Image Analysis: Basic Procedures for Description of Plant Structures - Plant Cell Morphogenesis: Methods and Protocols - page 74

9. Superimpose the sampling window on the image (Menu/
Plugins/Sampling_Window) at random position. Set the size,
color, line width and position of the frame in the dialog box.
10. Measurement: Set the parameters for analysis in Menu/Analyze/
Set measurements (e.g., Area, Shape descriptors, Perimeter,
Feret's diameter). Specify the minimal size of analyzed particles
in Menu/Analyze/Analyze Particles (e.g., 5-Infi nity), tick
Display results, Clear results, and Add to manager.

11. Save results for the cells selected by the counting frame—you
can browse from cell to cell in the ROI manager window to
see which cell corresponds to which number. Other possibility
is to measure the cells selected by the counting frame by the
Wand (tracing) tool and command Ctrl + M (or Analyze/
Measure). The selected cells are those which intersect the
frame rectangle (or are inside the rectangle) and simultane-
ously do not intersect the exclusion lines (Fig. 3d ).
12. Calculate the shape complexity of the object under study (ratio
of its perimeter to square root of its area: b / √ a , where b is

perimeter and a is area of the object, [ 11 ]. b / √ a is minimal

for circle: 2 = 3.54, for highly structured objects, such as

endoplasmic reticulum, it can exceed the value of 30.

π . √π

4 Notes

1. The real object size could change in consequence of specimen
processing. Therefore possible size artifacts, e.g., in structure
shrinking, collapsing, and distortion, should be identifi ed to get
proper information about real dimensions ( see, e.g., ref. [ 5 ]).
2. The pixels are commonly square shaped but some chips have
rectangular sensor elements. In case of non-square rectangular
pixels, the pixel aspect ratio (width/height).
3. Some parameters, such as circularity, can be defi ned in different
ways—thus it is recommended to check the formula in the pro-
gram manual.
4. Sequence of commands can be recorded as macro, saved to text
fi le, and repeatedly run. This can speed up routine analyses of
many images substantially. Saving the image analysis steps in
macro is also useful for documentation purposes. This tech-
nique is accessible also to non-experts as a sequence can be in
most programs “recorded” during work.
5. The new plug-ins can be written in JAVA using ImageJ applica-
tion interface. Extensive documentation as well as a lot of source
fi les of plug-ins published on ImageJ website can be used for study
of programming the image analysis modules. Some programming
experience and knowledge of JAVA and ImageJ API is necessary.