶的 CT 图像。该软件集成了一套图像处理管线, 用于对不同植物组织的2维 CT 图像进行高通量和自动分型分析。用自动图像处理方法检测、提取和识别玉米茎叶全断面的血管束。结果得到了玉米茎的31个显微表型和33个玉米叶片的显微表型。针对玉米根系的 CT 图像序列, 研制了一种图像处理方案, 以获得 metaxylem 血管的3维表型特征。与传统方法相比, 该方案具有更好的图像采集和重建效率。
结果表明, 考虑普通 X 射线显微 CT 成像特征的图像处理管线, 为血管束的显微分型提供了一种有效的方法;这极大地拓宽了 CT 技术在植物科学中的应用, 提高了细胞分辨率6、7的植物材料的自动分型。

<table border="0" cellpadding="0" cellspacing="0" style="width:841px;" width="841">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Skyscan 1172 X-ray computed tomography system</td>
			<td style="width:269px;">Bruker Corporation, Belgium</td>
			<td style="width:119px;">NA</td>
			<td style="width:237px;">For CT scanning</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">CO2 critical point drying system (Leica CPD300)</td>
			<td style="width:269px;">Leica Corporation, Germany</td>
			<td style="width:119px;">NA</td>
			<td style="width:237px;">For sample drying</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td>For FAA fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formaldehyde</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td>For FAA fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetic acid</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td>For FAA fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td>For cutting the sample sgements</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3D printer</td>
			<td style="width:269px;">Makerbot replicator 2, MakerBot Industries, USA</td>
			<td style="width:119px;">NA</td>
			<td>For printing the sample baskets of maize root, stem, and leaf</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge tube</td>
			<td style="width:269px;">Corning, USA</td>
			<td style="width:119px;">NA</td>
			<td>Place the root, stem, or leaf materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Solid iodine</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td>For sample dyeing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SkyScan Nrecon software</td>
			<td style="width:269px;">SkyScan NRecon, Version: 1.6.9.4, Bruker Corporation, Belgium</td>
			<td style="width:119px;">NA</td>
			<td>For image reconstruction</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">VesselParser software</td>
			<td style="width:269px;">VesselParser, Version: 3.0, National Engineering Research Center for Information Technology in Agriculture (NERCITA), Beijing, China</td>
			<td style="width:119px;">NA</td>
			<td>Image analysis protocol for single CT image of maize stem or leaf</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ScanIP</td>
			<td style="width:269px;">ScanIP, Version: 7.0; Simpleware, Exeter, UK</td>
			<td style="width:119px;">NA</td>
			<td>3D image processing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Latex gloves</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tweezers</td>
			<td style="width:269px;">Any</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

micron-scale phenotyping techniques of maize vascular bundles based on x-ray microcomputed tomography

基于高通量图像分析技术, 有必要对玉米材料的解剖结构进行准确量化。在这里, 我们提供了一个 "样品准备协议" 的玉米材料 (即茎, 叶, 和根) 适合普通 microcomputed 层析成像 (微 CT) 扫描。本文以玉米茎、叶、根的高分辨率 ct 图像为基础, 描述了两种用于血管束表型分析的协议: (1) 基于玉米茎叶 ct 图像, 研制出一种特殊的图像分析管道, 自动提取3133维管束的表型特征;(2) 基于玉米根系 CT 图像序列, 建立了 metaxylem 血管三维 (3 维) 分割的图像处理方案, 提取了二维 (2 维) 和3维表型特征, 如 metaxylem 血管的体积、表面积、等与传统的人工测量玉米材料的血管束相比, 该协议大大提高了微米尺度表型量化的效率和准确性。

适合于普通微 CT 扫描的样品制备方案不仅能防止植物组织的变形, 而且能提高 x 射线的吸收对比度。用微 CT 系统对预处理后的植物材料进行扫描, 得到高质量的切片图像, 最高分辨率可达2µm/像素。图 4显示了扫描的茎、叶和根的显微 CT 图像, 与从新鲜植物材料中扫描的结果相比, 图像对比度有显著的改善。在这些 CT 切片图像中, 在血管束、软组织细?...

Watch this Scientific Journal Video about Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography at JoVE.com

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

为提高适合普通 microcomputed 层析扫描的玉米组织 X 线吸收对比度提供了一种新的方法。在 CT 图像的基础上, 引入了一套不同的玉米材料图像处理工作流, 有效地提取了玉米血管束的显微表型。

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

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

该方法可以在规划的微米尺度表型技术构建中快速回答问题。如微CT扫描颗粒，以及玉米血管束的表型分析方法。该技术的主要优点是提供了新的、有效的方法，通过样品制备、CT扫描和方案分析来研究玉米血管束的表型特征。该技术的影响拓宽了普通微CT扫描和其他植物科学的应用。此协议可以很容易地修改，以适应其他植物材料，如脱水的干燥过程。首先，从新鲜的玉米植物中收集茎、叶和根。将它们分成三个样本组。接下来，使用手术刀片切割中茎中间茎的1至1.5厘米段。使用相同的手术刀片，从叶的最大宽度沿垂直方向与主静脉切割1/2至2厘米的段。使用手术刀片从接地根切开 1/2 厘米的段。在此之后，将样品段浸泡在 FAA 溶液中至少三天。在六个连续乙醇梯度中脱水样品段 30 分钟。然后将植物材料放在相应的样品篮中。快速将篮子转移到二氧化碳临界点干燥系统的样品单元。将干燥的植物材料放入含有两克固体碘的50毫升离心管中。然后将管子放在防光室四到五个小时。首先，根据文本协议设置CT扫描参数。然后，根据使用的工厂材料的不同尺寸和体积设置扫描范围。接下来，调整成像像素大小如下：根的两微米。6.77 微米用于茎。和10微米的叶子。要重建切片图像，请使用图像构造软件将原始 CT 数据转换为分辨率为 2K 的 CT 切片图像。首先，指定器官类型，在自动成像软件中初始化不同的算法管道。然后，单击方法参数按钮，在第一个下拉框中选择玉米茎或玉米叶。单击数据管理按钮，设置工作目录，并在目录中导入所有切片映像。然后在图像管道中选择单片或多切片图像。接下来，确定图像的像素大小。选择方法参数按钮，并在相应的编辑项中输入图像的像素大小。在此之后，单击表型计算按钮，自动提取所有选定切片图像的血管束表型特征。单击统计分析按钮，将这些分析的结果输出为文本或 CSV 文件。导入玉米根的重建图像，并确定精确的空间参数。然后使用递归高斯工具平滑图像，提高图像质量。调整阈值参数，对中轴器进行三维分割。这将为每个连接的梅塔西莱姆容器创建一个统一的颜色标签。然后，利用形态学和洪水填料操作，以交互方式改进和识别梅塔西勒容器。接下来，对容器进行体积可视化和表面重建。最后，使用掩码统计工具计算和测量容器在 2D 和 3D 级别的表型特征。利用协议中概述的分析，计算了玉米茎、叶子和根部血管束的表型特征。使用分段、重建和体积可视化的结果，创建了根和梅塔西勒血管生成的 3D 可视化。适当的样品制备方案显著提高了玉米茎、叶和根的微CT扫描质量。利用自动发射管道快速提取玉米茎和叶血管束的表型状状。并设置和发射处理扫描，分析玉米根系血管束的三维表型特征。该技术为准确、快速的玉米血管束鉴定提供了一条实用的方法。

Research

JoVE Journal

Biology

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

非侵入性的三维可视化与亚微米分辨率使用同步加速器X射线断层扫描

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

科研

生物学

Protein Crystallization for X-ray Crystallography

X射线晶体学的蛋白质结晶

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

基础X射线计算机断层数据的三维打印设置

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

人类诺沃克病毒凸，域的生产<em&gt;电子。大肠杆菌</em&gt; X射线晶体学

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

Techniques to Induce and Quantify Cellular Senescence

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

SA-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

使用矫形器（40-300 kV）X射线设施进行细胞辐照的测定

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose ...

Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

死后计算断层扫描中的图像渲染技术：受困鲸目动物的生物健康和特征评估

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

通过冷冻软X射线断层扫描对细胞进行3D制图描述

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...