imagej color threshold black pixels still selecting

The second option is what I think you mean, based on your other posts. I mean… stained area could mean stained blue or brown (in which case the ImmunoRatio plugin for ImageJ might be relevant), or it could mean ‘the area I’ve selected that could potentially be stained, even if not all of it is’. By using ROIs, you do not need convert your image to gray scale. You might then have less flexibility in selecting the truly brown pixels in the gray scale image than you might have in the color image. My concern is that in converting your image from RGB to 32-bit real, you convert your color image to a gray scale image. Similarly you can use the color threshold tool to select the brown pixels and then also add that to the ROI manager. You can then use the Measure feature to obtain the area of the ROI. Add the inverted ROI to the ROI manager.Selecting the combined ROI, go to Edit -> Selection -> Make Inverse.Add this combined ROI to the ROI manager.Selecting all the ROIs in the ROI manager.Once you have added all the individual exclusion ROIs to the ROI manager, combine them together by The keyboard shortcut to add an area to the ROI manager is Control-t. Rather than blacking out the areas as in your first image, add all the areas you want to exclude to the ROI manager: I would consider getting the total area using ROIs.

Imagej color threshold black pixels still selecting