Need help with analysing the colors of tomato seeds

I need to analyse different morphological properties of tomato seeds and create groups like:
seeds with a small area/Large area, oval shaped/round shaped (circularity) and light/darks seeds.
I already successfully analysed the area and cirularity with ImageJ, so I know how to select a segment of the full picture that I want to use, how to set the scale, covert the image to 8bit, adjust the threshold and analyse the particles. But I can’t seem to figure out how to get the color information of every individual seed so I can make 2 or 3 groups each within the different shades of brown present in the picture (ie. light/dark brown or light/medium/dark brown seeds).

The goal is to create 2 or 3 groups of color and sow these groups separately to see if there are any differences in seedling growth bewteen these groups. So any way to select the seeds by color is appreciated.

Thanks!

Hello Lorenzo -

From this, I assume that you do something like
Analyze Particles..., and get a bunch of
Rois for individual seeds in the ROI Manager.

Now take your original color image, and run
Image > Type > HSB Stack. For each slice of the stack (Hue,
Saturation, and Brightness) click Measure in the ROI Manager
(with the seed Rois still in the ROI Manager). For each seed
(Roi) you will now have the mean hue, saturation, and brightness
measured (to further process as you wish).

(You could also convert to a RGB Stack and measure the red,
green, and blue channels, if that makes more sense for your
use case.)

(If “Mean” is not being measured when you click Measure, run
Results > Set Measurements... from the Results-table
menu bar, select “Mean gray value,” and rerun the measurements.)

Thanks, mm

Thanks for the reply.

After I crop and duplicate the part of the image I’m using, I want to use “analyse particles” it says I have to change the picture to 8bit (black and white) to be able to set the threshold. So I turn the image into 8bit, set the threshold and analyse the particles. Now every seed is selected and provided with a number. The ROI opens with some info and I have the area of every selected particle (seed) in the results tab.
But from this point I’m lost because I now only have a black and white version of the image I’m using. If I duplicate the part of the full image that I’m using to get a color version again and I set it to RGB stack, it splits into 3 channels (rgb) but I can’t select anything here because it’s not set to 8bit (by which I’ll lose the colors).

I hope you could follow that a bit…
I don’t quite understand how to select the seeds without turning the image into 8bit (b/w).

PS: If I use 8bit color and set the threshold the selection is very bad.

Hello Lorenzo -

At this point I assume that you have a cropped section of your original
color image. Make a duplicate of this cropped image.

Perform this black-and-white, thresholded, analyze-particles
processing on your cropped duplicate, keeping your cropped
color image unchanged.

I assume that at this point, you also have the ROI Manager open
with a bunch of ROIs in it, one for each seed.

(If not, we have to sort this out. The ROI Manager should have
been opened and loaded when you ran Analyze Particles....)

Convert your cropped color image to HSB (or RGB) stack, make
sure that the stack (and desired slice (channel)) is selected, and
click on Measure in the ROI Manager. This will then use the ROIs
in the ROI Manager (your seeds) to make measurements of the
selected stack slice. You don’t need to select or re-threshold
anything – you will be using the ROIs that were created from your
black-and-white image and stored in the ROI Manager.

Thanks, mm

2 Likes

Thank you so much!

It worked, I got the mean brightness value per seed and used that information to differentiate between light and dark seeds. Exactly what I needed. Thanks again.