Differential and Simultaneous Visualization of Cells and Airspaces in Plant Leaves

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The Plant Journal
Jan 2015



This protocol is to differentially and simultaneously visualize both cells and airspaces in intact leaves and to create 3D structures of cells and airspaces from confocal images using the software DSLT_Demo (https://github.com/nslab2000/DSLT). Leaves stained with Nile Red in silicone-oil solution provide red color to cell membranes and green color to airspaces filled with silicone oil solution. This method is applicable to any tissues (except for dry seeds) of various plants including Arabidopsis, Nicotiana, Lemna and moss, and applicable even to hard leaves of plants such as switchgrass and Cinnamomum. Repeated use of this method enables time-lapse imaging of leaves over days and weeks because both Nile Red and silicone oil are harmless to plant tissues.

Materials and Reagents

  1. >100 ml container to hold the staining solution (see procedure A) during rotational incubation
  2. An incubation container for staining (e.g., 1.5 ml micro tube, Petri dish)
  3. Super frost slide glass (Matsunami Glass Ind., catalog number: S2441 )
  4. NEO microscope coverslip, 24 x 32 mm (Matsunami Glass, catalog number: C024321 )
  5. Parafilm M® (Bemis Company, Inc., catalog number: PM-996 )
  6. Pipetman p200 or 1 ml disposable transfer pipette
  7. Silicone oil (Shin-Etsu Chemical Co., catalog number: KF96L-1.0 cs or KF96L-1.5 cs )
    Note: It is named “Dimethyl Silicone Fluid” on Shin-Etsu Chemical Co. website.
  8. Nile Red (Invitrogen, catalog number: N-1142 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular Probes™, catalog number: N-1142”.


  1. Tube rotator or shaker
  2. Electronic scale
  3. Confocal microscope (Carl Zeiss Microscopy, model: LSM-780) or fluorescence microscope (Carl Zeiss Microscopy, model: Axioskop2 plus)
  4. Sonication bath (HONDA ELECTRONICS Co., model: W-113 )
    Note: This is not absolutely necessary.


  1. Preparation of staining solution
    1. Add 0.318 mg of Nile Red and 100 ml of silicone oil (KF96L-1.0 cs or KF96L-1.5 cs) to a container. The final concentration of this solution is 10 μM. About 100 samples can be prepared from this amount of staining solution if you use 1.5 ml microtubes for staining.
    2. Tightly seal the container with Parafilm M®.
    3. (Optional) Sonicate the suspended Nile Red using a sonication bath (10 min, 100 kHz) to make Nile Red dissolved faster.
    4. Wrap the container with aluminum foil, place the tube on a tube rotator and rotate for 1-2 d.
    5. (Optional) Undissolved dye can be removed via filtration or centrifugation.

  2. Staining
    1. Remove a small (5 x 5 ~ 10 x 10 mm) section of the leaf tissue to be examined.
      Note: When you conduct time-lapse imaging, skip this step and stain a whole plant.
    2. Add the specimen and staining solution to an incubation container.
    3. Incubate specimen in staining solution for 5 min (adaxial side) or 15 min (abaxial side).

  3. Slide preparation
    Prepare slides according to Figure 1.

    Figure 1. Procedure of slide preparation

  4. Microscopy
    The steps B-C fill airspaces with Nile Red in silicone oil. Image airspaces (excitation wavelength 488, emission wavelength 490-560) and cells (excitation wavelength 561, emission wavelength 563-639). Representative data is shown in Figure 2.

  5. (Optional) Procedures for time-lapse imaging
    After observation, shake the plants in silicone oil (KF96L-1.0 cs) gently at 30~60 rpm for 1 min twice to remove Nile Red. Put the plants on culture beds until the next observation. Repeat the steps B-E. Representative data of time-lapse imaging is shown in Figure 3.

  6. Image segmentation
    You can extract shapes of cells and airspaces from acquired 3D images by using DSLT_Demo. Step by step instructions for segmentation are available at http://dslt.bot.kyoto-u.ac.jp/DSLT_manual_v111.pdf.

Representative data

Figure 2. Visualizing cells and airspaces in an Arabidopsis thaliana leaf. Confocal images of the epidermis (left) and mesophyll (right) of an Arabidopsis thaliana leaf. The abaxial side of the leaf was stained with solution of Nile Red in silicone oil and imaged with 488 nm excitation for Nile Red in silicone oil (airspace, green) and 561 nm excitation for Nile Red in lipid (cellular membrane, magenta). Asterisks indicate airspaces. Scale bars: 50 μm.

Figure 3. Time-lapse imaging of a developing Nicotiana glauca leaf. Cross-sectional views of 3D images of the adaxial side of a Nicotiana glauca leaf. Confocal images were taken over 5 days of leaf development (times indicated in white above each image). Shown is the fluorescence of Nile Red in the lipid bilayer membrane. Bar is 50 μm.


This protocol was adapted from the research article Kawase et al. (2015). The work was supported by a Specially Promoted Research of Grant-in-Aid for Scientific Research to I. H. -N. (no.22000014) from the Japan Society for the Promotion of Science (JSPS).


  1. Kawase, T., Sugano, S. S., Shimada, T. and Hara-Nishimura, I. (2015). A direction-selective local-thresholding method, DSLT, in combination with a dye-based method for automated three-dimensional segmentation of cells and airspaces in developing leaves. Plant J 81(2): 357-366.


此协议是差异和同时可视化完整叶片的细胞和空间,并使用软件DSLT_Demo( https://github.com/nslab2000/DSLT )。 用尼罗红在硅油溶液中染色的叶子为细胞膜提供红色,并向填充有硅油溶液的空间提供绿色。 该方法适用于包括拟南芥 ,,, 和moss在内的各种植物的任何组织 硬叶植物如柳枝稷和樟脑。 重复使用这种方法使得叶片在几天和几周时间推移成像,因为尼罗红和硅油都对植物组织无害。


  1. >在旋转孵育期间保持染色溶液(参见程序A)的100ml容器
  2. 用于染色的孵育容器(例如,1.5ml微管,培养皿)
  3. 超级霜玻璃(Matsunami Glass Ind。,目录号:S2441)
  4. NEO显微镜盖玻片,24×32mm(Matsunami Glass,目录号:C024321)
  5. Parafilm M(bemis公司,目录号:PM-996)
  6. Pipetman p200或1ml一次性转移移液管
  7. 硅油(Shin-Etsu Chemical Co.,目录号:KF96L-1.0cs或KF96L-1.5cs)
    注意:在Shin-Etsu Chemical Co.网站上命名为"Dimethyl Silicone Fluid"。
  8. 尼罗红(Invitrogen,目录号:N-1142) 注意:目前,它是"Thermo Fisher Scientific,Molecular Probes™,目录号:N-1142"。


  1. 管旋转器或振动器
  2. 电子秤
  3. 共焦显微镜(Carl Zeiss显微镜,型号:LSM-780)或荧光显微镜(Carl Zeiss显微镜,型号:Axioskop2 plus)
  4. 超声波浴(HONDA ELECTRONICS Co.,型号:W-113)


  1. 染色溶液的制备
    1. 加入0.318mg尼罗红和100ml硅油(KF96L-1.0cs或 KF96L-1.5cs)。 该溶液的最终浓度 是10μM。 可以从该量的染色制备约100个样品   溶液,如果你使用1.5毫升微管染色。
    2. 用Parafilm M ®密封容器。
    3. (可选)使用超声波浴(10分钟,100 kHz)对悬浮的尼罗红进行声波处理,使尼罗红溶解得更快。
    4. 用铝箔包装容器,将管放置在管旋转器上并旋转1-2天
    5. (可选)未溶解的染料可以通过过滤或离心除去。

  2. 染色
    1. 取出一个小的(5×5〜10×10毫米)的待检查的叶组织 注意:当您进行时间推移成像时,请跳过此步骤并染色整株植物。
    2. 将样品和染色溶液加入培养容器中
    3. 孵育标本在染色溶液中5分钟(近轴侧)或15分钟(后轴侧)

  3. 幻灯片准备


  4. 显微镜
    步骤B-C用硅油中的尼罗红填充空间。 图像空间(激发波长488,发射波长490-560)和细胞(激发波长561,发射波长563-639)。 代表数据如图2所示。

  5. (可选)延时成像的程序
    观察后,将植物在硅油(KF96L-1.0cs)中以30〜60rpm轻轻摇动1分钟两次,以除去尼罗红。 将植物放在培养床上直到下一次观察。 重复步骤B-E。 延时成像的代表数据如图3所示。

  6. 图像分割
    您可以通过使用DSLT_Demo从已采集的3D图像中提取单元格和空间的形状。您可以访问 http://dslt.bot.kyoto- u.ac.jp/DSLT_manual_v111.pdf


图2.在拟南芥叶叶中可视化细胞和空间。 em>拟南芥叶。叶片的背面用尼罗红在硅油中的溶液染色并用在硅油(空间,绿色)中的尼罗红的488nm激发和在脂质(细胞膜,品红)中的尼罗红的561nm激发成像。星号表示空域。比例尺:50μm

图3.发育的烟草属glauca叶片的延时成像。烟草glauca 叶片的3D图像的横截面视图。叶。在叶发育5天(在每个图像上方以白色指示的时间)拍摄共焦图像。显示了尼罗红在脂质双层膜中的荧光。棒为50μm。


该协议改编自研究文章Kawase等人(2015)。 这项工作得到了特别推动的研究助理科学研究支持I. H. -N。 (日本科学促进会(JSPS))(日本科学技术研究所(No.22000014))。


  1. Kawase,T.,Sugano,S.S.,Shimada,T.and Hara-Nishimura,I.(2015)。 方向选择性局部阈值法(DSLT)与基于染料的自动化方法相结合 在发育叶中的细胞和空间的三维分割。植物J(81)(2):357-366。
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引用:Kawase, T., Sugano, S. S., Shimada, T. and Hara-Nishimura, I. (2016). Differential and Simultaneous Visualization of Cells and Airspaces in Plant Leaves. Bio-protocol 6(11): e1826. DOI: 10.21769/BioProtoc.1826.