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Centrifuge Microscopy to Analyze the Sedimentary Movements of Amyloplasts
离心显微镜检查淀粉体的沉淀   

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Abstract

A centrifuge microscope (CMS) functionally consists of a centrifuge producing a centrifugal force (hypergravity condition) and a microscope making an enlarged image of an object. This combination of equipment allows live-cell imaging during centrifugation. We have developed a new CMS (NSK Ltd.) to observe movements of the plant organelles such as amyloplasts, under hypergravity conditions (Toyota et al., 2013). This CMS is distinct from previously designed CMSs in terms of spatio-temporal resolution, ease of use and compactness. Here, we show a quick protocol to prepare a specimen of Arabidopsis inflorescence stem, use the CMS, obtain imaging data and analyze them using a single tracking method.

Materials and Reagents

  1. Arabidopsis thaliana inflorescence stems
  2. MS salt mixture (Wako Pure Chemical Industries, catalog number: 392-00591 )
  3. 1% (w/v) sucrose
  4. 0.05% (w/v) MES
  5. 0.1% (w/v) agar
  6. Growth media (see Recipes)

Equipment

  1. Fine tweezers
  2. Scissors
  3. Razor blade (Electron Microscopy Sciences, catalog number: 72000 )
  4. Kimwipes
  5. Aluminum chamber (custom built) (NSK Ltd.)
  6. Silicone rubber (thickness: 0.5 mm) (AS ONE Corporation, catalog number: 6-611-01 )
  7. Round cover glass (diameter: 12 mm) (Matsunami Glass, catalog number: CO12001 )
  8. CMS system (Figure 1, not commercially available) (NSK Ltd., http://www.nsk.com/)
    CMS is a newly designed compact centrifuge microscope, 30 cm in height and 20 cm in diameter. CMS consists of a direct-drive motor (NSK Ltd., MEGATORQUE MOTOR™, model: M-PS1006KN002 ) and optics including a 50x objective lens with a working distance of 18 mm (SLMPLN 50x, 0.35 NA, OLYMPUS), LED light source (SCHOTT MORITEX Corporation, model: MEBL-CW25 ) and a CCD camera (SENSOR TECHNOLOGY, model: STC172C ).


    Figure 1. Overview of the CMS system

  9. Windows computer [minimum computer requirements: Windows® XP or later, Pentium M 778 (1.6 GHz), RAM 1024 MB or higher]
    Note: To install the software below, Windows computers are highly recommended.

Software

  1. MEGATORQUE MOTOR™ controller (EDC megaterm software) (NSK Ltd., http://www.nsk.com/)
    Note: This is free software to control the motor and is available only for Windows.
  2. Video capture software (COREL, http://www.corel.com/)
    Note: You can use any video capture soft/hardware that converts analog video signal into digital signal and stores the data in a computer.
  3. G-Track spot-tracking software (G-Angstrom, http://www.g-angstrom.com/eng/products/index.php)
    Note: This is a piece of commercial software to trace fluorescence/bright spots and available only for Windows.

Procedure

  1. Excise an approximately 1-cm-long segment of an inflorescence stem at 1-2 cm from the apex of the primary stem.
  2. Place the stem segment onto a hand-made stem holder and slide a razor blade to split the stem longitudinally (Figure 2) (Saito et al., 2005; Nakamura et al., 2011).


    Figure 2. Making longitudinal sections of Arabidopsis inflorescence stems. A. Place the stem segment onto a hand-made stem holder. B. Close the holder and slide a razor blade in the direction of the arrow. C. Open the holder and retrieve the longitudinally-sectioned stem segments.

  3. Drop a small amount of growth medium onto the sectioned side of the stem to prevent the tissue from drying out.
  4. Keep the sectioned side up and put this segment into a slit in a round silicone rubber sheet (0.5 mm deep) on the bottom glass of an aluminum chamber (Toyota et al., 2013).
  5. Pour growth medium into this slit, put a cover glass onto the silicone rubber and remove spilt growth media with Kimwipes.
  6. Mount the aluminum chamber in a holder under an objective lens of the CMS.
  7. Turn on the LED light to illuminate the specimen and the CCD camera to acquire images.
  8. Acquired bright-field images are transmitted through a radio system and shown on the video capture software in the computer.
  9. Adjust focus and field of view in the CMS while monitoring the computer.
  10. Set a centrifugal acceleration between 0 to 33 x g in the MEGATORQUE MOTOR™ controller of the computer. In case of wild-type Arabidopsis stems, maximum gravitropic responses are seen at 10 x g.
  11. Start video capture and run MEGATORQUE MOTOR™ (Videos 1 and 2).

    Video 1. Side view of the CMS during operation

    Video 2. Top view of the CMS during operation

  12. Monitor real-time images (30 frames per sec) on the computer during centrifugation.
  13. Stop the motor and video capture and save the images as an avi file in the computer.
  14. Open this file in G-Track spot-tracking software.
  15. This software automatically recognizes many white or black spots (amyloplasts) and traces them (Video 3; Figure 2). If necessary, you can modify image parameters such as gain or contrast, and invert brightness.

    Video 3. Single-particle tracking analysis of amyloplast movement during centrifugation at 10 x g (Toyota et al., 2013). Most amyloplasts are automatically recognized by the G-Track spot-tracking software and traced while they are recognized as white or black spots. Please note that this software does not precisely recognize an amyloplast (spot) with weak contrast nor aggregated amyloplasts. Video duration = 152 s (8 x speed).

  16. Run the tracking program. You can automatically get data [i.e., mean square displacement (MSD) of an amyloplast, Table 1] and then calculate velocity and displacement.
  17. Export the data to Excel/CSV file (Table 1).

Representative data


Figure 3. Representative tracking image (Toyota et al., 2013). Movement of an amyloplast (arrow head) is successfully traced by the G-Track spot-tracking software during centrifugation.

Table 1. Mean square desplacement (MSD) of the amylplast for 1 sec of centrifugation. MSD of the amyloplast traced in Figure 3 is automatically calculated by the tracking program. X, Y and 2D denote movement in the horizontal (10 x g) and vertical directions and in a two-dimensional (2D) plane, respectively. For downloading data, please click the image below.

Recipes

  1. Growth media (pH 5.1)
    1x MS salts
    1% (w/v) sucrose
    0.05% (w/v) MES
    0.1% (w/v) agar

Acknowledgments

We thank Professor T. Mimura (Kobe University), Professor Y. Yoshimoto (Kansai Medical University) and Professor T. Shimmen (University of Hyogo) for valuable information about centrifuge microscopes and Y. Ishibashi for technical assistance. This work was supported in part by TOYOBO Biotechnology Foundation (to M. Toyota); Grant-in-Aid for JSPS Fellows (to M. Toyota), for JSPS Fellows for Research Abroad (to M. Toyota) and for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (16085205 to M.T.M.); and grants from the Bioarchitect Project of RIKEN (to M.T.M.) and PREST (to M. Toyota and M.T.M.).

References

  1. Nakamura, M., Toyota, M., Tasaka, M. and Morita, M. T. (2011). An Arabidopsis E3 ligase, SHOOT GRAVITROPISM9, modulates the interaction between statoliths and F-actin in gravity sensing. Plant Cell 23(5): 1830-1848.
  2. Saito, C., Morita, M. T., Kato, T. and Tasaka, M. (2005). Amyloplasts and vacuolar membrane dynamics in the living graviperceptive cell of the Arabidopsis inflorescence stem. Plant Cell 17(2): 548-558.
  3. Toyota, M., Ikeda, N., Sawai‐Toyota, S., Kato, T., Gilroy, S., Tasaka, M. and Morita, M. T. (2013). Amyloplast displacement is necessary for gravisensing in Arabidopsis shoots as revealed by a centrifuge microscope. Plant J 76(4): 648-660.

简介

离心显微镜(CMS)在功能上包括产生离心力(超重力条件)的离心机和使物体放大的显微镜。 这种设备的组合允许离心期间的活细胞成像。 我们已经开发了一种新的CMS(NSK有限公司)观察植物细胞器的运动,如在超重力条件下的淀粉体(Toyota et al。,2013)。 该CMS与先前设计的CMS在空间 - 时间分辨率,易用性和紧凑性方面不同。 在这里,我们显示准备拟南芥花序茎的标本的快速方案,使用CMS,获得成像数据并使用单一跟踪方法分析它们。

材料和试剂

  1. 拟南芥 花序茎
  2. MS盐混合物(Wako Pure Chemical Industries,目录号:392-00591)
  3. 1%(w/v)蔗糖
  4. 0.05%(w/v)MES
  5. 0.1%(w/v)琼脂
  6. 生长培养基(参见食谱)

设备

  1. 精细镊子
  2. 剪刀
  3. 剃刀刀片(Electron Microscopy Sciences,目录号:72000)
  4. Kimwipes
  5. 铝室(定制)(NSK有限公司)
  6. 硅橡胶(厚度:0.5mm)(AS ONE Corporation,目录号:6-611-01)
  7. 圆形盖玻璃(直径:12mm)(Matsunami Glass,目录号:CO12001)
  8. CMS系统(图1,不可商购)(NSK有限公司, http://www.nsk.com/
    CMS是一种新设计的紧凑型离心显微镜,高30厘米,直径20厘米。 CMS由直接驱动电动机(NSK有限公司,MEGATORQUE MOTOR TM,型号:M-PS1006KN002)和包括工作距离为18mm的50倍物镜(SLMPLN 50x,0.35NA,OLYMPUS)的光学器件,LED光源(SCHOTT MORITEX公司,型号:MEBL-CW25)和CCD照相机(SENSOR TECHNOLOGY,型号:STC172C)。

    图1. CMS系统概述

  9. Windows计算机[最低计算机要求:Windows ® XP或更高版本,Pentium M 778(1.6 GHz),RAM 1024 MB或更高版本]
    注意:要安装以下软件,强烈建议使用Windows计算机。

软件

  1. MEGATORQUE MOTOR™控制器(EDC大型软件)(NSK有限公司, http://www.nsk.com/
    注意:这是用于控制电机的免费软件,仅适用于Windows。
  2. 视频捕获软件(COREL, http://www.corel.com/
    注意:您可以使用将模拟视频信号转换为数字信号并将数据存储在计算机中的任何视频捕获软件/硬件。
  3. G-Track跟踪软件(G-Angstrom, http://www.g -angstrom.com/eng/products/index.php
    注意:这是一个用于跟踪荧光/亮点的商业软件,仅适用于Windows。

程序

  1. 在距主要茎干顶点1-2厘米处取消大约1厘米长的花序茎节。
  2. 将茎段放置在手工茎支架上,并滑动剃刀刀片以纵向分裂茎(图2)(Saito等人,2005; Nakamura等人 >,2011)。


    图2.制作拟南芥花序茎的纵向部分。 A。将茎段放置在手工制作的茎柄上。 B.关闭夹具并沿箭头方向滑动剃须刀刀片。 C 。打开支架并取回纵向切片的茎段。

  3. 将少量生长培养基滴在茎干的切片侧,以防止组织变干
  4. 保持剖切面朝上,并将此段放入铝制室底部玻璃上的圆形硅橡胶片(0.5mm深)的狭缝中(Toyota等人,2013年)。
  5. 将生长介质倒入该狭缝,将盖玻片放在硅橡胶上,用Kimwipes清除溢出的生长培养基。
  6. 将铝室安装在CMS的物镜下的支架中。
  7. 打开LED灯照亮样品和CCD相机获取图像。
  8. 获取的明场图像通过无线电系统传输并显示在计算机的视频捕获软件上。
  9. 在监视计算机时调整CMS中的焦点和视野。
  10. 在计算机的MEGATORQUE MOTOR™控制器中设置0到33 em之间的离心加速度。 在野生型拟南芥茎的情况下,最大引力反应见于10×g 。
  11. 开始视频捕获并运行MEGATORQUE MOTOR™(视频1和2)。

    视频1.操作期间CMS的侧视图
                                       <! - [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] - >

    视频2.操作期间CMS的顶视图
                                          <! - [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] - >

  12. 在离心过程中在计算机上监视实时图像(每秒30帧)。
  13. 停止电机和视频捕获,并将图像保存为计算机中的avi文件。
  14. 在G-Track点跟踪软件中打开此文件。
  15. 此软件自动识别许多白色或黑色斑点(淀粉体)并跟踪它们(视频3;图2)。如果需要,您可以修改图像参数,如增益或对比度,以及反转亮度
    视频3.离心10分钟后 (Toyota ,2013)的淀粉体运动的单粒子跟踪分析。 点跟踪软件和跟踪,而他们被认为是白色或 黑点。请注意,此软件不能精确识别  具有弱对比度的淀粉体(斑点),也不是聚集的淀粉体。 视频时长= 152秒(8倍速)。
                                       <! - [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] - >

  16. 运行跟踪程序。您可以自动获取数据[即,一个淀粉体的平均位移(MSD),表1],然后计算速度和位移。
  17. 将数据导出到Excel/CSV文件(表1)。

代表数据


图3.代表性跟踪图像(Toyota等人,2013年)。由G-Track点跟踪软件成功追踪淀粉体(箭头)的移动离心
表1.淀粉质离心1秒的平均平均位移(MSD)。图3中追踪的淀粉体的MSD由跟踪程序自动计算。 X,Y和2D分别表示在水平(10×g)和垂直方向上以及在二维(2D)平面中的移动。要下载数据,请点击下面的图片。

食谱

  1. 生长培养基(pH 5.1)
    1×MS盐
    1%(w/v)蔗糖 0.05%(w/v)MES
    0.1%(w/v)琼脂

致谢

感谢神户大学教授T. Mimura,关西医科大学Yoshimoto教授和兵库大学教授T. Shimmen教授有关离心显微镜的有价值的信息和Y. Ishibashi的技术帮助。 这项工作得到东洋生物基金会(丰田)的部分支持。 JSPS研究员(对M.丰田),JSPS研究员(M.丰田)和科学研究的奖学金 教育,文化,体育,科学和技术部(16085205至M.T.M.);和来自RIKEN的生物架构项目(授予M.T.M.)和PERST(授予M.丰田和M.T.M.)的赠款。

参考文献

  1. Nakamura,M.,Toyota,M.,Tasaka,M。和Morita,M.T。(2011)。 拟南芥E3连接酶,SHOOT GRAVITROPISM9,调节植物细胞 23(5):1830-1848。
    植物细胞
  2. Saito,C.,Morita,M.T.,Kato,T.and Tasaka,M。(2005)。 拟南芥花序的活重免疫细胞中的成纤维细胞和液泡膜动力学茎。植物细胞 17(2):548-558。
  3. Toyota,M.,Ikeda,N.,Sawai-Toyota,S.,Kato,T.,Gilroy,S.,Tasaka,M.and Morita,M.T。 淀粉体移位对于在拟南芥苗中的重力感应是必要的,如通过离心显微镜。植物J [76](4):648-660。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Toyota, M., Ikeda, N., Tasaka, M. and Morita, M. T. (2014). Centrifuge Microscopy to Analyze the Sedimentary Movements of Amyloplasts. Bio-protocol 4(17): e1229. DOI: 10.21769/BioProtoc.1229.
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