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Using Silicon Polymer Impression Technique and Scanning Electron Microscopy to Measure Stomatal Aperture, Morphology, and Density
使用硅聚合物印模技术和扫描电子显微镜技术测量气孔的开度、形态和密度   

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Abstract

The number of stomata on leaves can be affected by intrinsic development programming and various environmental factors, in addition the control of stomatal apertures is extremely important for the plant stress response. In response to elevated temperatures, transpiration occurs through the stomatal apertures, allowing the leaf to cool through water evaporation. As such, monitoring of stomata behavior to elevated temperatures remains as an important area of research. The protocol allows analysis of stomatal aperture, morphology, and density through a non-destructive imprint of Arabidopsis thaliana leaf surface. Stomatal counts were performed and observed under a scanning electron microscope.

Keywords: Arabidopsis thaliana(拟南芥), Heat stress(热胁迫), Non-destructive imprint(非破坏性印记), Stomata(气孔), Scanning electron microscope(扫描电子显微镜检查)

Background

The different techniques have been explored to study stomatal density and patterns in variety of plants. It can be broadly grouped into two classes including direct observation of fresh materials and the preparation of replicas, or castings (Gitz and Baker, 2009). These methods have included the use of clear nail varnish, which is a traditional method used to analyze stomata density, however, surfaces of leaves can be damaged by the solvent in the nail varnish. Here we use a non-destructive imprint of Arabidopsis thaliana leaves for stomata visualization, which can directly observe freshly collected materials and assess stomata density for sequential measurements. Additionally, it can simplify the observation and the measurement of stomatal density, and facilitate the analysis of several lines or different treatments in parallel.

Materials and Reagents

  1. Arabidopsis thaliana
  2. Glass slide
  3. Lens cleaning tissue (GE Healthcare, Whatman, catalog number: 2105-862 )
  4. Toothpick
  5. Double-sided tape
  6. Dental silicon (vinyl polysiloxane; VPS) impression materials: EXAFINE VPS impression material injection type, with low viscosity (GC America, catalog number: 138120 )
    Note: Two components–base (A) and catalyst (B).
  7. High strength 5-min Epoxy gel (ITW Devcon, catalog number: 14210 )
    Note: Two components–resin (C) and hardener (D).
  8. Ultrafine threads used in ophthalmologic surgery (Fine Science Tools, catalog number: 18020-03 )

Equipment

  1. Forceps (Fine Science Tools, catalog number: 00108-11 )
  2. Forced convection oven (YIHDER Technology, model: DK-600D )
  3. Stereo microscope (ZEISS, model: Stemi DV4 )
  4. Scanning electron microscopy (SEM; Inspect S, FEI)
  5. Sputtering coater (EIKO Engineering, model: IB-2 )

Software

  1. ImageJ software (http://rsb.info.nih.gov/ij)

Procedure

  1. Select leaves for observation
    1. 3- to 4-week-old mature, fully expanded and flat Arabidopsis leaves.
    2. The leaves at same developmental stage were collected after treatments immediately for use.
      Note: Thermotolerance test as described previously by Huang et al. (2017).
    3. Gently and thoroughly remove any moisture and any other surface contamination with a piece of lens cleaning tissue from the sample tissues.
      Note: Do not wipe it.

  2. Impression mold preparation
    1. The silicone impression material was applied on the lower surface (abaxial side) of leaves to obtain the negative impression mold (Figure 1A).
      1. Mix two equal-volume pea size drops of the dental polymer pastes [A (base) + B (catalyst)] with a toothpick on a glass slide.
        Note: This step should be completed in less than 10 sec to avoid the impression material to thicken.
      2. As soon as possible, apply a small amount of the polymer to the leaf surface using a toothpick, and allowed it to harden for 3 to 5 min.
        Note: The silicone forms a mold that is less than 1/2 inch.
      3. When the polymer is not sticky, carefully remove the mold with a toothpick by gently pushing at one of the edges until the mold gradually peels away, do not attempt to remove it completely in one go.
        Note: If experiencing difficulties, introduce a small amount of water under the edge of the mold to help loosen it from the leaf.
      4. Place the mold upside down on double-sided tape on a glass slide, and examine under a dissection microscope to check for air bubbles and foreign particles that might obstruct analysis.
        Note: Orientating the mold horizontally ensures the Epoxy applied in the next step will not flow out.


        Figure 1. Silicon polymer impression technique and scanning electron microscopy (SEM) to measure stomatal apertures. A. (a) Mix silicon polymer (reagents A + B) with a toothpick on a slide; (b) Apply on the lower surface of leaf; (c) Negative impression mold made with silicon polymers; (d) The mold seated on a double-sided tape on top of the glass slide. B. (a) Mix Epoxy gel (reagents C + D) with a toothpick; (b) Fill the impression mold with freshly mixed Epoxy gel; (c) Thoroughly harden the Epoxy gel at room temperature (RT) for overnight, or in a 60 °C oven for 1 h; (d) Once hardened, observe using SEM examination. C. The example of image was taken using an SEM. The round shapes on the SEM picture could be air bubbles and foreign particles.

  3. Cast preparation
    1. Fill the top of the impression mold with an Epoxy gel to prepare the cast (Figure 1B).
      1. Mix directly two equal-volume pea size drops of the Epoxy gel [C (resin) + D (hardener)] on a glass slide using a toothpick. Air bubbles that were introduced during mixing need to be removed with the toothpick.
      2. After the gel becomes viscous (it usually takes 10 sec), apply to the surface of the mold.
        Note: Make sure that the resin is not too thin, and applying additional amounts if necessary. For easily removing the cast from the mold, you can attach ultrafine threads at the edge of the cast if necessary (see Kwiatkowska and Burian, 2014).
      3. Leave the molds filled with Epoxy at room temperature overnight or in a 60 °C oven (not under vacuum) for 1 h to thoroughly harden the cast.
      4. Gently remove the resin using forceps, and examine first under the dissection microscope, then place on a SEM stub.

  4. SEM observation
    1. The cast was fixed on metal stubs, and coated with a thin gold-palladium (1:1) film to 3 min in an automated sputter coater with a rotating stage.
    2. Follow the SEM manual to visualize the cast.
      1. Setting the parameters for stomata visualization.
        Note: Non-biological samples, it is not necessary to consider the loss of water, the intensity of electron flow, etc., so there are no special conditions on the setting.
      2. Setting high accelerating voltage to 15 kV (as a guideline) and photographed at 500x magnification.
        Note: Less resolution and noise images may occur at low accelerating voltages. Due to non-plane of stomata has the existence of an angle, try to acquire images with cells within the focal plane to avoid error of measurement.

Data analysis

All images were saved in TIFF format, and stomatal apertures were analyzed using the ImageJ software. Stomatal density is determined by counting the number of stoma per unit of leaf surface area. The width and the length of the stomatal aperture were measured. Three leaves per each treatment at different time points were used, at least 150 stomata per leaf were measured for statistical analysis (Huang et al., 2017). See the details in Figure 2.


Figure 2. The measurement of stomata aperture using the image processing software ImageJ. The basic procedures of image analysis are showing step-by-step for analyzing the objects in the SEM image. Step 1. Download and Run ImageJ program; Step 2. Open stomata image via select File, Open sample and click OK; Step 3. For image processing: (a) Convert image to 8-bit grayscale mode, and then (b) Crop a meaningful part of the image (draw a rectangular shape). (c) It can use a sharpened version of the image before the threshold setting. (d) The threshold new image of stomata was chosen by manually defining the histogram. (e) and (f) Process binary to make black and white images. To refine shapes of samples in this step. (g) The particle size range (minimum size and maximum pixel area size) can be determined for your objects of interest, and therefore to exclude anything that is not interested in the image. Step 4. Results window opens automatically.

Acknowledgments

The protocol presented here was based on previous work of William and Green (1998), and Kwiatkowska and Burian (2014). The authors are grateful to Dr. Chin-Ho Lin (National Chung Hsing University) for his helpful advice on this impression technique, and Lynne Stracovsky for English editing. This work was supported by grants from National Taiwan University (101 to 105R892003, and 105R89203) and Ministry of Science and Technology, Taiwan (102-2311-B-002-031, 103-2311-B-002-008 and 104-2311-B-002-007) to T.L.J.

References

  1. Gitz, D. C., and Baker, J. T. (2009). Methods for creating stomatal impressions directly onto archivable slides. Agron J 101: 232-236.
  2. Huang, Y. C., Wu, H. C., Wang, Y. D., Liu, C. H., Lin, C. C., Luo, D. L. and Jinn, T. L. (2017). PECTIN METHYLESTERASE34 contributes to heat tolerance through its role in promoting stomatal movement. Plant Physiol 174: 748-763.
  3. Kwiatkowska, D. and Burian, A. (2014). Sequential replicas for in vivo imaging of growing organ surfaces. Methods Mol Biol 1080: 99-110.
  4. William, M. H. and Green, P. B. (1988). Sequential scanning electron microscopy of a growing plant meristem. Protoplasma 147: 77-79.

简介

叶片上的气孔数量可能受到内在发育规划和各种环境因素的影响,此外,气孔开孔的控制对植物胁迫反应极为重要。 响应于升高的温度,通过气孔开孔发生蒸腾,允许叶子通过水分蒸发而冷却。 因此,将气孔行为监控到升高的温度仍然是研究的重要领域。 该方案允许通过拟南芥叶片表面的非破坏性印记分析气孔孔径,形态和密度。 在扫描电子显微镜下进行气孔计数并观察。
【背景】已经探索了不同的技术来研究各种植物的气孔密度和模式。 它可以大致分为两类,包括直接观察新鲜材料和制备复制品,或铸件(Gitz和Baker,2009)。 这些方法包括使用透明的指甲油,这是用于分析气孔密度的传统方法,然而,叶子的表面可能被指甲油中的溶剂损坏。 在这里,我们使用拟南芥叶片的非破坏性印记进行气孔可视化,其可以直接观察新鲜收集的材料并评估气孔密度用于顺序测量。 此外,它可以简化气孔密度的观察和测量,并且有助于并行分析几条线或不同的处理。

关键字:拟南芥, 热胁迫, 非破坏性印记, 气孔, 扫描电子显微镜检查

材料和试剂

  1. 拟南芥
  2. 玻璃幻灯片
  3. 镜片清洁纸(GE Healthcare,Whatman,目录号:2105-862)
  4. 牙签
  5. 双面胶带
  6. 牙科硅(乙烯基聚硅氧烷; VPS)印模材料:EXAFINE VPS印模材料注射型,粘度低(GC美国,目录号:138120)
    注意:两个组分 - 基(A)和催化剂(B)。
  7. 高强度5分钟环氧胶(ITW Devcon,目录号:14210)
    注意:两种组分 - 树脂(C)和硬化剂(D)。
  8. 用于眼科手术的超细线(Fine Science Tools,目录号:18020-03)

设备

  1. 镊子(精细科学工具,目录号:00108-11)
  2. 强制对流烤箱(YIHDER技术,型号:DK-600D)
  3. 立体显微镜(ZEISS,型号:Stemi DV4)
  4. 扫描电子显微镜(SEM;检查S,FEI)
  5. 溅射涂布机(EIKO Engineering,型号:IB-2)

软件

  1. ImageJ软件( http://rsb.info.nih.gov/ij

程序

  1. 选择叶观察
    1. 3至4周龄的成熟,完全扩张和平坦的拟南芥叶片。
    2. 在处理后立即收集相同发育阶段的叶子使用。
      注意:如Therm等人先前所述的耐热测试(2017)。
    3. 轻轻地彻底清除样品组织中的一块镜片清洁组织中的任何水分和任何其他表面污染物。
      注意:不要擦拭。

  2. 印模准备
    1. 将硅印模材料施加在叶片的下表面(背面)上以获得负模具(图1A)。
      1. 用玻璃滑块上的牙签将两个等体积的豌豆大小的牙科聚合物糊剂[A(底物)+ B(催化剂)]滴下来。
        注意:此步骤应在不到10秒内完成,以避免印象材料变稠。
      2. 尽快使用牙签将少量的聚合物涂在叶面上,使其硬化3〜5分钟。
        注意:硅胶形成的模具小于1/2英寸。
      3. 当聚合物不粘时,请用牙签轻轻取出模具,轻轻推入其中一个边缘,直到模具逐渐脱落,不要一次完全移除。
        注意:如果遇到困难,请在模具边缘引入少量水以帮助将其从叶子上松开。
      4. 将模具倒置在玻璃载玻片上的双面胶带上,并在夹层显微镜下检查以检查可能阻碍分析的气泡和异物。
        注意:水平定位模具确保下一步中应用的环氧树脂不会流出。


        图1.硅聚合物印模技术和扫描电子显微镜(SEM)来测量气孔孔。A.(a)将硅聚合物(试剂A + B)与载玻片上的牙签混合; (b)施用于叶的下表面; (c)用硅聚合物制成的负压模具; (d)模具坐在载玻片顶部的双面胶带上。 B.(a)将环氧胶(试剂C + D)与牙签混合; (b)用新混合的环氧胶填充印模; (c)在室温(RT)下将环氧凝胶彻底硬化过夜,或在60℃烘箱中持续1小时; (d)硬化后,用SEM检查。 C.使用SEM拍摄图像的例子。 SEM图片上的圆形可能是气泡和异物。

  3. 铸造准备
    1. 用环氧凝胶填充压模的顶部以制备铸件(图1B)。
      1. 使用牙签将两个等体积的豌豆大小的环氧树脂凝胶[C(树脂)+ D(硬化剂)]滴在玻片上。在混合过程中引入的气泡需要用牙签去除。
      2. 凝胶变得粘稠(通常需要10秒)后,涂于模具表面。
        注意:确保树脂不要太薄,如有必要,额外添加量。为了方便地从模具中移除铸件,您可以在铸件的边缘附加超细线(见Kwiatkowska和Burian,2014)。
      3. 将室温下放置环氧树脂的模具放置过夜或在60℃烘箱中(不在真空下)1小时以彻底硬化铸件。
      4. 使用镊子轻轻取出树脂,首先在夹层显微镜下检查,然后放在SEM存根上
  4. SEM观察
    1. 将铸件固定在金属短棒上,并在具有旋转台的自动溅射涂布机中用薄金钯(1:1)薄膜涂覆3分钟。
    2. 按照SEM手册可视化演员表。
      1. 设置气孔可视化参数。
        注意:非生物样品,没有必要考虑水的损失,电子流动的强度等,所以没有特殊的条件设置。
      2. 将高加速电压设置为15 kV(作为指导),并以500倍放大倍数拍摄。
        注意:较低的分辨率和噪点图像可能会在低加速电压下发生。由于非平面的气孔有角度的存在,请尝试用焦平面内的细胞获取图像,以避免测量误差。

数据分析

所有图像均以TIFF格式保存,并使用ImageJ软件分析气孔。通过计算每单位叶面积的造口数确定气孔密度。测量气孔孔的宽度和长度。使用不同时间点每次处理三叶,每叶至少150个气孔进行统计分析(Huang et al。,2017)。请参见图2中的详细信息

图2.使用图像处理软件ImageJ测量气孔孔径图像分析的基本步骤正在逐步显示,用于分析SEM图像中的对象。步骤1.下载并运行ImageJ程序;步骤2.通过选择文件打开气孔图像,打开样品,然后单击确定;步骤3.对于图像处理:(a)将图像转换为8位灰阶模式,然后(b)裁剪图像的有意义部分(绘制矩形)。 (c)在阈值设置之前,可以使用图像的锐化版本。 (d)通过手动定义直方图来选择气孔的阈值新图像。 (e)和(f)处理二进制以制作黑白图像。在此步骤中细化样品的形状。 (g)可以为您感兴趣的对象确定粒度范围(最小大小和最大像素面积大小),因此排除图像中不感兴趣的任何内容。步骤4.结果窗口自动打开。

致谢

这里提出的方案是基于William和Green(1998)和Kwiatkowska和Burian(2014年)的以前的工作。作者非常感谢Chin-Ho Lin博士(国立中兴大学)对这种印象技术的有益建议,以及Lynne Stracovsky进行英语编辑。这项工作得到了台湾大学(101〜105R892003,105R89203)和台湾科技部(102-2311-B-002-031,103-2311-B-002-008和104-2311)的资助。 -B-002-007)到TLJ

参考

  1. Gitz,DC和Baker,JT(2009)。将气孔曝光直接制成可归档幻灯片的方法。 Agron J 101:232-236。
  2. Huang,YC,Wu,HC,Wang,YD,Liu,CH,Lin,CC,Luo,DL和Jinn,TL(2017)。 PECTIN METHYLESTERASE34通过其促进气孔运动的作用有助于耐热性。植物生理学174:748- 763.
  3. Kwiatkowska,D.和Burian,A.(2014)。 生长器官表面的体内成像的顺序复制物。方法Mol Biol 1080:99-110。
  4. William,MH和Green,PB(1988)。 顺序扫描电子生长中的植物分生组织的显微镜。原生质体 147:77-79。
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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. Wu, H., Huang, Y., Liu, C. and Jinn, T. (2017). Using Silicon Polymer Impression Technique and Scanning Electron Microscopy to Measure Stomatal Aperture, Morphology, and Density. Bio-protocol 7(16): e2449. DOI: 10.21769/BioProtoc.2449.
  2. Huang, Y. C., Wu, H. C., Wang, Y. D., Liu, C. H., Lin, C. C., Luo, D. L. and Jinn, T. L. (2017). PECTIN METHYLESTERASE34 contributes to heat tolerance through its role in promoting stomatal movement. Plant Physiol 174: 748-763.
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