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An Arabidopsis leaf blade is composed of many layers that are sandwiched between two layers of tough skin cells (called the epidermis). Four layers (adaxial epidermis, palisade layer, spongy mesophyll and abaxial epidermis) contain specialized cells. Here we describe a quick and simple method for analyzing the size, number and density of different types of cells in an Arabidopsis leaf blade. This method would be of interest to people who would like to investigate cell size and number changs in different cell layers in leaves or leaf-like organs without having to dissect the samples.

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Analyses of Plant Leaf Cell Size, Density and Number, as Well as Trichome Number Using Cell Counter Plugin
采用细胞计数器插件分析植株叶片细胞大小、密度和数量以及表皮毛数量

植物科学 > 植物细胞生物学 > 细胞结构
作者: Yan Cheng
Yan ChengAffiliation: National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a1481
Ling Cao
Ling CaoAffiliation: National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a1482
Sheng Wang
Sheng WangAffiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
Bio-protocol author page: a1483
Yongpeng Li
Yongpeng LiAffiliation: National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a1484
Hong Wang
Hong WangAffiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
For correspondence: hong.wang@usask.ca
Bio-protocol author page: a1485
 and Yongming Zhou
Yongming ZhouAffiliation: National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
For correspondence: ymzhou@mail.hzau.edu.cn
Bio-protocol author page: a1486
Vol 4, Iss 13, 7/5/2014, 7330 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1165

[Abstract] An Arabidopsis leaf blade is composed of many layers that are sandwiched between two layers of tough skin cells (called the epidermis). Four layers (adaxial epidermis, palisade layer, spongy mesophyll and abaxial epidermis) contain specialized cells. Here we describe a quick and simple method for analyzing the size, number and density of different types of cells in an Arabidopsis leaf blade. This method would be of interest to people who would like to investigate cell size and number changs in different cell layers in leaves or leaf-like organs without having to dissect the samples.

[Abstract]

Materials and Reagents

  1. Dissected rosette leaves from Arabidopsis
  2. Petals from the well-opened flower
  3. Ethanol (Sigma-Aldrich, Catalog number: 459844 )
  4. Chloral hydrate (Sigma-Aldrich, Catalog number: 15307 )
  5. Glycerol (Sigma-Aldrich, Catalog number: G7757 )
  6. Washing solution (see Recipes)
  7. Clearing solution (see Recipes)

Equipment

  1. Stereomicroscope (Zeiss, model: Zeiss Stemi 2000 )
  2. Digital Cameral (Canon, model: Canon powershot S5IS )
  3. Glass slides and thin coverslips
  4. Differential interference contrast (DIC) microscope (Nikon Corporation, model: ECLIPSE 80i) with CCD camera (Nikon Corporation, model: DS-Ri1 )

Software

  1. Image J software (http://rsbweb.nih.gov/ij/index.html).
  2. Cell Counter plugin (http://rsbweb.nih.gov/ij/plugins/cell-counter.html)

Procedure

  1. Dissect cotyledons, leaves and petals from plants at the specific stages of study interest.
  2. Measure the areas of the dissected cotyledons and leaves (Figure 1a).


    Figure 1. The images used for area measuring and trichome number counting. (a). Cotyledons laid out on a white surface are shown, bar = 1 cm. The photo was taken with a digital camera. (b). Trichomes on the adaxial surface of an Arabidopsis leaf are shown. The photo was taken with a digital camera through a stereomicroscope.

    1. Label the cotyledons/leaves from the same plant, place the samples on a white surface, and try to keep them flat.
    2. Set a 1 cm scale in the middle (if possible) of the surface and take the photo with a digital camera.
    3. Measure the area of individual cotyledon or leaf blade using Image J software (Flash 1).

    Flash 1. The flash showing the steps of measuring areas of cotyledons in Figure 1a using ImageJ

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

    Get Adobe Flash Player



  3. Trichome number and density.
    1. Take photos of individual leaf with a scale under a stereomicroscope and make sure the trichomes are clear (Figure 1b).
    2. Measure the area of the leaf blade with Image J software (Flash 2) and count the total trichome numbers using Cell Counter plugin in Image J software (Flash 3). 
    3. The trichome densities are calculated from dividing the total cell number of trichomes by leaf area.

      Flash 2. The flash showing the steps of measuring an individual leaf using ImageJ

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

      Get Adobe Flash Player

      Flash 3. The flash showing how to use cell-counter plug-in in ImageJ for counting

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

      Get Adobe Flash Player

  4. Fix the samples by soaking in 70% ethanol overnight; clear off chlorophyll by washing the sample 2~3 times till washing solution (70% ethanol) becomes clear. Each time, soak the sample in the washing solution for 1-2 h.
    Note: Handle the sample gently to avoid the destruction of cell structure, no vortexing.
  5. Soak the sample in clearing solution-Hoyer’s solution to make the tissue transparent. Change the clearing solution 1-2 times if necessary.
    Notes:
    1. Handle the sample gently to avoid the destruction of cellular structures, no vortexing.
    2. The Hoyer’s solution makes the sample transparent and soft. In this method, different layers of tissue could be observed under a DIC microscope without dissection of different layers. For good observation, the tissue sample should be transparent. Softening the sample by Hoyer’s solution is good for sample mounting.
  6. Prepare microscope slide: Add a few drops of Hoyer’s solution onto a glass slide; place a piece of sample (treated cotyledon, leaf or petal) flat on the surface of the slide; cover the slide with a coverslip glass carefully and try to avoid the air bubbles.
  7. Measurement of petal areas.
    1. Take photos of the whole petal under a 4x differential interference contrast (DIC) optics, make sure the edge of the petal is clear.
    2. Measure the area of the petal using Image J software.
  8. On a DIC microscope, take the images of the center areas in different cell layers of the cotyledon, leaf or petal blade between the mid-vein and the margin.
    Note: For the cotyledons and leaf blades, three layers could be observed clearly under the DIC optics: The adaxial epidermis layer, the palisade mesophyll layer and the abaxial epidermis layer; for the petals, the cell in the adaxial epidermis and abaxial epidermis could be easily observed under DIC microscope (Figure 2).


    Figure 2. Three layers of the Arabidopsis leaf are visible under differential interference contrast (DIC) microscope. Images show adaxial epidermis, palisade mesophyll and abaxial epidermis cells in the second pair of leaves from 25-day-old Wt (row a) and a mutant (row b) lines. Scale bar: 100 μm (reproduced from Reference 1; Figure 4a in the manuscript: http://onlinelibrary.wiley.com/doi/10.1111/tpj.12228/full)

  9. Count the numbers of different types of cells and stomata in 1024 x 768 pixel areas using Cell Counter plugin (http://rsbweb.nih.gov/ij/plugins/cell-counter.html) in Image J software (the same as shown in Flash 3).
    Note: For the epidermis of cotyledon and leaf blade, we can count both the stomatal and pavement cell numbers in the same image. For the pavement cells or stomata located in the margins of the image, if over half of the cell/stomata area is within the microscope viewfield, count it in; otherwise, don’t count it. Use the same standard for the same batch of assay.
  10. Measure the actual areas of a 1024 x 768 pixel area of the image in different DIC optics.
  11. Calculate cell/stomatal densities and cell size.
    1. For the cotyledon and leaf blade, cell/stomatal densities can be calculated from the total number of the cells and stomata in the image divided by the actual area of the image.
    2. For the petal, the cell size and cell density can be calculated from the total cell number in the image and the actual area of the image.

Recipes

  1. Washing solution
    75% ethanol
  2. Clearing solution (Hoyer’s solution)
    Chloral hydrate/water/glycerol (8 W: 2 V: 1 V)

Acknowledgments

This protocol was developed in association with the study as previously published (Cheng et al., 2013). It was expanded and the full protocol with additional data is only described here. Y.Z. thanks the Ministry of Science and Technology and National Natural Science Foundation of China, and H.W. thanks the Natural Sciences and Engineering Research Council of Canada for financial support.

References

  1. Anderson, L. E. (1954). Hoyer's solution as a rapid permanent mounting medium for bryophytes. Bryologist: 242-244.
  2. Cheng, Y., Cao, L., Wang, S., Li, Y., Shi, X., Liu, H., Li, L., Zhang, Z., Fowke, L. C., Wang, H. and Zhou, Y. (2013). Downregulation of multiple CDK inhibitor ICK/KRP genes upregulates the E2F pathway and increases cell proliferation, and organ and seed sizes in Arabidopsis. Plant J 75(4): 642-655.

材料和试剂

  1. 解剖的玫瑰花叶从拟南芥
  2. 来自开放花的花瓣
  3. 乙醇(Sigma-Aldrich,目录号:459844)
  4. 水合氯醛(Sigma-Aldrich,目录号:15307)
  5. 甘油(Sigma-Aldrich,目录号:G7757)
  6. 洗涤液(见配方)
  7. 清除解决方案(参见配方)

设备

  1. 立体显微镜(Zeiss,型号:Zeiss Stemi 2000)
  2. 数码相机(佳能,型号:Canon powershot S5IS)
  3. 玻璃载玻片和薄盖玻片
  4. 具有CCD照相机(Nikon Corporation,型号:DS-Ri1)的微分干涉对比(DIC)显微镜(尼康公司,型号:ECLIPSE 80i)

软件

  1. Image J软件( http://rsbweb.nih.gov/ij/index.html )。
  2. 单元计数器插件( http://rsbweb.nih.gov/ij/plugins/cell -counter.html

程序

  1. 在研究的特定阶段从植物中解剖子叶,叶和花瓣
  2. 测量解剖的子叶和叶的面积(图1a)

    图1.用于面积测量和毛孔数计数的图像(a)。 显示了布置在白色表面上的子叶,条= 1cm。 照片是用数字照相机拍摄的。 (b)。 显示拟南芥叶的近轴表面上的毛状体。 照片是通过立体显微镜用数码相机拍摄的。

    1. 标记来自同一植物的子叶/叶,将样品置于白色表面上,并尽量使其保持平坦。
    2. 在中间(如果可能)设置1厘米的标度,并用数码相机拍摄照片。
    3. 使用Image J软件(Flash 1)测量单个子叶或叶片的面积。

    闪光灯1.闪光灯显示使用ImageJ 测量图1a中子叶面积的步骤
                                        <! - [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] - >


  3. Trichome数量和密度。
    1. 用立体显微镜下的鳞片拍摄单个叶子,并确保毛状体清晰(图1b)。
    2. 用Image J软件(Flash 2)和测量叶片的面积  使用Image J中的Cell Counter插件计算总毛数 软件(Flash 3)。
    3. 毛状体密度是通过毛状体的总细胞数除以叶面积计算的
      Flash 2.闪光灯显示使用ImageJ测量单个叶子的步骤
                                         <! - [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] - >
      Flash 3.闪光灯显示如何在ImageJ中使用单元格计数器插件
                                            <! - [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] - >
  4. 通过在70%乙醇中浸泡过夜固定样品; 通过清洗样品2〜3次清除叶绿素,直到清洗液(70%乙醇)变清。 每次,将样品浸泡在洗涤液中1-2小时 注意:轻轻拿取样品以避免细胞结构破坏,无涡流。
  5. 将样品浸泡在透明溶液-Hoyer溶液中,使组织透明。 如有必要,请更改清除溶液1-2次。
    注意:
    1. 轻轻处理样品,避免细胞结构破坏,无涡旋。
    2. Hoyer的解决方案使样品透明和柔软。 在这里 方法,在DIC下可观察到不同层的组织 显微镜无解剖不同层。 为了良好的观察,   组织样品应该是透明的。 软化样品 Hoyer的解决方案适合样品安装。
  6. 准备显微镜载玻片:在玻璃载玻片上加入几滴Hoyer溶液; 将一片样品(处理的子叶,叶或花瓣)平放在载玻片的表面上; 小心地用盖玻片覆盖载玻片,并尽量避免气泡
  7. 花瓣区域的测量。
    1. 在4x微分干涉下拍摄整个花瓣的照片 对比度(DIC)光学,确保花瓣的边缘清晰。
    2. 使用Image J软件测量花瓣的面积。
  8. 在DIC显微镜上,拍摄中间静脉和边缘之间的子叶,叶或花瓣叶片的不同细胞层中的中心区域的图像。
    注意:对于子叶和叶片,在DIC光学下可以清楚地观察到三层:近轴表皮层,栅状叶肉层和背面表皮层;对于花瓣,在DIC显微镜下可以容易地观察到在正轴表皮和远轴表皮中的细胞(图2)。


    图2.显示在差异干涉对比(DIC)显微镜下的三层拟南芥叶。图像显示第二表面中的近轴表皮,栅栏叶肉和背面表皮细胞 来自25日龄Wt(行a)和突变(行b)行的叶对。比例尺:100μm(从参考文献1中再现;图4a在手稿中: http: //onlinelibrary.wiley.com/doi/10.1111/tpj.12228/full

  9. 使用Image J软件中的Cell Counter插件(http://rsbweb.nih.gov/ij/plugins/cell-counter.html)计数1024×768像素区域中不同类型的细胞和气孔的数量(与所示的相同在Flash 3)。
    注意:对于子叶和叶片的表皮,我们可以计算同一图像中的气孔和铺路细胞数目。对于位于图像边缘的路面细胞或气孔,如果超过一半的细胞/气孔区域在显微镜视野内,则计数;否则,不要算。对同一批次的测定使用相同的标准。
  10. 在不同的DIC光学中测量图像的1024 x 768像素区域的实际面积。
  11. 计算细胞/气孔密度和细胞大小。
    1. 对于子叶和叶片,细胞/气孔密度可以是 从图像中的细胞和气孔的总数计算 除以图像的实际面积。
      来自25日龄Wt(行a)和突变(行b)行的叶对。比例尺:100μm(从参考文献1中再现;图4a在手稿中: http: //onlinelibrary.wiley.com/doi/10.1111/tpj.12228/full

    2. 使用Image J软件中的Cell Counter插件(http://rsbweb.nih.gov/ij/plugins/cell-counter.html)计数1024×768像素区域中不同类型的细胞和气孔的数量(与所示的相同在Flash 3)。
      注意:对于子叶和叶片的表皮,我们可以计算同一图像中的气孔和铺路细胞数目。对于位于图像边缘的路面细胞或气孔,如果超过一半的细胞/气孔区域在显微镜视野内,则计数;否则,不要算。对同一批次的测定使用相同的标准。
    3. 在不同的DIC光学中测量图像的1024 x 768像素区域的实际面积。
    4. 计算细胞/气孔密度和细胞大小。
      1. 对于子叶和叶片,细胞/气孔密度可以是 从图像中的细胞和气孔的总数计算 除以图像的实际面积。
        ... Chloral hydrate/water/glycerol (8 W: 2 V: 1 V)

      Acknowledgments

      This protocol was developed in association with the study as previously published (Cheng et al., 2013). It was expanded and the full protocol with additional data is only described here. Y.Z. thanks the Ministry of Science and Technology and National Natural Science Foundation of China, and H.W. thanks the Natural Sciences and Engineering Research Council of Canada for financial support.

      References

      1. Anderson, L. E. (1954). Hoyer's solution as a rapid permanent mounting medium for bryophytes. Bryologist: 242-244.
      2. Cheng,Y.,Cao,L.,Wang,S.,Li,Y.,Shi,X.,Liu,H.,Li,L.,Zhang,Z.,Fowke,LC,Wang, ,Y.(2013)。 下调多种CDK抑制剂ICK/KRP基因上调E2F通路并增加细胞增殖,器官和 种子大小在拟南芥中。植物J 75(4):642-655。
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How to cite this protocol: Cheng, Y., Cao, L., Wang, S., Li, Y., Wang, H. and Zhou, Y. (2014). Analyses of Plant Leaf Cell Size, Density and Number, as Well as Trichome Number Using Cell Counter Plugin. Bio-protocol 4(13): e1165. DOI: 10.21769/BioProtoc.1165; Full Text



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