How to Count and Measure Roots Using ImageJ: A Protocol for Auxin Experiments

https://www.youtube.com/watch?v=PqQIvCWd6Dg

https://youtu.be/PqQIvCWd6Dg Summary

Technical training session established standardised ImageJ measurement protocols for quantifying auxin experiment root growth and length. ImageJ Setup and Scaling: Participants installed the ImageJ software and configured image scaling by setting rulers to establish consistent pixel-to-millimetre units. Counting and Measuring Roots: Researchers utilised multi-point tools for root counting and freehand tracing for curvature measurement. Each recorded segment is mapped to data tables for analysis. Data Compilation Workflow: Teams exported measurement results to Excel spreadsheets to calculate total root lengths. The process requires repetition for all 5 experimental specimens.

• Image Processing Preparation: The session began with an overview of using ImageJ software to conduct measurements on rooted plants for an auxin experiment. Participants were instructed to install the software, open their respective experimental images within the application, and zoom into the region of interest using the control key and mouse scroll function (00:12:20).

• Setting Image Scale: To ensure accurate measurements, participants must establish a scale for each image. This process involves creating a line over a ruler depicted in the photograph, selecting the "Set Scale" tool under the "Analyze" menu, entering the known distance (e.g., 1 millimeter), and checking the "Global" box to apply the settings (00:17:09). It was noted that this scaling step must be repeated for every new image, as pixel dimensions vary between files (00:24:44).

• Counting Roots: The "Multi-point" tool is utilized to count the number of roots on the plant specimens (00:18:07). After clicking on each root to mark it, the total count should be recorded by pressing "M" to generate a data table, which can then be transferred to a folder for the auxin experiment data (00:19:13).

• Measuring Root Length: To measure the length of curved roots, the "Freehand line" tool is selected by right-clicking the straight-line icon. Researchers should trace each root carefully, pressing "M" after each trace to record the length in the specified units (00:21:09). This procedure must be repeated for every root identified in the initial counting phase (00:22:06).

• Data Compilation: Upon completing the measurements, all data points should be copied from the ImageJ results table and pasted into an Excel spreadsheet. The final step involves calculating the summation of all individual root lengths to determine the total root length for the plant specimen (00:23:25). This entire measurement process must be repeated for each of the five experimental plants and for both current and future measurements (00:24:44).

Apr 30, 2026

Meeting Apr 30, 2026 at 08:51 GMT+08:00 - Transcript

00:12:20

Oops. Yes. Yes. Okay, let's do this. Okay. Uh let's do this. So uh we'll have a look how to use image J uh to do the counting and measurement for your rooted plants uh in your oxen experiment. Let me share the screen. Just share the entire screen and the sound screen share. Right. Okay. So this is the uh image provided by one of you yesterday. So you need to um have your image ready. So just in here I already got it installed um my PC. So you have that little tabs there. Get your image. Just drop it in there.

00:17:09

So number one, when it's ready, it's going to look like this. You have your image and then you have your image J uh tab. Number one, we need to set the uh the scale for for this image. This is why you need to take your image uh the subject with a ruler. So what you need to do is we need to zoom in to this bit here. Press control and then use your mouse scroll up. So that will automatically scroll and zooming in into the region that you are interested with. So we want to set the scale now. Go to analyze set the scale. Oh sorry um we need to create a line first. We need to create a line. So you can see that this is worth 1 mm.

00:18:07

Right? So starting from here all the way to here middle to the middle. So go to analyze go to set scale and that is the non uh pixel which is this line here. The non distance is one. The unit is millimeter because this is the ruler that you use. Just make it global. Okay. So once uh the scale has been set uh we can unzoom control scroll down. Let's proceed with the counting of your root. Right. Uh to do the root you need uh a different tool. Can use the tool here. It's called multi-point tool. So this image uh it seems like it has got a number of roots here small roots big roots.

00:19:13

So we're going to count them all. So use those multipoint just simply click on your root. So you click here one two. You see as you click the number is going to be left uh on the image. clear this um numbering later. Every single one of them. Okay. If you if you want to move for a bit, so you can use this um scrolling tool. It does one 12. Okay. Okay. That's that's all. Uh let's say that that's all that you have. You have 12, right? So you need to press M so that you do not forget the number that you have just counted which is 12. So you got the 12. Go to your communial plus data pool for oxygensation in your folder.

00:20:17

So this row number 12, this is my row specifically for this demonstration today. So the root car here put 12 because that's what you get. What about the root length? So the root the root length is the total length of all of these 12 root. So how do you do that? Yeah. So you need to close this. Don't worry about that because we're done with that. We need to clear all of these. Um shift A. Yeah. Now we need to use a different tools. Use a different tool. Um actually if you didn't if you do not want to remove the numbering that's also fine. Okay. Uh I just I just like to keep it clear.

00:21:09

So you need to go to this uh straight line. You need to change it to um right click. change it to uh freehand line so that you can measure the root because your root is not completely straight. It's a bit curved uh from here and there. So you can let's say that you want to come you want to measure this. So this that's the first number one after you have traced the root press M immediately that is the leg remember the unit that you set earlier for the scale setting. So this root here is 1.3 mm. Then you move with the uh uh second one. Okay, that doesn't look good. Doesn't look good. You can redo it again. You can zoom in. Okay, if that helps you with the tracing, do it slowly and carefully.

00:22:06

But that's number two. And then clear that again. You can move it here. back to the free line. So you want to do it for how many time? For all the 12 roots that you have um created or counted earlier, right? So I'm just doing it quickly because this is just for the demonstration. Um every time you're done, press M. Press M. So we're going to get the total root length for these all 12 roots that you have. Um right then this one is very long nine and I think this was somewhere that way. What else? In this one. Let's see that that's the other one. Uh yeah. Oh, where's the other one?

00:23:25

I think I missed one. Uh where is it? Uh I think it should be somewhere. Maybe that guy here. Okay, this is clearly demonstration to waste too much time uh making this video, but you get the point. Okay. So let's say that this is all the result that you have. So you need to copy all of this. You can ctrl a copy. Go to your Excel. You can quickly paste it in there. So this is the leng right in millimeter. Um we need to change this in millimeter because just now I use millimeter. Millimeter. And the summation of this you need to get the average for uh the the no no you you do not need to get the the uh average you just need the summation of this which is 147659 147.659 65 147 I forgot 65 66 run it off.

00:24:44

Okay. Right. Uh total. Okay. Total right now. You're done. Okay, remember this is only for one because remember you got five, right? So do that again for the rest of the cutting in your cut. And that's all you need to do. Okay, you you want uh if you want to go back to your image A, you can close this. Uh you have to save. You can drag a new picture. But every time you drag a new picture, remember to set the new scale because the pixels is not the same. You see the pixel here. It's got it's got the pixel reading. It's got not going to be the same for for each image. Okay. So, uh do that uh for the rest of these for yesterday. um measurement and also the next week measurement. Then you are good to go. Good luck and I'll see you again uh next week. Right. Oh, that's all. Bye.