Biology Reference
In-Depth Information
3. Run macro. Drag and drop txt fi le of the macro into ImageJ.
Then select “run macro” from the “macro” menu in the new
ImageJ macro window.
4. Input you image scale. Once the macro begins it will fi rst ask
the user “What is the image scale in pixels per micrometer?” In
the text box the user must input a numeric value to specify the
number of pixels it takes to yield one micrometer on the
image. This will ensure that the calculations output is in the
correct units in the results data fi le.
5. Input a value for saturation tolerance (percent of pixels). In
order for the macro to execute its computations on the entire
plant, the plant must be thresholded from the rest of the
image. The macro uses the maximum entropy algorithm to
threshold the plant of interest. Unfortunately, there may be
some cases where dim images are not thresholded properly.
For this reason the macro automatically enhances the contrast
for each image from the stack using a histogram stretching
procedure. The value entered determines the percent of pixels
that are allowed to be saturated in the whole image; the default
value of 0 % should be tested fi rst, but values between 0.1 and
1 % may improve image thresholding.
6. Enter the type of image. Select the option if your image is
color (RGB) or black and white (
see
Note 13
).
7. Shave images. At this point the macro will produce a window
that states, “Finish shaving and press OK to continue.” This
indicates that the program is waiting for the user to determine
which plant on each image the program should perform its
calculations. This is done primarily because there are cases
when multiple plants appear on the screen. To select the plant
of interest, the user must completely encase the plant within
the selection bounds. This is done using the mouse to select
an area around the plant. Once the plant is selected, the user
can press OK to continue to the next image. This process will
continue for all the images in the stack.
8. Saving fi le path. After the shaving process is completed, two
windows will appear: a window called Results and a window
called Phenotype Results. The Results window displays the
calculations of area and perimeter for each plant as they are
computed, while the Phenotype Results window is a table
where all the data is stored. Once the calculations are com-
plete, another saving message will appear. This one asks if you
want to save the fi les in the same place. (Depending on the
processing power of your computer and the size of your data
set, it may take a moment for the second saving message to
appear.) If “yes” is selected, all of the fi les selected in the next
step will be saved to the same fi le path. If not, the user will
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