I would like to cut out a couple regions in an image that are unfortunately not usable for my analysis. I am trying to determine %area of the usable sample that displays intensity values above a threshold value. I have been trying to do this using the free hand tool and then selecting Clear from the Edit menu. My issue is that this does not remove these pixels from the total number of pixels in the image. This causes erroneous calculations (in my situation) for the % of pixels above/below a certain threshold value (%area in the set measurements menu).
To test this, I made an image that is exactly 50% black (left half) and 50% white (right side). When I clear an area from the white side prior to thresholding, I see a reduction in % area. However, when I cut a region from the black side (prior to thresholding) I see no change in %area above threshold. I would have thought this value should be much higher, since I exclusively removed only black pixels. However, I learned that clearing a region simply fills the area with background (black) pixels.
How can I remove multiple regions within an image so that these pixels don’t exist in subsequent calculations, i.e, they won’t be considered in % area calculations?
As you've noted, imageJ does not remove pixels from your image (e.g. as you might be used to seeing in Photoshop or GIMP, where pixels can be eliminated in a layer).
If your image is one such that you could measure within an ROI instead of the whole image, this would eliminated the need to clear the noise(?) that you currently remove. You would then use the area from the ROI as your total area, instead of the total image frame.
If not, then measure the area you delete (add them to the ROI manager and use the measurement function for example) and subtract it from the total negative area.
% Area = [above threshold] / ([below threshold] - [deleted area])