Original research article

The authors used this protocol in:
Aug 2013

Navigate this Article


Analyses of Plant Leaf Cell Size, Density and Number, as Well as Trichome Number Using Cell Counter Plugin    

How to cite Favorites 2 Q&A Share your feedback Cited by


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.

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)


  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 )


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


  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

  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

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

  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.
    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.


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


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.


  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.
Please login or register for free to view full text
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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.

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data including images for the troubleshooting.

Hong Wang
Department of Biochemistry, University of Saskatchewan, Canada
The protocol is still working. For the trichome counting, first, we export the .tiff image into ImageJ and start the cell counter plug-in, then we have to recognize and label the targets manually. During the labelling progress, the software will show the number of the target we have labelled. What the software does here is only counting the number.
10/14/2019 1:37:00 AM Reply
Peter Apicella
Will the protocol still work if I use the DIC microscope for trichome counting?
10/1/2019 7:37:07 AM Reply
Jee Yan Chu
Brock University

It should. You just have to capture the image using the software that controls your microscope and save it as a TIFF file so that you can import it into ImageJ to use the plug-in.

10/8/2019 11:39:27 AM

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.