Multiplex-immunofluorescence whole slide data analysis

Hi !
I need to analyse whole slide image with tissue stained with 4 fluorescent tags. I want to count the number of cells with each tag or a combination of two or more tags. I also want to measure the intensity of each fluorescent tag.
I have developed a pipeline:

  • First to identify the nuclie - this will be the cell count.
  • Each channel is save separately
  • Each channel is identified as a primary object
  • Each fluorescent spot is counted and the count and intensity is exported as a spreadsheet.

Is this approach correct?

Thanks in advance
IF_pipeline.cpproj (1.2 MB)
PCE-TDTG-T6_CRE _GFP_TDtomato_CC1_olig2_caudal_009_slice21.tif (2.0 MB)
PCE-TDTG-T6_CRE _GFP_TDtomato_CC1_olig2_rostral_006_slice16.tif (2.0 MB)
PCE-TDTG-T6_CRE _GFP_TDtomato_CC1_olig2_rostral_007_slice9.tif (2.0 MB)


I am not sure I completely understand the pipeline and have no experience with CellProfiler, but due to the lack of other responses I figured I would mention that…
If you have a whole slide image (not a pile of tiffs), have you tried dropping it directly into QuPath (with BioFormats!) to try something like this. As a note, QuPath doesn’t have a great way to trace the cytoplasmic outline of a cell, so accurately measuring total intensity is difficult with closely packed or strangely shaped cells. If calculating the number of cells of particular classes is most important though, it might be a fairly quick and easy way to go.

Hi ! Thank you so much for your reply. Will definitely look into this.

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