MeasureFluorescentIntensityDistribution Data Output for Single Cells

Green.tif (2.6 MB)

Hi guys. So it’s my first time working with Cell Profiler after bingeing all the tutorials and examples from the Broad Institute lab and I need a couple clarifiers and possibly some help.

I am attempting to run two analyses on the cells that I labelled “Green” which are just COS-7 cells transfected with a GFP-tagged protein.

  1. The first analysis is to assess the fluorescence intensity’s in different regions in the cells going outwards from the nucleus. My CellProfiler pipeline goes Images > Metadata > NamesAndTypes > Groups > Identify Primary Objects > MeasureFluorescentIntensityDistribution > Export to Spreadsheet

Now, at the moment, I’m attempting to identify cells using “IdentifyPrimaryObject” which is working okay. Ideally I would have the nuclei stained with DAPI and use “IdentifyPrimaryObject” for those and “IdentifySecondaryObject” for the cells, but the nuclei don’t make a difference in the way I plan to analyze my data and I also do not have, nor have the time, to generate that data. I’m having issues with my pipeline excluding those little protrusions in my cells which you can see have a high concentration of GFP however, I’m less worried about that as I believe I can solve that by working on the thresholding.

**What worries me at the moment is following the “MeasureFluorescenceDistribution” module, I get an output of the fluorescence intensities per bin, however it seems to be averaging out all identified cells as opposed to showing me the fluorescence intensity distributions per cell which is what I’d want. Is there any way to see this information?

  1. The second analysis seems to be more tricky as I don’t know where to start. I want to quantify all the GFP aggregates at the peripheries of the cells, which tends to work on ImageJ with the function Local Maxima’s, but I can’t seem to find an equivalent in CellProfiler. Now I do understand you can import the scripts from ImageJ into Cell Profiler however, I want to adapt this to a high-throughput screening platform eventually and would like to stick to one program, ie CellProfiler. Ideally, I’d want to use the bins from the “MeasureFluorescenceIntensityDistribution” module and quantify GFP aggregates and their intensities in there, however, I don’t believe that’s possible. Any tips?

Thank you, and if I missed any crucial information or my information is unclear, please let me know!

Hi @jliebert,

At least for question one the cell level measurements will be in the object spreadsheets. So whatever you named your primary objects (e.g. nuclei), you will have a spreadsheet in that name where all detected objects will be measured. Some of the columns will be called something like “RadialDistribution_MeanFrac_GFP_1of4”

Re: question 2, it might be useful to see what the results would look like when you detect the maxima in ImageJ. Pretty sure you are right that CellProfiler doesn’t have a direct function but there will very likely to be another way to get similar results.

And the second part of your question 2, I think you are right that you can’t access the bins created by the Intensity Distribution module but there may be a sufficient alternative, for example using the RelateObjects module you can measure the distances between the parent and child centroids so you would be able to get an idea of how far the maxima are from the centre of the cells.