Probably, you’ve heard about the battle between Petiole Pro and ImageJ. However, if not – soon you will get more details about this. As of now, it is the right time to start with ImageJ. However, for any researcher, who would like to use this software it would be a problem to understand how to measure leaf area with ImageJ without a step-by-step instruction. Also, there are no full guidelines for ImageJ leaf area index measurement. Hence, this article will tell you in details how to measure leaf area with program ImageJ.

The basis of the ImageJ experiment

ImageJ is a program for personal computer, which is available for Linux, Windows and Mac. It was developed at the University of Wisconsin-Madison in the United States for processing and analyzing images.

For today’s experiment we will have three green squares to measure the “leaf area index“. We intentionally use the shapes of known size to give you the overall understanding of the process of measurement leaf area with ImageJ and Petiole Pro.

To measure leaf area with ImageJ we need to have a photo of the green squares or in real life it has to be green squares but also, you must have a ruler on the same photo in order to set the scale. Examples of measuring leaf area index with ImageJ and Petiole Pro on different crops such as cucumber, strawberry, tomato and pepper. Photo credit: University of Florida Examples of measuring leaf area index with ImageJ on different crops such as cucumber, strawberry, tomato and pepper. Photo credit: University of Florida

The length of each side of the square is approximately two centimetres. As you can see by grid paper and thanks to the ruler which we have.

In our case we measure these three green squares, the photo is taken with a smartphone and then transferred to the desktop of the personal computer.

In our case we use ImageJ for Linux so if you have any differences in the interface, they can be caused by the operational system. And we suggest they are not impacting the precision.

Knowing the size of each side, we can suggest and visually see that the area of each square is 4 square centimetres. Or not four? This is a question for ImageJ today.

Step 1. Launch of ImageJ and open the photo

First of all, we need to launch an ImageJ application on your computer. Then you need to open this photo in the ImageJ program. Go to File and choose the submenu Open.

As you can see now the photo with green squares is open within the program and we can place this window upon our convenience.

Step 2. Setting a scale

The next step for leaf area measurement with ImageJ is setting a scale. For this task you need to go to choose the Line instrument and make a line on the ruler between 10 and 15.

This means our line will have a length of 5 centimetres and we can use distance for setting scale. Go to menu Analyze and choose submenu Set Scale.

In the new window you need to put number 5 next to the field Known distance and write “centimetres” next to the Unit of length.

Then you need to press OK to apply the changes.

Step 3. Image preparation for leaf area measurement

After we set a scale for estimation of leaf area we can start work with squares. Go to menu Image and choose Type 8-bit.

This will make your photo black and white.

The next step is to go to menu Image, choose submenu Adjust and in the available options press Threshold. You can see that the appearance of your squares has changed. For more advanced levels you can regulate some of the features to change the contour of leaves. However, as of now, we will leave the squares as they are. You can press Apply and then your squares will get black colour but for our experiment we will leave them red for visual differentiation. You can leave the squares in red just closing the window and not pressing Apply.

Step 4. Image analysis for estimating leaf area

After this step, you need to go to menu Analyze, choose submenu Tools and then go to ROI Manager.

We will move the new window to make it convenient for further work.

The next important thing is to check the two options – Show All and Labels. You can see these options in the lower corner of the ROI Manager window.

For the next step you need to choose the Wand (Tracing) Tool from the panel of instruments. Click on the first leaf, press the button Add [t] in ROI Manager and you will see number 1 on the first leaf.

Do the same with the second and the third leaf and you will get their numbers respectively.

Then press the button Measure in the ROI Manager window and you will get the area of the squares. For our case we have the first square with 4.2 square centimeters, the second square with 4.91 square centimeters and finally the third square with 4.12 square centimeters.

Also you can notice the rough sides of each square and this aspect, probably, creates some inaccuracy. At the same time, the plant leaves in most cases have different edges, hence, it may impact on the precision of the measurements.

Step 5. Export of ImageJ Data

Finally, you can export your measurements in a .CSV file for further analysis.

Video instruction how to measure leaf area?

As you can see, we got more specific and accurate results with ImageJ than estimating leaf area visually. Hence, now I am curious to compare the results of leaf area measurement by ImageJ and Petiole Pro. This will be a goal for my next experiments :).

Also, you can compare leaf area measurement with Petiole Pro. Soon we will prepare more detailed overview about the advantages of both methods – PC software ImageJ and a mobile application Petiole Pro.