Hi, I have just begun using CP so I have downloaded the latest version of CP (1.0.7522) running on a Mac Pro at home (where I’m developing the pipeline) and will be running the pipeline eventually on a PC running Windows XP. The images are from an ImageXpress Micro (epifluorescent microscope). The object is to count cells in a population (8 images to cover entire well) that fall in to the following 3 different phenotypes:
- unchanged, normal nuclei
- mitotic nuclei (higher intensity nuclei but not bar-shaped because treatment causes breakage of DNA into high intensity spots within the nuclei)
- multinuclei (intensity typically low like normal nuclei but borders of several nuclei hopefully can be traced within the outer nuclear boundary)
Here is an image containing a mix of these cells.
This image has all three cell types, hopefully I’ve described them well enough for you to pick them out. I couldn’t create good images for each of the 3 types using the software I have at home.
Here is the pipeline that I’ve been working on, everything up to the PauseCP step.
I basically feel pretty good about defining the outer nuclear boundary for all of the cells in the first IdentifyPrimAutomatic. Then I thought that I could Crop so I can identify each cell and then associate (children, parent) further objects within each nucleus as either being mitotic or multinuclear. If there are no further objects found in the nuclei (or 1) then it would be counted as a normal (planning to use CellAnalyzer for final counting).
So, I’m able to get a good outline (and measurement) of mitotic using a threshold within the second IdentifyPrimAutomatic, but can’t figure out best way to get the multinuclear population outlined. I had an idea that I could expand the mitotic until it reached the background intensity and then exclude these nuclei from the next IdentifyPrimAutomatic but it is not working and I feel this is not the correct approach anyway. The tough thing is that the intensities of the 2 phenotypes are so different that I assume I need to deal with each one separately. With your expertise, I’m hoping that you can steer me in the right direction but giving me some potential module/pipeline steps. I have a short timeline on developing this so I’m hoping that I can get through this and devote much more time in understanding the nuances to this flexible analysis tool. Thank you for your help! And please feel free to send private messages to me if you need more information.
One thing I’ve discovered is that the background is currently very high and the outer boundary of the nuclei is only 1.5 or so above background. We are going to work on trying to modify final media conditions to bring that down (to improve the outer nuclei detection). The general pipeline development is still valid using this image though!
ExampleShannonPIPEspecklNoIllcorr_expand_excl.mat (2.01 KB)