Counting oocytes

Hi !
I’m working on a pipeline to count the number of primordial follicles in mice ovaries. I’m used to count fluorescent granulosa cells with for example DAPI/PCNA or DAPI/BrdU. The main difference here is that I did whole mount immunofluorescence so as it’s the entire ovary I can’t use DAPI that only results in a very blurry signal due to the very high density of cells.
Moreover, several oocytes are overlapping and as there are different stages of development I would like to be able to differentiate the oocytes based on their size but it might not be essential. If I can get it working for the primordial follicles it would already be great.
I attached a picture and the (very simple) pipeline I’m using for now and that I’m sure can be improved.

Thanks for your help !

Marie

Oocytes_Counting.cpproj (113.4 KB)

Stitch_Zstack.tif (3.4 MB)

Hi Marie

The first step is to identify what is not tissue and mask those pixels
37%20PM

Without a DAPI it is difficult to properly segment the cells.
The left image is the identification using adaptive OTSU with shape segmentation (blue).
The right image is the identification after enhancing for circles before OTSU shape segmentation (yellow).
You have other options to enrich for cells and changing threshold correction values.

10%20PM

Cellprofiler v3.5.1 Oocytes_Counting.cpproj (652.4 KB)

Best
Lee

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Hi Lee !

Thank you so much. It’s much better than what I was doing. I’ll try to play with the diameter of objects threshold now see if I can get it to independently count the primary and secondary follicles too.

Thanks again,

Best,

Marie

1 Like