Hi, I need your help with an analysis
I need to measure intensity of cells stained in GFP, and compare the intensity between samples (or genotypes). The images are tiff, 8bit, RGB color, and green if I look at image mode when I open them with photoshop. I have also the corresponding DAPI images in blue. I generated a pipeline where I load the images, and I use the module ColorToGray for both images (GFP and DAPI), then I use IdentifyPrimaryObjects with the DAPI image in gray and then I use modules for MeasureObjectIntensity and MeasureObjectSize using all the time the gray images.
I know that my 2 genotypes have different levels of my protein stained in GFP, but when the pipeline shows me the original image in green and the change to gray, the genotype with lower levels of GFP shows what it looks like an autoscaling of intensity, because the background doesn’t look as negative as it looks in photoshop. My question is: is the pipeline doing an autoscaling? how can I compare between genotypes? If it is autoscaling, can I use rescaleIntensity dividing by image minimum? Will that be equivalent to substracting the background?
Thanks in advance