Hello CellProfiler Staff,
I am new to CellProfiler; I have been using ImageJ up to this moment. I need to analyze fluorescence intensity at a single cell level over time. The cells do not move during the experiment. Thanks to this forum, I have managed to set up the required pipelines in order to do such analysis. The analysis seems to work. However, the intensity values I obtain are completely different from what I would obtain using ImageJ (manual selection of cells). This is probably (and most likely) due to the fact that I did not subtract the background intensity in my image sequences.
FYI, we stain the cells with PI to mark their location. This is an example:
After reading the manual and many relevant posts on this forum, I have managed to come up with some different pipelines to correct for the background fluorescence. I’d like to share such pipelines with you and ask for your opinion. Am I approaching this the right way? Which pipeline do you think is best tackling this problem? Do you suggest something different?
Masked_Background.cpproj (1.1 MB) In this pipeline, I followed an old post (2009) from this forum that suggested identifying the background as an object if my objects were very clumsy (see first photo above). However, it seems like nothing changes when I do this.
Background_Median_Manual(8).cpproj (1.1 MB) In this pipeline, I used CorrectIlluminationCalculate:Background, Median, Manual. FYI, my objects are in the 05-10 range.
Background_Median_Object(8).cpproj (1.1 MB) : This pipeline is similar to the one above, I used CorrectIlluminationCalculate:Background, Median, Object(8).
I hope you guys can help me solve this issue as well. Particularly, I’d like to understand how to properly judge whether or not the background intensity of an image has been properly removed.
Thank you in advance for your time, I really appreciate it.