I’m using CellProfiler to quantify nuclear import of pDNA. Therefore, my cells are incubated with labeled pDNA, afterwards the nuclei are stained and images are taken with the confocal microscope. The images are divided into the green channel for the nuclei staining and the red channel for the pDNA.
Image analysis is done like that:
- Images are loaded into CellProfiler and converted into grayscale images.
- Nuclei are recognized in the green picture with the “Identify primary automatic”-module, are then shrunken to be sure to “examine” only nuclearly accumulated pDNA and not pDNA at the outside of the nuclei.
- Then the intensity of the identified and shrunken objects are measured in the pDNA-picture
What I analyse is the integrated intensity.
I’m now wondering if my image-analysis makes sense, respectively if it is correct, because I read in a paper that direct quantification of fluorescence intensity is not possible, they speak about saturation of fluorescence intensity. I think my images are not saturated.
As I’m the only one in my working group who is working with this program, I would be glad if you could help me.