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[Bio101] Leaf Clearing Protocol to Observe Stomata and Other Cells on Leaf Surface
[Bio101] 用于观察叶面气孔和其他细胞的叶片透化方案   

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Abstract

In this protocol, leaves are cleared and fixed in an ethanol and acetic acid solution, and mounted in Hoyer’s solution. The cleared leaves are imaged under differential interference contrast (DIC) microscope. This protocol is beneficial for studying stomata, hair cells, and other epidermal cells in plants.

Keywords: Leaf clearing(叶片透化), Stomata(气孔), DIC microscopy(DIC显微镜检查), Hoyer’s solution(Hoyer’s 溶液)

Background

There are multiple ways to observe stomata and other epidermal cells such as hair cells on plant leaf surface. Traditionally a clear nail polish or wood glue is applied to the leaf surface and let dry. The leaf is peeled and observed under the microscope. Alternatively, scotch tape is applied to the leaf and removed to observe an imprint of the leaf surface. These traditional methods can be used for thicker leaves that are sturdy but the images are generally not of the highest quality. Small and delicate leaves such as Arabidopsis leaves require a more advanced method. A fresh Arabidopsis or Brachypodium leaf may also be observed directly under the microscope; however, the thickness and pigments in the leaf pose difficulties in viewing the stomata and other epidermal cells clearly. This protocol describes a method clearing of leaves for visualizing stomata including other epidermal cells and obtaining good quality images for publishing in peer-reviewed journals (Anderson, 1954).

Materials and Reagents

  1. Multi-well plates (Greiner Bio One International, catalog number: 665102 )
  2. Parafilm (Bemis, catalog number: PM996 )
  3. Microscope slides, 25 x 75 x 1.0 mm (Fisher Scientific, catalog number: 12-550-A3 )
  4. Cover slips (Fisher Scientific, catalog number: 12-544D )
  5. Aluminium foil
  6. Arabidopsis or Brachypodium leaf
    Note: You may cut the Brachypodium leaf into 1 cm pieces to fit into wells.
  7. Ethanol (Sigma-Aldrich, catalog number: 459844 )
  8. Acetic acid (Sigma-Aldrich, catalog number: AX0077-1 )
  9. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
  10. Gum Arabic (Sigma-Aldrich, catalog number: 30888-1KG )
  11. Chloral hydrate (Sigma-Aldrich, catalog number: C8383 )
  12. Glycerol (Sigma-Aldrich, catalog number: G5516-1L )
    Note: Sterile is not required.
  13. Distilled water
  14. Clearing solution (see Recipes)
  15. 1 N KOH (see Recipes)
  16. Hoyer’s solution (see Recipes)

Equipment

  1. Fume hood
  2. Forceps (Fisher Scientific, catalog number: 22-327379 )
  3. Microscope (Leica, model: Leica DM6 B )
  4. 1 L glass beaker (Sigma-Aldrich, catalog number: Z169161 )
  5. Magnetic stirrer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: S194615 )

Software

  1. MS Excel
    (https://support.office.com/en-us/article/STDEV-function-51fecaaa-231e-4bbb-9230-33650a72c9b0)
    Note: Calculate Standard Deviation in MS Excel.

Procedure

  1. Fill multi-well culture plate wells with 500 μl-1 ml clearing solution (work in a fume hood to avoid the smell of acetic acid, see Recipes). Place the whole seedling or a leaf (about 1 cm) in the clearing solution and seal the plate with Parafilm (Figure 1). Leave the tissue in clearing solution overnight at room temperature. Younger seedlings (2-4 d old) only need a few hours to clear. For bigger leaves (6 d old or more), change the clearing solution every 4 h until the leaves become transparent (for faster clearing, otherwise overnight incubation is fine). You may keep the leaves in clearing solution for about a week.


    Figure 1. Leaf clearing and fixing. The ethanol:acetic acid solution fixes the tissue and the tissue becomes transparent.

  2. Remove the clearing solution and refill the wells with 500 μl-1 ml 1 N KOH (see Recipes) until the tissue becomes transparent (Figures 2C-2D).
    Note: Usually takes 30 min, you may leave them longer (5-6 h) in 1 N KOH but overnight incubation is not advised.
  3. Remove 1 N KOH and refill the wells with 500 μl-1 ml water to wash the cleared leaves (one wash is enough) and remove excess KOH.
  4. Take a clean microscope slide and place 2 rows of Hoyer’s solution (see Recipes) drops (~10 μl each for Arabidopsis cotyledons). You may put 4-6 drops per row. Thicker leaves require more Hoyer’s solution (Figure 2E).


    Figure 2. Washing the tissue. Cleared tissue (A, B) is washed in 1 N KOH (C, D). Tissue is washed in water (E, top slide) and placed in Hoyer’s solution (E, bottom slide).

  5. Place the seedling on a cover slide and dissect out the cleared leaves (you may use magnifying glasses), so that you have at least 2 leaves (cotyledons or true leaves) and hold them with abaxial side up using forceps (Figure 3).


    Figure 3. Slide preparation. Individual leaves form a precipitate when they are placed in Hoyer’s solution.

  6. Place the leaves in Hoyer’s solution droplets (Figure 3).
    Note: KOH reacts with Hoyer’s solution to form a precipitate that can guide you to place one leaf in each droplet. The precipitate will dissolve away quickly for young seedlings and may require more time for larger leaves.
  7. Repeat the above steps for 3-4 seedlings per slide.
  8. Gently place coverslip and press to flatten the leaf for better visualization (Figure 3).
  9. Leave the slides to dry overnight at room temperature. You may image the young seedlings in 3-4 h but longer drying is advised for bigger leaves (1-5 days).
  10. Apply additional Hoyer’s solution on the edges of the coverslip to fill any air bubbles formed during drying period.
  11. Image your cleared seedling under a microscope with DIC filter and document the phenotype (Figure 4).


    Figure 4. Same leaf imaged with Brightfield or DIC filters. DIC image (B) gives better resolution than Brightfield (A) to observe different cell types. Arrows: stomata (young and mature).

Data analysis

Different cell types e.g., young vs. mature stomata (pores on leaf surface); pavement cells vs. stomata; or the number of hair cells per surface area can be measured through the images obtained in this protocol (Figure 5). Typically at least three leaves (N = 3) are observed for each line. E.g.,
Count No. of stomata = X1, X2, X3
Count No. of pavement cells = Y1, Y2, Y3
Average X and Y values respectively.
Calculate Stomatal Index: X/(X + Y)
Calculate standard deviation (Calculate Standard Deviation in MS Excel) and standard error (standard deviation/) for each cell type and plot in a graph.


Figure 5. Quantification of stomata. Number of stomata (X) and pavement cells (Y) are counted to calculate stomatal index.

Recipes

  1. Clearing solution
    7:1 solution of 95% ethanol:acetic acid
    7 ml 95% ethanol + 1 ml acetic acid
  2. 1 N KOH
    56.11 g KOH in 1 L water
    Note: The dissolution of KOH in water is exothermic, hence the solution will get warmer and it is important to add KOH slowly.
  3. Hoyer’s solution (prepare in a fume hood)
    1. Add 15 g of Gum Arabic to 25 ml of H2O in a 1 L glass beaker (cover the beaker with aluminium foil)
    2. Heat to 60 °C
    3. Stir overnight on a magnetic stirrer in the fume hood at room temperature
    4. Add 200 g of chloral hydrate until dissolved
    5. Add 20 ml of glycerol and mix
    Notes:
    1. Store the solution at room temperature for about a year.
    2. Pellets appear after long storage but they do not cause any problems in tissue preparation or imaging.

Acknowledgments

I would like to thank Dr. Dominique Bergmann and her lab members for fine-tuning and implementing this protocol in their publications. I sincerely thank Graham Dow for providing the initial protocol.

References

  1. Anderson, L. E. (1954). Hoyer’s solution as a rapid permanent mounting medium for bryophytes. Bryologist 57(3): 242-244.

简介

在该方案中,将叶子清除并固定在乙醇和乙酸溶液中,并安装在Hoyer溶液中。 清除的叶片在差分干涉对比(DIC)显微镜下成像。 该方案有利于研究植物中的气孔,毛细胞和其他表皮细胞。
【背景】观察气孔和其他表皮细胞如植物叶片表面毛细胞有多种方法。 传统上,将明亮的指甲油或木胶施加到叶子表面并使其干燥。 将叶片剥离并在显微镜下观察。 或者,将透明胶带施加到叶子上并去除以观察叶表面的印记。 这些传统方法可用于坚固的较厚的叶子,但是图像通常不是最高质量的。 小而脆弱的叶如拟南芥叶需要更先进的方法。 还可以在显微镜下直接观察到新鲜的拟南芥或Brachypodium叶。 然而,叶片中的厚度和颜料在观察气孔和其他表皮细胞方面是困难的。 该协议描述了一种清除叶片,用于可视化包括其他表皮细胞在内的气孔的方法,并且获得了在同行评审期刊中出版的优质图像(Anderson,1954)。

关键字:叶片透化, 气孔, DIC显微镜检查, Hoyer’s 溶液

材料和试剂

  1. 多孔板(Greiner Bio One International,目录号:665102)
  2. 石蜡膜(Bemis,目录号:PM996)
  3. 显微镜载玻片,25 x 75 x 1.0 mm(Fisher Scientific,目录号:12-550-A3)
  4. 封面(Fisher Scientific,目录号:12-544D)
  5. 铝箔
  6. 拟南芥或
    叶子
    注意:您可以将枝条切成1厘米,以适应井。
  7. 乙醇(Sigma-Aldrich,目录号:459844)
  8. 乙酸(Sigma-Aldrich,目录号:AX0077-1)
  9. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:221473)
  10. 胶阿拉伯胶(Sigma-Aldrich,目录号:30888-1KG)
  11. 水合氯仿(Sigma-Aldrich,目录号:C8383)
  12. 甘油(Sigma-Aldrich,目录号:G5516-1L)
    注意:不需要无菌。
  13. 蒸馏水
  14. 清除解决方案(请参阅食谱)
  15. 1 N KOH(参见食谱)
  16. Hoyer的解决方案(见配方)

设备

  1. 通风柜
  2. 镊子(Fisher Scientific,目录号:22-327379)
  3. 显微镜(Leica,型号:Leica DM6 B)
  4. 1升玻璃烧杯(Sigma-Aldrich,目录号:Z169161)
  5. 磁力搅拌器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:S194615)

软件

  1. MS Excel
    https ://support.office.com/en-us/article/STDEV-function-51fecaaa-231e-4bbb-9230-33650a72c9b0
    注意:计算MS Excel中的标准差。

程序

  1. 用500μl-1ml澄清液填充多孔培养板孔(在通风橱中工作以避免乙酸的气味,参见食谱)。将整个幼苗或叶(约1厘米)放置在澄清溶液中,并用Parafilm密封板(图1)。将组织在室温下离开清除溶液过夜。幼苗(2-4岁)只需要几个小时才能清除。对于较大的叶子(6 d以上),每4 h更换清除液,直到叶片变得透明(为了更快的清除,否则过夜孵育很好)。你可以把叶子留在清理液中约一周。


    图1.叶清除和固定。乙醇:乙酸溶液固定组织,组织变透明。

  2. 取出清除溶液,并用500μl-1 ml 1N KOH重新填充孔(参见食谱),直至组织变透明(图2C-2D)。
    注意:通常需要30分钟,您可以在1 N KOH中留下较长时间(5-6小时),但不建议过夜孵育。
  3. 取出1N KOH,并用500μl-1ml水重新填充孔,以洗涤清除的叶子(一次洗涤足够)并除去过量的KOH。
  4. 取一个干净的显微镜载玻片,放置2排Hoyer's溶液(参见食谱)滴(约10μl,用于拟南芥子叶)。你可以每排放4-6滴。较厚的叶子需要更多的霍耶解决方案(图2E)

    图2.洗涤组织清除的组织(A,B)在1N KOH(C,D)中洗涤。将组织在水中洗涤(E,顶部载玻片)并置于Hoyer溶液(E,底部载玻片)中。

  5. 将幼苗放在盖子上,将清除的叶子(您可以使用放大镜)进行解剖,以便至少有2个叶子(子叶或真叶),并使用镊子将其背面朝上(图3)。


    图3.幻灯片准备。个人叶片放置在Hoyer's解决方案中时会形成沉淀物。

  6. 将叶放在霍伊氏溶液中(图3) 注意:KOH与Hoyer溶液反应形成沉淀物,可引导您在每个液滴中放置一片叶子。沉淀物将迅速溶解幼苗,并可能需要更多的时间用于较大的叶子。
  7. 每个幻灯片重复上述步骤3-4个幼苗。
  8. 轻轻放置盖玻片并按压以使叶片变平,以便更好地观察(图3)。
  9. 将载玻片在室温下干燥过夜。您可以在3-4小时内形成幼苗,但长时间干燥建议较大的叶子(1-5天)。
  10. 在盖玻片的边缘附加Hoyer溶液,以填充干燥期间形成的任何气泡。
  11. 使用DIC过滤器在显微镜下将您清除的幼苗图像,并记录表型(图4)

    图4.用Brightfield或DIC滤镜成像的相同叶片。 DIC图像(B)比Brightfield(A)提供更好的分辨率,以观察不同的细胞类型。箭头:气孔(年轻和成熟)。

数据分析

不同的细胞类型,例如,年轻对成熟气孔(叶表面的孔);路面细胞与气孔;或者每个表面积的毛细胞数量可以通过本方案获得的图像来测量(图5)。通常对于每一行观察到至少三叶(N = 3)。 ,
气孔计数= X1,X2,X3
计数路面细胞数= Y1,Y2,Y3
平均X和Y值分别为 计算气孔指数:X /(X + Y)
计算标准偏差(计算MS Excel中的标准偏差)和标准误差(标准偏差/ ),并在图表中绘制。


图5.气孔的定量。 计数气孔数(X)和路面细胞(Y),以计算气孔指数。

食谱

  1. 清算解决方案
    7:1 95%乙醇:乙酸溶液
    7 ml 95%乙醇+ 1 ml乙酸
  2. 1 N KOH
    56.11克KOH在1升水中 注意:KOH在水中的溶解是放热的,因此溶液将变得更暖,并且缓慢加入KOH是重要的。
  3. 霍耶的解决方案(在通风橱中准备)
    1. 在1升玻璃烧杯(用铝箔覆盖烧杯)上,加入15g的阿拉伯胶25ml的H 2 O 2。
    2. 加热至60°C
    3. 在室温下通风橱中的磁力搅拌器上过夜搅拌
    4. 加入200克水合氯醛直至溶解
    5. 加入20ml甘油并混合
    注意:

    1. 将解决方案存放在室温下约一年

致谢

我要感谢多米尼克·伯格曼博士及其实验室成员在其出版物中微调和实施该协议。我真诚地感谢Graham Dow提供了最初的协议。

参考

  1. Anderson,LE(1954)。 Hoyer的解决方案作为快速永久性的安装介质 苔藓植物。 57(3):242-244。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sharma, N. (2017). Leaf Clearing Protocol to Observe Stomata and Other Cells on Leaf Surface. Bio-protocol Bio101: e2538. DOI: 10.21769/BioProtoc.2538;
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