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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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<…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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)。
Materials
| Skyscan 1172 X-ray computed tomography system | Bruker Corporation, Belgium | NA | For CT scanning |
| CO2 critical point drying system (Leica CPD300) | Leica Corporation, Germany | NA | For sample drying |
| Ethanol | Any | NA | For FAA fixation |
| Formaldehyde | Any | NA | For FAA fixation |
| Acetic acid | Any | NA | For FAA fixation |
| Surgical blade | Any | NA | For cutting the sample sgements |
| 3D printer | Makerbot replicator 2, MakerBot Industries, USA | NA | For printing the sample baskets of maize root, stem, and leaf |
| Centrifuge tube | Corning, USA | NA | Place the root, stem, or leaf materials |
| Solid iodine | Any | NA | For sample dyeing |
| SkyScan Nrecon software | SkyScan NRecon, Version: 1.6.9.4, Bruker Corporation, Belgium | NA | For image reconstruction |
| VesselParser software | VesselParser, Version: 3.0, National Engineering Research Center for Information Technology in Agriculture (NERCITA), Beijing, China | NA | Image analysis protocol for single CT image of maize stem or leaf |
| ScanIP | ScanIP, Version: 7.0; Simpleware, Exeter, UK | NA | 3D image processing software |
| Latex gloves | Any | NA | |
| Tweezers | Any | NA |
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