搜索

Imaging and Quantitative Analysis of Size and Distribution of Spherical Bodies, e.g. Embryonic Oil Bodies
胚胎油体等球状体大小、分布的成像和定量测定   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Oil bodies (OBs) are seed-specific lipid storage organelles that allow the accumulation of neutral lipids that sustain plantlet development after the onset of germination. Using fluorescent dyes and confocal microscopy, we monitored the dynamics of OBs in living Arabidopsis (Arabidopsis thaliana) embryos at different stages of development (Miquel et al., 2014). Image acquisition was followed by a detailed statistical analysis of OB size and distribution during seed development in the four dimensions (x, y, z, and t).

Keywords: Oil body(油体), Embryo(胚胎), Size(尺寸), Imaging(成像), Statistical analysis(统计分析)

Materials and Reagents

  1. Plant material
    1. Arabidopsis thaliana plants, wild type or mutant or transgenic plants.
    2. Developing siliques between 6 and 11 days after pollination of plants grown in a greenhouse under the following conditions (13 h of light, diurnal temperature of 25 °C, and nocturnal temperature of 17 °C), and irrigated twice per week with mineral nutrient solution.
  2. Nile Red (a neutral lipid stain, at 2 μg/ml final in acetone) (Sigma-Aldrich, catalog number: 72485 )

Equipment

  1. Confocal microscope
    Note: In this study an inverted LEICA SP2-AOBS spectral confocal laser microscope (Leica Microsystems) equipped with an HCX PL APO CS 40 x 1.25 objective and a multiline argon laser was used.
  2. Forceps (Dumont No.5) (Sigma-Aldrich)
  3. Scalpel (11 P, blade) (Swann Norton)
  4. Glass slides or glass-bottom dish, cover slips
  5. Binocular (Nikon Corporation, model: SMZ1000 ) (Champigny sur Marne with a led ring - Shott easyledTM for illumination)

Software

  1. ND-SAFIR (http://serpico.rennes.inria.fr/doku.php?id=software:nd-safir:index)
  2. AVIZO® FIRE (http://www.vsg3d.com/avizo/fire)

Procedure

  1. Sample preparation
    1. Remove developing seeds from 2-3 siliques under the binocular with a sharp scalpel and forceps (DUMONT N°5) and place them on a glass slide or in a glass-bottom dish.
    2. Add a small volume (between 20 and 30 µl) of Nile Red solution and cover with a cover slip. Remove embryos from the seed teguments by gently pressing seeds between the slide and cover slip with the rounded end of the handle of the scalpel and observe after 30 min of incubation in the dark at room temperature (see Video 1).

      Video 1. Dissecting embryos out of Arabidopsis developing siliques

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

      Note: You can use a 60% (v/v) glycerol solution or 0.4 % (w/v) of low melting agarose as mounting medium for your staining solution to better immobilize the embryos for long observation periods.

  2. Confocal microscopy
    1. Find appropriate embryos and adjust the focus on cotyledon edges using a 40x objective.
    2. Set optical parameters to optimize signal to noise ratio.
      1. Excitation laser: the argon laser is set at 10% of 488 nm for Nile Red excitation.
      2. Emission band of 550 to 650 nm.
      3. Capture the images (90 to 100 optical sections of 0.16 µm thickness) with a 40x/1.25 oil objective and 8x digital zooming. Each section is the average of two scans conducted at the resolution of 512 x 512 pixels with a spatial resolution of 0.09 mm x 0.09 mm x 0.16 µm in the x, y, z referential.
        Notes:
        1. The Nile Red stain is highly fluorescent when associated with neutral lipids. Nevertheless you can bleach it if using too high laser power for excitation. According to our experience, reducing the laser power to its minimal value and laser excitation to 10% of laser power is far enough for excellent observation and preserves fluorescence for up to one night.
        2.  As the fluorescence signal is usually very strong, you can decrease the pinhole’s size to 0.7 Airy to reduce the thickness of the optical section.
        3.  Increasing scanning speed as well as using bidirectional-scanning mode will speed up acquisition.

  3. Quantitative analysis
    1. Obtain images at a similar location from 3 different embryos for statistical analysis.
    2. Analyze the images using the following pipeline:
      1. The crude image stacks present several characteristics that disturb direct automated counting of the oil bodies (Figure 1). To circumvent this problem, the procedures for image segmentation by watershed and data extraction were implemented in AVIZO® FIRE in a semi-automated pipeline. A specific module (WshedBinSeparate) has been developed in TCL language (Tool Command Language). The outline of the procedure is described in Figure 2. A movie shows the sequence of operations (Video 2).


        Figure 1. 2D/3D view of an image stack with various problems preventing direct use in image analysis. A. Images with high noise; B. Non homogenous objects in size; C. Variable grey levels; D. Objects with touching edges.

        A

        B

        C

        Figure 2. Image analysis pipeline.
        A. Generating binary images with double thresholding; B. Generating watershaded images; C. Individual labelling of objects and 3 D rendering. The segmentation chain in Avizo (left) and the resulting images (right).

        Video 2. OB image processing pipeline

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player

      2. The WshedBinSeparate module allows segmentation of oil bodies images stacks by watershed methods.
      3. The input image is a 3D stack in grey scale. The images are first de-noised with a Gaussian filter using ND-SAFIR. Two thresholding operations are then performed with threshold values manually indicated by the operator. The best value is chosen by visual test of various threshold values.
      4. The high threshold allows the detection of large objects without worrying about over- or under-estimating them but without consideration of small objects.
      5. The low threshold allows the detection of small objects without worrying about the over- or under-estimation of the large objects. The images are combined to produce a thresholded “binary image”.
      6. The 3D distance transform of this binary image is computed (a value is given for each pixel of the image according to its distance to the border of the closest object in the vicinity).
      7. The maximum of the distance function produces the “marker image” i.e. the centre of the segmented quasi-spherical filled object. The complement of the distance transform (CDT), with the marker image is used to create the “watershed lines”: the watershed algorithm expands the markers (like inflating a balloon) toward increasing value of the CDT. The watershed lines represent the boundaries separating the objects. Finally, the watershed lines are subtracted from the binary image to obtain separated and individualized objects (“segmented image”). The image of separated objects is then converted to a spread sheet document where each item in the spread sheet is a labeled object volume.

Acknowledgments

This work was funded by INRA and, in particular, by the divisions BAP (Plant Biology and Breeding) and MIA (Applied Mathematics and Informatics) for G. Trigui’s Ph.D. This work was adapted from Miquel et al. (2014).

References

  1. Miquel, M., Trigui, G., d'Andrea, S., Kelemen, Z., Baud, S., Berger, A., Deruyffelaere, C., Trubuil, A., Lepiniec, L. and Dubreucq, B. (2014). Specialization of oleosins in oil body dynamics during seed development in Arabidopsis seeds. Plant Physiol 164(4): 1866-1878.
  2. Pierre, S. (2003). Morphological image analysis: Principles and Applications. 2nd edition. Springer.

简介

油体(OB)是种子特异性脂质储存细胞器,其允许中性脂质的积累,其在萌芽发生后维持小植物发育。 使用荧光染料和共聚焦显微镜,我们监测在不同发育阶段的活拟南芥(拟南芥)胚胎中的OB的动力学(Miquel等人,2014)。 图像采集之后,在四个维度( x , y , z )中种子发育期间的OB大小和分布的详细统计分析 t )。

关键字:油体, 胚胎, 尺寸, 成像, 统计分析

材料和试剂

  1. 植物材料
    1. 拟南芥植物,野生型或突变体或转基因植物
    2. 授粉后6至11天开发长角果 植物在温室中在以下条件下生长(13小时 光照,日间温度为25℃,夜间温度为17℃ ℃),每周用矿物质营养液灌溉两次。
  2. 尼罗红(中性脂质染色,在丙酮中最终为2μg/ml)(Sigma-Aldrich,目录号:72485)

设备

  1. 共焦显微镜
    注意:在本研究中,使用装备有HCX PL APO CS 40×1.25物镜和多线氩激光器的倒置LEICA SP2-AOBS光谱共聚焦激光显微镜(Leica Microsystems)。
  2. 镊子(Dumont No.5)(Sigma-Aldrich)
  3. Scalpel(11 P,刀片)(Swann Norton)
  4. 玻璃载玻片或玻璃底盘,盖玻片
  5. 双眼(尼康公司,型号:SMZ1000)(Champigny sur Marne带有led环 - 照明 TM 用于照明)

软件

  1. ND-SAFIR( http://serpico.rennes.inria .fr/doku.php?id = software:nd-safir:index
  2. AVIZO ® FIRE( http://www.vsg3d.com/avizo/fire

程序

  1. 样品准备
    1. 删除发育中的种子,从2-3长角双体下与a 锋利的手术刀和镊子(DUMONT N°5),并将它们放在载玻片上 或在玻璃底盘中
    2. 添加一个小卷(介于20和30之间 μl)尼罗红溶液并盖上盖玻片。 删除胚胎 从种子的表面,通过轻轻地压在幻灯片和之间的种子 盖玻片与手柄的手柄的圆形端部并观察   在室温下在黑暗中温育30分钟后(见视频 1)。

      视频1.剖析拟南芥发育长角果的胚胎
                                                                                                                                                                                                                                            <! - [if!IE]> - > <! - <![endif] - >                                                                                                                                                                                                                                                          

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      获取Adobe Flash Player

      <! - [if!IE]> - >
      <! - <![endif] - >
                                                                                                                                                                                                                       <! - [if!IE]> - > <! - <![endif] - >                                                                                                                                                                                                                                                                                   

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      获取Adobe Flash Player

      <! - [if!IE]> - >
      <! - <![endif] - >
      注意:您可以使用60%(v/v)甘油溶液或0.4%(w/v)的低浓度 熔化琼脂糖作为封固剂,为您的染色溶液更好 固定胚胎长期观察期。

  2. 共焦显微镜
    1. 找到合适的胚胎,并使用40倍的目标调整焦点在子叶边缘
    2. 设置光学参数以优化信噪比。
      1. 激发激光:氩激光设置为488nm的10%,用于尼罗红激发
      2. 550至650nm的发射带。
      3. 拍摄图像(90到100个厚度为0.16μm的光学切片)   具有40x/1.25油镜物镜和8x数字变焦。 每个部分 在512×512像素的分辨率下进行的两次扫描的平均值   在 x , y , z 参考中的空间分辨率为0.09mm×0.09mm×0.16μm。 注意:
        1. 尼罗河红染色很高 当与中性脂质相关时是荧光的。 不过你可以 如果使用太高的激光功率激发,则漂白它。 根据我们   经验,将激光功率降低到最小值和激光 激发到10%的激光功率足够好,可以进行优良的观察   并保存荧光最多一晚。
        2.  作为 荧光信号通常很强,可以减少 针孔尺寸为0.7透气以减小光学厚度 部分。
        3. 增加扫描速度以及使用双向扫描模式将加快采集速度。

  3. 定量分析
    1. 从3个不同的胚胎在类似的位置获取图像进行统计分析
    2. 使用以下管道分析图像:
      1. 粗图像堆叠存在干扰的几个特性 直接自动计数油体(图1)。规避 这个问题,图像分割程序通过分水岭和 数据提取在AVIZO ® FIRE中在半自动化中实施 管道。在TCL中开发了一个特定的模块(WshedBinSeparate)  语言(工具命令语言)。程序的大纲是 如图2所示。电影显示操作序列(视频 2)。


        图1.具有各种问题的图像堆栈的2D/3D视图 防止图像分析中的直接使用。 A.具有高噪声的图像;乙。 尺寸不均匀的物体; C.可变灰度级; D.对象  触摸边缘
        A

        B

        C

        图2.图像分析流程 A.使用双阈值生成二进制图像; B.产生水印图像; C.对象的单独标记和3D渲染。 Avizo中的分段链(左)和生成的图像(右)。

        视频2. OB图像处理管道
                                                                                                                                                                                                                         <! - [if!IE]> - > <! - <![endif] - >                                                                                                                                                                                                                                                                                   

        要播放视频,您需要安装较新版本的Adobe Flash Player。

        获取Adobe Flash Player

        <! - [if!IE]> - >
        <! - <![endif] - >
                                                                                                                                                                                                                         <! - [if!IE]> - > <! - <![endif] - >                                                                                                                                                                                                                                                          

        要播放视频,您需要安装较新版本的Adobe Flash Player。

        获取Adobe Flash Player

        <! - [if!IE]> - >
        <! - <![endif] - >
      2. WshedBinSeparate模块允许通过分水岭方法分割油体图像堆栈。
      3. 输入图像是灰度级的3D堆栈。 图像是第一 使用ND-SAFIR使用高斯滤波器去噪声。 两个阈值 然后用手动指示的阈值执行操作 由操作者。 通过各种视觉测试来选择最佳值 阈值。
      4. 高阈值允许检测 大对象,而不必担心过高或低估它们,但是 而不考虑小物体。
      5. 低阈值允许   检测小物体而不用担心过度或 大对象的低估。 图像被合并 产生阈值"二进制图像"。
      6. 3D距离变换 (对于每个像素给出一个值   图像根据其到最近对象的边界的距离 附近)。
      7. 距离函数的最大值   "标记图像"即分割的准球面的中心 目的。 距离变换(CDT)的补码,带有标记 图像用于创建"分水岭线":分水岭算法 扩展标记(如充气气球)朝向增加的值 的CDT。 分水岭线表示分隔的边界 对象。 最后,从二进制中减去分水岭线 图像以获得分离的和个性化的对象("分割的" 图片")。 分离的对象的图像然后被转换为扩展 其中所述电子表格中的每个项目是标记的对象 卷。

致谢

这项工作由INRA资助,特别是由G. Trigui博士的BAP(植物生物学和育种)和MIA(应用数学和信息学)部门资助。 这项工作改编自Miquel等人(2014)。

参考文献

  1. Miquel,M.,Trigui,G.,d'Andrea,S.,Kelemen,Z.,Baud,S.,Berger,A.,Deruyffelaere,C.,Trubuil,A.,Lepiniec,L.and Dubreucq,B (2014)。 拟南芥种子种子发育过程中油体动力学中油质蛋白的专业化。 Plant Physiol 164(4):1866-1878。
  2. Pierre,S。(2003)。 形态学图像分析:原理和应用。 2 nd 版本。 Springer。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Miquel, M., Trigui, G., Trubuil, A. and Dubreucq, B. (2015). Imaging and Quantitative Analysis of Size and Distribution of Spherical Bodies, e.g. Embryonic Oil Bodies. Bio-protocol 5(1): e1369. DOI: 10.21769/BioProtoc.1369.
  2. Miquel, M., Trigui, G., d'Andrea, S., Kelemen, Z., Baud, S., Berger, A., Deruyffelaere, C., Trubuil, A., Lepiniec, L. and Dubreucq, B. (2014). Specialization of oleosins in oil body dynamics during seed development in Arabidopsis seeds. Plant Physiol 164(4): 1866-1878.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。