I used CellProfiler to quantify the number of Lipid droplets are contained per cell (I used LD540 for lipid-droplet staining and my sample was Brown adipocytes). However, when I compared the intensity of my differentiated adipocytes (that have more and bigger Lipid droplets) to the intensity of the non-differentiated adipocytes (that is mostly Background), I observed that cell profiler gives me the same intensity for both pictures/samples and in the end I get same results of amount of Lipid droplets for both samples. One can clearly see that the differentiated adipocytes have more Lipid droplets and also I openned the same pictures with PhotoShop and it gave me more intensity for the differentiated adipocytes (as expected).
Could you please help me with this and suggest me a way that I can adjust my settings in order to identify the difference between the intensities?
I attach two pictures (differentiated vs. non-differentiated cells) so that you can also see the differences and check if there is anything that can be done.
Thank you very much.