(Note: I moved this from the CP1 forum to the CP2)
First, a couple pieces of advice:
- if you can, avoid the JPG image format. They are a lossy – TIFs are a better choice, or PNG as they produce smaller files.
- Save the images in separate channels as grayscale. It speeds processing time and disambiguates the color unmixing step.
Re: the pipeline:
(1) LoadImages is looking for files with “30” in them. I needed to change this to something common to all files, like “14-12-2010” . Presumably this is just an oversight for these examples.
(2) Use Test Mode for debugging. Mine shows “Caution” symbols where settings are obviously incorrect.
(3) CorrectIllumCalculate, in the “Each” mode, almost certainly should have some smoothing, and not all of yours do.
(4) CorrectIllumApply should use the “Divide” option, not Subtract, when using the “Each” mode and especially when using “Rescale” in CorrectIllumCalculate. This is a common mistake, and likely the cause of your blank images. This is documented in the Help buttons (labeled with “?”).
(5) You are not using the Corrected images (e.g. “CorrRed”) anywhere later in your pipeline. You likely should change the inputs of IDPrimaryObjs to input the Corr images
(6) I would try some other smoothing options in CorrIllumCalc, since the Splines option gives some odd looking illumination functions. Too peaked?
(7) Most of the green cells are being excluded because the size range of 32-60 pixels was too large. Lower it, or don’t exclude objects outside this diameter range.
(8) You can add a CalculateMath module to calculate the ratio that you desire.
I am attaching my pipeline. Let us know how it looks.
pipeline_FDA_DL.cp (10.5 KB)