Hi, Integrated density is the product of area and mean value, while raw integrated density is simply the sum of all pixel values. As such integrated density depends on the scaling of the pixel size (whether one pixel is 0.1 or 0.5 um will make a difference) while raw integrated density doesn't. If you are acquiring images under identical settings (the pixel size is always the same) you can use either value. Both values depend on the size of the imaged area (assuming a uniform cell density, there will be 4x higher integrated density in an image of a 4x larger area) as well as on the cell density (empty areas contribute little to the integrated density, so assuming identical cells, the integrated density will be about 4x higher when there are 4x more cells in the image). So when evaluating the intensities you need to consider what type of information you want to extract. If you for example want to compare cell fluorescence intensities and individual images differ largely in the number of cells present, you'd need to do some segmentation (e.g. thresholding) first to find the area occupied by the cells and then compare integrated intensity from the areas occupied by cells rather than from the whole images. See also https://www.researchgate.net/post/Mean_gray_intensity_value_or_the_integrated_density_value_which_among_the_two_value_is_more_appropriate_for_quantifying_the_fluorescence_image_data

Maybe you will find this page useful

https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.htmlhttps://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html