Image Analysis: Basic Procedures for Description of Plant Structures - Plant Cell Morphogenesis: Methods and Protocols - page 72

brightness values by the ruler under the histogram. Automatic
segmentation is offered. Several values can be sampled from
the image and used for segmentation. At the end the leaves
shall be masked with color, while the background shall be
unchanged. When you have set the threshold, press “Select”
and then “Sample” to get the binary image.
3. Specify which geometrical characteristics you intend to mea-
sure (Analyze/Set Measurements). For their explanation, see
the manual parameter description. This applies particularly, for
example, to the parameter of circularity, formula of which can
vary in different image analysis software.
4. To exclude thresholded tiny objects in the background, set a
minimal area of a measured object (Analyze/Set Measurements).
5. Measure the objects by command Analyze Particles (Menu/
Analyze/Analyze Particles).
6. Save the acquired results (File/Save as in the Results window).

1. Install the Sampling_Window plug-in to ImageJ: Download
the fi le “Sampling_Window.class” from http://rsbweb.nih.
gov/ij/plugins/sampling-window/index.html and copy it to
the folder (Program fi les\…) ImageJ\plugins. Restart ImageJ.
2. Open your image of an epidermal peel, which was sampled in
a uniform random way, and set right calibration.
3. Superimpose the counting frame on the image (Menu/
Plugins/Sampling_Window). Set the size, color, line width,
and position of the frame in the dialog box. Note the size of
the frame and calculate its area.
4. Count the stomata ( see Note 7 ) which are selected by the
counting frame according to the following rules: stomata fully
inside, stomata laying partly inside, and intersecting the dashed
line of the sampling frame. Do not count stomata, which lay
partly inside and simultaneously intersect full exclusion lines of
the frame (Fig. 3c ). You can use the Multipoint selection tool
(Tools/Multipoint selection tool) to mark the counted sto-
mata by a point, which are then numbered. You can undo the
selection by Alt + left click.
5. Relate the recorded number of stomata to the area of the
counting frame and get the estimation of stomatal density.

3.5 Estimation
of Stomatal Density

1. Install the Sampling_Window plug-in to ImageJ ( see
Subheading 3.5 , step 1 ).
2. Open your image of an epidermal peel, which was sampled
in a uniform random way and acquired in such way to get a
high contrast of cell walls (e.g., using cell wall polyphenolic
fl uorescence or stained cell walls with toluidine blue). Set right
calibration.

3.6 Analysis of
Epidermal Cell Shape