Measure Cell Fluorescence

Hello everyone,

based on my DAPI/nuclei signal, I would like to measure the signal of a fluorescent channel I have recorded. As my cells are fairly dense, I assume, that some Voronoi-estimation/propagation for estimating the corresponding total cells would be sufficient. However, while all the necessary theoretical steps appear clear to me (having worked with Fiji/ImageJ successfully), I seem to fail at a very early point in developing a practical pipeline:

I cannot even segment my nuclei correctly! Much less measuring my additional fluorescent channel per cell. (Having tried quite a few pipelines I found online, I assume that there must be something fundamentally wrong either with my understanding of how to identifyprimaryobjects for a nuclear stain, or with my images per se.)

I would appreaciate some beginners help if possible

blueStone




CP_CellFluorescence.cpproj (455 KB)

I believe what’s happening is the same as what is reported here: Merging objects. In short, the max intensity sum isn’t indicated by the image format, and the usual scaling done in CellProfiler isn’t occurring.

The easiest workaround is to use ImageMath to multiply each image by 0.00000000023283064365386962890625 (i.e, divide by 32 bits = 4294967296 which is what CellProfiler thinks it is).
-Mark

Awsome, thank you, Mark. That works just like a charm! :smiley: