Protocol

Author

Amy Davidson

Overview

Measuring leaf perimeter and area using digital images and Image J software. NOTE: A similar protocol can be used to measure seed size (see Seed scans using Photoshop and ImageJ ).

Background

• Leaf area measures are commonly used to calculate Specific Leaf Area Specific leaf area SLA or Leaf mass per area LMA (area/dry weight) or LMA (the inverse of SLA).
• Leaf perimeter can be divided by the square root of leaf area to provide relative information on the complexity of the leaf shape (a high leaf perimeter:area ratio indicates a more complex leaf shape).
• The approach described here can also be used to measure leaf length and width, and applied to measuring other objects using imageJ.

Materials/Equipment

• Scanner attached to computer or digital camera and computer
• Image J software (shareware: http://rsbweb.nih.gov/ij/download.html )
• Fresh leaves
• Florist foam or plant press or ziplock bag and coolbox
• Circular sticker of known diameter, or object of known dimensions, for scale reference when analysing image

Units, terms, definitions

Specific leaf area SLA or Leaf mass per area LMA – Specific Leaf Area mm²= square millimeters

Procedure

Step 1 Leaf harvest

• • Harvest leaves of interest and either place in wet florist foam, or place in ziplock bag with moist paper towel and lie flat in a cooler/icebox/esky. Do not freeze leaves. The main thing is that leaves are kept cool and moist, in the dark and not allowed to shrivel up and contort. Leaves in a plant press will dry and shrink somewhat, so this is a less desirable approach, but can be used if there’s no alternative.

Step 2 Scan or photograph leaves

• • The important thing here is to make sure there is some scale indicator in your image. A ruler works, but a circular sticker (a sticky dot) of known area is ideal.
    • If scanning: Lie leaves flat on scanner (ensuring you have recorded which is which/location on scanner), spread out any lobes to ensure no overlap.
    • If photographing: you may need to flatten the leaves under a sheet of perspex/plexiglass to avoid shadows. Photograph in high but diffuse light to avoid reflections and your own shadow.
  • If the lobes still overlap you may need to scan the leaf twice, once with the leaf intact to get the projected leaf area’s perimeter and then again with the leaf cut into pieces so that there is no more overlap to get the full leaf area.
  • Include an object of known dimension (e.g. sticky dot) in the scan to use to set scale later.
  • You may want to include a label with details about your leaf as well
  • Scan the image in full colour or grayscale and save to your computer.
    • If photographing: download images and save to computer.

Step 3 Analyse scanned leaves

• • Open the scanned image in Image J.
  • Open the image menu and select “type” then select “8-bit”.
  • 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. Click apply. You should now have a black and white image.
    • This can be tricky. If your image does not have enough contrast, or your leaves are very pale, you may have problems and need to use drawing tools to darken the leaf. In the worst case scenario, print the image and colour in the leaves. This should only be necessary for very dissected or pale leaves. (You can also scan/photograph pale leaves against a black background to improve contrast)
  • Set the scale on your image. There are 2 ways to do this.
    • If your obejct is square you can use the straight-line tool from the toolbar draw a line across the object of known dimensions. Open the analyse menu and select set scale, type the known length into the “known dimensions” box, tick the global box and set the units.
    • Alternatively, and certainly better if your object is round, select the entire object of known dimension with the magic wand and enter the known area into appropriate box.
      
  • Measure the area of your leaf(leaves)
    • To analyse leaves one at a time, select the box or polygon tool from the toolbar and draw a box or outline around the leaf of interest, a single click will place a new line in the shape and double click to close the shape.
      • You can also analyse multiple leaves at a time in a given image, this saves time but requires more cross checking to be sure that each of the measured objects is actually a leaf. Select the area containing all the leaves using the box selection tool.
    • Open the analyse menu and select “set measurements”, make sure the area and perimeter boxes are ticked.
    • Open the analyse menu again and select “analyse particles”, a dialogue box should open. Select the minimum size of an object where it says “size (Pixels^2):”. Select show outlines if you want to be able to look back and see which area corresponds to each object in the image.
      
    • 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.
      • If multiple areas are shown check that you have as many objects as leaves. If there are more you may need to increase the minimum object size or edit your image to remove noise. You can do this manually using the paintbrush tool in the toolbar or by opening the image menu and selecting noise then “remove outliers” adjust the threshold size for outliers until you image is smooth. You may also like to experiment with the smooth and sharpen tools, remember to save your image between each step so that you can go back to an earlier version if need be.

Step 4 Calculate dissection index

• • Dissection index is simply the perimeter/(square root(area)). If your leaf is complex you will need to get the perimeter from the intact leaf scan and the area from the total area of the cut pieces of leaf. If the leaf is a simple shape both perimeter and area can be taken form the intact leaf scan. Ensure both measures are in equivalent units i.e. mm and mm².

Step 5 Effective leaf width

• • Effective leaf width is the diameter of the largest circle that can be inscribed within the area of the leaf.
    • Select the circle tool in ImageJ and stretch to the size of the largest circle that can be fit within the leaves.
    • Constrain the tool to be circular (as opposed to oval) by holding down the shift key while drawing your circle. Once drawn your circle can be resized by dragging on the little box “handles” around the margin of the circle. The circle and be moved by clicking the edge and dragging

Notes and trouble shooting tips

• If your leaf has interior holes, or lobes that overlap and leave a hole, you need to be careful with the area and perimeter measures. There is a box in the ‘set measurements’ box called ‘include holes’ which you need to be aware of

Preparation time: 5 min (time required to harvest all target leaves). Measuring time: ~ 5 min per scan (depending on leaf size it is possible to fin multiple leaves on the one scan). Analyzing time: ~3 min/leaf

• When collecting leaves decide if you want to include the petiole or not and make sure this is consistent for all leaves.
• Remember the scanner will take a mirror image of what you see on the scanner so be careful when recording plant locations on the scan screen that you note what is on your left when viewing it on the scanner will be on the right side in the scanned image.

Links to resources and suppliers

Related protocols: Leaf shape analysis

References

McLellan T, Endler JA (1998) The relative success of some methods for measuring and describing the shape of complex objects. Systematic Biology 47:264-281

Health, Safety & Hazardous Waste Disposal Considerations

• Keep scanner lid closed when scanning is in progress