I am currently during experiments where I am comparing transient transfected/untransfected NIH3T3 cells with various mutants using cellprofiler. The transfected cells are coloured green by an HA-tag in the GFP channel. Everything in the pipeline works fine except for this:
I am currently seperating the transfected and untransfected cells by the ‘FilterObjects’ module and then defining transfected cells as objects with a minimum mean HA-tag intensity of X.
The problem is that pictures from the same experiment are not correctly filtered by the same intensity threshold; if it’s a low minimum intensity value, it works fine in some pictures but includes too many clearly untransfected cells in other pictures. If it’s higher, the untransfected cells are filtered out in some pictures (as they should be), but so are clearly transfected cells in other pictures.
I have tried playing around with the correctillumination calculate/apply modules, rescale intensity and enhance/supress features modules but nothing has worked for me yet. It might be that I just don’t know how to apply them correctly
I have attached the pipeline as well as examples of pictures that don’t add up. For the first image (01) the minimum mean intensity need to be 1.5 to filter correctly, while the second image set (05) it needs the minimum to be at 1.2.
Pipeline JNO 2.6 changed for 20201109 2 with no background editing.cpproj (2.2 MB) NIH3T3 D1 RhoA E40Q 01-Image Export-08_b0c0x0-1388y0-1040.tif (4.1 MB) NIH3T3 D1 RhoA E40Q 01-Image Export-08_b0c1x0-1388y0-1040.tif (4.1 MB) NIH3T3 D1 RhoA E40Q 01-Image Export-08_b0c2x0-1388y0-1040.tif (4.1 MB)
NIH3T3 D1 RhoA E40Q 05-Image Export-12_b0c0x0-1388y0-1040.tif (4.1 MB) NIH3T3 D1 RhoA E40Q 05-Image Export-12_b0c1x0-1388y0-1040.tif (4.1 MB) NIH3T3 D1 RhoA E40Q 05-Image Export-12_b0c2x0-1388y0-1040.tif (4.1 MB)