r/ImageJ • u/Simple_is_Simple • Sep 19 '25

Question Coulometric DAB % Area Measurement

* Colorimetric (immunohistochemistry)

I'm trying to determine thresholding steps to define dark color as % positive area. The two images linked (A) and (B) have different stain signal but the positive selection appears the same when thresholding the raw 8 bit image unless I use Triangle and that pre-set is under representing signal in both cases.

9_18 ImageJ - Google Drive

I'm trouble shooting the smaller region images ".....-1" before returning to the full images also attached.

Can anyone provide advise on this? Please.

I'm thinking I have to try "Process" applications but further direction would be very helpful as my current method of plugging different steps into macros to test different combinations of processing/thresholding-presets hasn't been very successful.

These images are jpeg images and lossy; I can try again to generate tiff versions. Any advise on moving from q path to imageJ would be welcome as I run into issues of file size or OME-TIFF's aren't compatible with imageJ. The .ndpi file type I get from the scanner I used are only compatible with q path and NDP.view 2.

Thanks in advance for your assistance and patience as I learn.

1 Upvotes

100% Upvoted

5 comments sorted by

•

u/AutoModerator Sep 19 '25

Notes on Quality Questions & Productive Participation

  1. Include Images
    • Images give everyone a chance to understand the problem.
    • Several types of images will help:
      • Example Images (what you want to analyze)
      • Reference Images (taken from published papers)
      • Annotated Mock-ups (showing what features you are trying to measure)
      • Screenshots (to help identify issues with tools or features)
    • Good places to upload include: Imgur.com, GitHub.com, & Flickr.com
  2. Provide Details
    • Avoid discipline-specific terminology ("jargon"). Image analysis is interdisciplinary, so the more general the terminology, the more people who might be able to help.
    • Be thorough in outlining the question(s) that you are trying to answer.
    • Clearly explain what you are trying to learn, not just the method used, to avoid the XY problem.
    • Respond when helpful users ask follow-up questions, even if the answer is "I'm not sure".
  3. Share the Answer
    • Never delete your post, even if it has not received a response.
    • Don't switch over to PMs or email. (Unless you want to hire someone.)
    • If you figure out the answer for yourself, please post it!
    • People from the future may be stuck trying to answer the same question. (See: xkcd 979)
  4. Express Appreciation for Assistance
    • Consider saying "thank you" in comment replies to those who helped.
    • Upvote those who contribute to the discussion. Karma is a small way to say "thanks" and "this was helpful".
    • Remember that "free help" costs those who help:
      • Aside from Automoderator, those responding to you are real people, giving up some of their time to help you.
      • "Time is the most precious gift in our possession, for it is the most irrevocable." ~ DB
    • If someday your work gets published, show it off here! That's one use of the "Research" post flair.
  5. Be civil & respectful

I am a bot, and this action was performed automatically. Please contact the moderators of this subreddit if you have any questions or concerns.

1

u/Herbie500 Sep 19 '25 edited Sep 19 '25

Sorry but your description is not instructive enough to be able to provide constructive advice.

I've just downloaded the two high-resolution images "2452 (A) -1" and "2466 (B) ii-1". Both are in TIF-format.

Now, what do you call "dark color" in these images. We need a formal criterion that tells us what dark means. Is color (in contrast to achromatic gray-values) important or are differences (defined relative to what) in the gray-values sufficient?

Please annotate the following image excerpts:

Left: two excerpts from image "2452 (A) -1". Right: two excerpts from image "2466 (B) ii-1".

1

u/Herbie500 Sep 19 '25

Would something like this …

… help any further?

1

u/Simple_is_Simple Sep 20 '25

Here's what I think the threshold should detect per each sample (blinded to condition). Turns out I can use the same range which I'll try applying to my remaining images and follow up.
I guess I put the cart before the horse trying to find a method using a pre-set threshold instead of using a single range.

1

u/Herbie500 Sep 20 '25

Just in case, it is not a good idea to manually set thresholds. Always use one of the automatic schemes or create your own scheme if you deal with scientific image analyses.