Thresholding background to zero

Hello,
Thank you for developing this program. I am still learning a lot about its applications(which thankfully are numerous). I am currently working on colocalization analysis between three dyes per cell(2 channel comparison for each). The main problem is with the background intensity differences across the three different filters.I would like to be able to eliminate the background intensities as much as possible for each channel(to zero,if possible) so that a pixel based correlation does not spuriously include background noise in my data. The background intensities also make it harder to perform an object identification(cell identification), and perform a correlation of fluorescent marker inside the cells. I end up having errors of boundaries being drawn around certain bright spots as cells even though they are just background. I tried to perform my background thresholding in metamorph before performing my analysis in CP but that also posed a problem. After thresholding, the images had extremely low background intensities(below 0.01) in cell profiler, and my pipeline could not run them no matter how much i tweaked the settings to match the intensities inside the cells. I am desperately in need of technical assistance. Please find attached my pipeline and sample images(originals) and edited in metamorph(background corrected). Thank you for your assistance.

Francis.
fbugyei@bgsu.edu










FrancisWorkingPipeline.cp (26.8 KB)

Hi,

Did my answer to this thread help here?

Best,
David

Yes it did.Thank you.

Francis.