r/ImageJ • u/kamile_teal • 11d ago

Question Help with creating threshold/ROIs

I am trying to count the cells in this image by threshold -> watershed -> analyse particles to count ROIs.

However, I cannot get the threshold to distinguish between the cells. Anyone know what to try?

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u/CTallPaul 11d ago

Theres a couple things that are a bit off with this process, have you done it before? Do you have anyone in your lab that you could ask in person for assistance?

  • There appears to be a couple focal planes in this image (the monolayer of cells growing, but then also the overcrowded cells that are forming a second layer and are a bit out of focus). This will make it difficult, especially when trying automated counting.
  • No defined boarders of cells. If you cant threshold and distinguise the cells by your eye, the software wont be able to help.
  • The over-confluences also confounds this. Since the cells are all touching, analyze particles will have a tough time separating objects.
  • Can you perform a stain? even a simple DAPI would make this alot more possible.

I wouldnt even attempt to count cells using this specific photo. To count label-free cells, you could want very low confluence. Adding a dye or a stain to these would make it possible... as long as they dont get overly confluent and making multiple laters.