Speckle Counting Help!

Hi everyone,
I am new to CellProfiler, but am loving it so far despite being a complete idiot when it comes to computers!

I am looking to use its Speckle Counting function, but with a bit of a twist: I want to compare the number of speckles in transduced cells expressing a protein of interest versus the number of speckles in neighboring untransduced cells.

So, image-wise, I have:

  1. DAPI
  2. Cye3 (stains protein of interest, is positive in ~60% of the cells within a given field)
  3. FITC (stains speckles)

And I want to see if the number of speckles is greater in that ~60% transduced cells compared to the remaining 40% untransduced.

Anyone have a good pipeline, or any suggestions to do this?
Thank you in advance!


Briefly, I would use
(1) IdentifyPrimaryObjects - ID nuclei
(2) IdentifySecondaryObjects - with the Distance-N option, simply growing out a certain number of pixels as a “cytoplasm”
(3) MeasureObjectIntensity in the secondary “cytoplasm”
(4) FilterObjects - filter your cytoplasm objects with a mean intensity > some value that you determine as constituting Cy3 positive cells.
(5) Identify and count the number of speckles in each population (filtered and unfiltered). I’m paraphrasing here, but you can use the Speckle Counting pipeline here for reference.