I am new to this community and am writing for great help in my project.
We want to compare the structural change of the retinal pigmented epithelial cells (RPE) after drug treatment. We try to analyze the difference in some parameters, e.g., form factor, solidity, roundness, eccentricity, neighbors, nuclear number within the cells. Hence we stained RPE cells with ZO1 antibody for the tight junction protein and DAPI for nucleus. Because the staining is very dirty, there are a lot of staining debris within the cells. I have to remove the dirt within the cells using Photoshop before I can run in Cellprofiler, which is very time-consuming and frustrated. After I ran the files into Cellprofiler, most of the cells can be counted correctly, but there are about 1-2% cells were recognized as two cells as indicated in my files. You can access my original confocal images, edited files and pipeline file in my Dropbox (https://www.dropbox.com/sh/u1p7whrcyd1n2cb/AABSr91ueZMC9AMsFNedO5fFa?dl=0). The cells which were recognized as two cells were labeled with blue or black dots in the .png files. I have the following questions for help from experts working in Cellprofiler.
- How can use the documents directly without editing within Photoshop.
- How can I avoid cells being split.
- Is there any other parameters can be used for distinguishing the structural difference amount these cells? A recent paper (https://www.biorxiv.org/content/early/2018/11/21/372755) indicated that the method in the paper maybe better than the parameters (e.g., form factor, eccentricity, solidity and roundness) listed in Cellprofiler. But I have no idea on how to use the method in Cellprofiler.
Any help and recommendation from the forum would be greatly appreciated!