Problem with measuring areas within ROI

I’m a newbie here and use ImageJ just for basic stuff. I found this forum because I have a small but irritating problem that I cannot resolve myself. I’m trying to analyze the amount of collagen from the trichrome stained heart cross-sections with ImageJ. Collagen stains blue and is easily distinguishable from red muscle tissue, which makes it easy to select with “color threshold”. But then: I want to measure the amount of collagen only from the left ventricle wall, not from the right ventricle or the areas without cardiac tissue (there might some fatty tissue etc.). I threshold the image, draw a ROI with “freehand selection” and then select either “measure” or “analyze particles”. The program measures the thresholded areas but ignores my ROI and I get results from the whole image. I have tried to use “ROI manager” and selected “Limit to threshold” from “Set measurements”, but still get the same results. What am I doing wrong? I noticed that ROI measurement functions correctly with 8-bit images, but there I cannot really make a difference between e.g. light purple and light blue.

Any help is appreciated :slight_smile:

@TainaV -

The only thing I can think of quickly at the moment… is that you need to ensure your ROI is selected in the ROI Manager - ie, it should be highlighted in ‘blue’. Then you know that ROI is active, and Measure should be restricted to that ROI.

Perhaps you can post an original dataset and your ‘protocol’ more specifically here… to be sure we can reproduce your workflow and potential error?