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NIH Image/ Scion Image Protocol for
Milkweed Damage Analysis

Image processing (IP) is the analysis and manipulation of pictures on a computer screen. Two IP software programs, NIH Image (Macintosh®) and Scion Image (Windows® 95), have been used throughout the world by scientists for many years. These public domain tools currently are indispensable tools for research in planetary science, medicine, dentistry, materials science, environmental science, biotechnology, and agriculture. One of these two programs must be on your computer to use the procedure below. Your teacher can help you download these programs if you do not have one of them on your computer.

  1. Scan the milkweed leaves on a flat bed scanner with a white background, a piece of plain white paper works well.

  2. Save the scanned image as a .tif or .bmp image. Make sure to keep track of the leaves and which plant they come from.

  3. Open NIH Image on the Mac, or Scion Image on the P.C.

  4. Go to File on the menu bar - Open the image of the leaf to be analyzed in Image

  5. When the milkweed leaf is open you should have the image of one leaf on a white background. Go to Options, on the menu bar - Turn on, Density Slice

  6. A red color will appear on the LUT. Move the Density Slice (red color) down to the bottom of the LUT. You should be in the black area.

  7. Starting in the black stretch the Density Slice red color up the LUT until the entire leaf is red. None of the background should be red.

  8. Use the magic wand on the tool bar on the left (next to the eye dropper) and click on the red leaf. The entire milkweed leaf should be selected. Go to Edit and Copy Selection.

  9. Go to File in the menu bar and pen a New window - Paste the image of the milkweed leaf into the new window. This will make sure that only the pixels in the leaf will be counted. If these steps are skipped then the pixels in the white background will also be counted.

  10. Go to Options and turn on Density Slice for the new image.

  11. Move the Density Slice (Red) down to the bottom of the LUT.

  12. Starting in the black move the Density Slice up until the entire leaf is red

  13. Go to Analyze - Measure. From the Info Box, record the number of square pixels listed as Area. (To make sure the Area option is on, go to Analyze - Options, and make sure the box next to Area is checked)

  14. Reduce the coverage of the Density Slice (the red) in the LUT until only the ozone damage on the leaf is marked in red.

  15. Go to Analyze - Measure. From the Info Box, record the number of square pixels listed as Area. This number will represent the pixels covering the ozone-damaged part of the leaf. Do not include damage not due to ozone. Pixels representing damage due to fungus or insect damage may be removed from the count by zooming into the image and erasing the pixels that should not be counted.

  16. Make a very careful count of the ozone damage. You might want to do step 14 twice to be sure you are only counting the ozone damage.

  17. Divide the ozone damage by the total.

  18. Repeat this for every leaf from each milkweed plant. Calculate the average ozone damage for each milkweed plant in the sample. Enter the average percent damage for each of the 10 milkweed plants on the data submission page.

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