I’m an old user of Cellprofiler for some months. Nowadays, I use it analysis the intensity and rate of GFP’s expression in chicken myoblast cells and whole embryonic fibroblast cells.
We have blue photos in which nuclei are stained with Hoechst33258 and green photos that are GFP’s expression. But there are only some cells expressing GFP rather than all. Besides, the cells’outlines are long and irregular, the intensity of cells are discrepant, so we don’t get perfect cell’s outline although we adjust the Threshold correction factor in IdentifySecondary.
Here is an example of pictures:
I have created a pipeline about it. Here is my current pipeline:
Call the resulting greyscale image: BlueGray, gfpGray
Size of smoothing filter: 9
Suppress local maxima within this distance: 10
Set interactively (in intensity distribution, I choose 0.08 of image 1, 2 and 0.195 of image 3, 4)
Call the grayscale images: gfpGray
Call the objects: cells
In the Excel of the cells’intensity, there are some cells whose mean intensity is higher than 0.08 in image of 1, 2 and 0.195 in image of 3, 4, others are lower. So I choose the former as GFP-positive cells. I want to know whether the way is right.
And there is some diversity about the intensity of different images, even though the backgrounds’ intensity is different, so I can’t compare with the mean intensity of many images. I think I can adjust them through some coefficient, but I don’t know. How shall I do?
I look forward to your prompt help.
Thanks a lot.