Analyze scanned leaves with ImageJ software

Last modified by Xwiki VePa on 2024/02/07 07:36

When analyzing leaf area from aquatic macrophytes

Step-by-step guide

  1. Open the scanned image in Image J.
  2. Set scale: measure a known distance on the ruler e.g. 10 mm by zooming in on the ruler and using the line tool (See below screenshot -5th tool from the left with line).
  3. When a line has been drawn, select “Analyze” and “Set scale”.
  4. Type the known distance (e.g. 10 for 10 mm) in “Known distance” and mm in “Unit of length”. Click “Global” to set the scale for all pictures analyzed during a session with Image J.


    NB! For every new image, the scale needs to be checked every time. When a new image is analyzed use again the line tool and set scale to see if the newly drawn line is close to 10 mm (“known distance”).


  5. Open the Image menu and select “type” then select “8-bit”.
  6. Open the image menu again and select “adjust” then select “threshold”. Adjust the sliding bars until your leaf area is all red and the background is white. For certain leaves, e.g. Potamogeton perfoliatus, shaded areas around the leaf might be covered as well. In that case, use the white paintbrush (double-click and the thickness and color can be adjusted) and manually draw the outlines of the leaf before clicking apply You should now have a black and white image (and if you drew outlines manually with white, you’ll see the correct edge of the leaf).image2020-11-30_11-6-48.png
  7. If you have outliers (black dots etc. close to the edges of the leaf) you can remove them with the paintbrush in WHITE. The same goes for uneven leaf edges and holes inside of the leafà fill them with the BLACK paintbrush. Be careful not to use a too thick paintbrush. You can only undo one move if you draw wrongly.
  8. When the leaf is ready to be analyzed, select the box tool (first tool in the toolbar) and draw a box around the leaf (try not to include any dots or other outliers).
  9. Open the analyze menu and select “analyze particles”. A new window pops upà click “show” outlines to see which area corresponds to each object in the image
  10. Click OK and you should see a results box with one line for each object and a new window with your objects appearing as outlines. There are numbers in each object, but you may need to zoom in to see them. Preferably there won’t be too many objects (which is why we try to trim the image before analyzing it, point 7.) If there are many that interfere with the actual size, try to trim the image anew.
  11. Save the black-and white picture (with appropriate label and species name) and record the area of the leaf and perimeter in an Excel sheet (be sure to record the sample number and species).