How to handle manual segmentation WITHIN a ROI?

Dear community,

In summary, I am selecting circular ROIs containing Golgi-specific structures from transmission electron microscopy images. WITHIN these circular ROIs, I want to do manual segmentation (outline tracing) of Golgi structures. I have approximately 50 images with approximately 30-150 objects to segment in each. (I later want to sort and count these object into categories based on their morphology and e.g. distance to each other, but I will leave that out from this topic.)
I add an image to clarify a bit more what I am aiming at here:

I spent a lot of time trying to automate the segmentation, and I also tried Ilastik for that. I then concluded that this was probably one of these times where you just have to do the hard work, instead of looking for clever workarounds…

Now, what is constantly interrupting my process is this: As I segment the object inside the ROIs sometimes I would accidentally click in a way that will move the circular ROI, and then the segmentation I was making at that moment would be lost, and I would have to start over. This impacts the progression speed a lot. I would assume that there is a simple way to get around this.

Do you have any thoughts of how to improve the workflow and get rid of these constant interruptions? Can I burn the circular ROIs into the image in some way? Is there a better program for this than ImageJ? Is there something easier than the polygon tool? Any suggestions are welcome.

This post is doing something similar, but with a plugin I have no knowledge of. Also, the problem does not seem to be the sam. I include the link anyway: Select and measure multiple ROIs within the bigger ROI

This is my first post here, and I tried to follow all guidelines (but if you notice something, please tell me - I always have room for improvement).

Thanks a lot in advance!


I use the selection brush tool for selections of this kind. It is very useful as you can easily adjust the form of you selection. You can adjust the size of the brush by double clicking on the symbol. If I got you right you would like to make selections within a selection. Wouldn’t it be possible to “inverse” the first selection (circle ROI) before adding the new selections by hand?
Screenshot 2021-03-19 at 12.41.13

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Dear Charlotte,

On top of the Brush tool suggested, you can help yourself in several ways:

  1. Select all the circles in the ROI Manager and save them: ROI Manager>More>Save so you can always restart from there
  2. Select a circle at a time and duplicate: you will make a new image that contains only the content of the circle where you can then annotate your golgi
  3. Select all circles, combine them (ROI Manager>More>OR (Combine)) and then clear outside of the selection: Edit>Clear Outside. Everything outside the circles will turn black. Clear the ROIManager and then annotate your Golgi with the brushtool, save the ROIs per circle.
  4. Select all the circles (as in 3), add them to the overlay (Image>Overlay>Add Selection). The outline of the circles will be burned in the overlay which you can show or not.




QuPath is a great program for manual segmentation. Not only are all of the annotations individual objects - they can be sent to ImageJ later for other, ImageJ specific analysis, there is an Undo button, plus many editing options like-
Alt+Brush or Alt+Wand to erase parts of ROIs, even well after creating them.
Locking ROIs so you do not accidentally edit or move them
CTRL+SHIFT brush or wand to draw right up to but not overlap previously drawn ROIs.
Specify annotation to create annotations of a fixed size
And most standard annotation adjustments like Expansions, Erosions, fill in holes and the like.

I really like having the whole project saved, so I can jump between images and check what I manually annotated in a previous image quickly, but without needing to leave all of the images open.
All of the objects are just… objects in QuPath, so moving the outer circular annotation would not really impact the inner ones.

Alternatively, there is a different version of the Wand tool here by… @jerome I think?

that may speed things up within ImageJ


Hi Brauer!

Thank you showing the selection brush option to me! I was not aware that it existed, and it is very helpful!
(I will look into the suggestion of reversing the selection, and also going in-depth in the helpful suggestions from Matthieu and Research_associate as well.)

Thaks a lot, and have a nice Easter!

Dear Matthieu,

Thank you for your clear and constructive answers! I am especially fond of alternative 3, since has the same dimensions as the original image making it possible to show the ROIs on the original image as well.

This vas very helpful,
Have a nice Easter!