I recently met Cell Profiler which seems great! I need to prepare a pipeline for myself but I couldn’t figure out a few things. I seed two different types of cells in my plates and need to count only one type which is detected by a cytoplasmic marker (Alexa 555,). Among the cells I need to count, I also need to separate them as positive or negative against a second marker (Alexa 488). In short, I need to count how many nuclei (seen in DAPI) are present under the red mask (555) and how many of them are green (488). My first problem is identifying the red area, and cropping the rest of the image where there are other DAPI labelled nuclei which I’m not interested in. I attached one image. In this one there are two DAPI labelled nuclei which I want to exclude, one is outside of the red mask while the other is on top of the red area. How can I do these two types of exclusions?
The order of my pipeline is
ColorToGray (my images are in RGB) --IDPrimAuto – IDSecAuto – IDTert – OverlayOutline
Can I do this with the crop module? If so, how can I specify the red areas?
Secondly in the case of thresholding, I tried but Otsu and MoG but they basicly divide a single nuclei into lots of different regions even though I chose “shape” for distinguishing clumped objects. Then, I tried interactive settings and set up an absolute threshold. Do I need to set the threshold each time or am I not using Otsu/MoG right?
Final question is if I have images with different magnifications, do I need to change the diameter range of nuclei in the module?
Many thanks in advance!