Counting Seeds using ImageJ
Preparation: Taking the photo
1. Use a white paper as your background
2. Make sure the seeds are widely spread
3. Label the seeds
4. Try to minimize reflections/shadows
Counting seeds:
1. Open the seed photo through ImageJ
2. Through the ImageJ menu, click on “Image” –> “Type” –> “8 Bit” checkbox. This
converts the photo into Black-and-White.
3. Convert this into a Binary image by clicking on “Process” –> “Binary” –> “Make
Binary”. Verify that the image looks to be OK; sometimes if the lighting is uneven across
the image, the binary crops off some parts. In this case, you may want to change the
thresholds.
4. Convert the image into a Binary Mask by clicking on “Process” –> “Binary” –> “Convert
to Mask”.
5. In some cases, ImageJ might consider adjacent seeds as one object. You can check if this
is the case by zooming into a clump of seeds and checking whether each seed is identified
separately.
6. To avoid the issue described above, click on “Process” –> “Binary” –> “Watershed”.
This process draws boundaries between clumps of objects that are likely to be
composites.
7. To count seeds, draw a bounding box around the area of the photo with seeds
8. After ensuring that the bounding box encompasses all seeds, click on “Analyze” –>
“Analyze particles”. Make sure to select “Overlay” under the “Show” checkbox. Also
select “Display Results”, “Summarize”, and “Exclude on edges”
9. Check using the overlay that (a) all seeds in the image are identified as objects, and (b) no
debris is identified as a seed. If you do notice debris identified as seeds, close the analysis
results, and re-click “Analyze particles”. Give a minimum size threshold, and reanalyze
particles. The right size threshold will depend on the species you are working with, and
on your photographs. For the B. rapa image above, a threshold of 400 seems to work
well.
10. Iterate step 11 until you feel confident that you are identifying an appropriate threshold.
11. Save the output as desired in the CSV datasheet
Video tutorial: [Link]
