However, if you’re associating the spots with a filtered object set then sometimes the cell it was touching most was already filtered out, and so that particular spot then gets related to a different parent cell. When a spot is on the border between two cells, it should be associated with whichever cell more of it is touching. However, your cells are quite dense and often end up touching. In your pipeline you’re using the spot counts to generate filtered object sets like “Red cells”, then you run RelateObjects again to associate the spots with that filtered set of “Red cells”. I think what is happening here is as follows:Įach spot gets related to a cell using RelateObjects. Replying here in the main thread, but sent over some images. RNAscopeCellprofilertroubleshooting.pdf (412.3 KB) It claims in the filter objects module that cells are filtered so that only three dots are accounted for, but I cannot figure out where this measure is set, where I can adjust this, or if this is even the problem.Īny help would be appreciated! Many thanks in advance. However I am having difficulty figuring out where in the pipeline I can adjust to fix this. As you can see by the red transcript, there is a considerable number of red pixels in the green cell. I am trying to figure out why this cell is filtered out. In the third image, you can now see this same green cell, but it does not colocalize with the red cell. When you look at the second image, you can see this same cell (red arrow) is detected as a red cell. I have pointed out one green cell (red arrow) that has red transcripts. I have zoomed in on one area- You can see green cells in the first picture along with the red transcript. For instance, I have attached a document containing a few example images generated in this process. I have the pipeline working for the most part, however I am finding it difficult to get the colocalization that I expect. 2017 in CellProfiler to try and automatically quantify the number of cells containing green, red or white transcripts, as well as the number of green cells containing white or red, none or both transcripts (a colocalization analysis). I am currently using the pipeline by Erben et al. My images are fluorescent and detected using epifluorescent microscopy. I have performed RNAscope for three RNA transcripts and have used DAPI to identify cells.
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