I am coming here for expertise on pipeline optimization for cell counting, specifically counting fluorescing adherent cells. My cells overlap quite a bit, and this can make the cell counting procedure a bit tricky. I have attached a result of the pipeline I have made, but as you can see, it is forgetting to count quite a few cells in the middle… I believe that this is due to the threshold that is automatically generated, at 0.186.
Here a the set parameters of the IdentifyPrimaryObjects module that I have been using (I have also attached my pipeline if it makes it easier):
- Typical diameter: 30-180 pixel units, Discard objects outside range YES.
- Threshold strategy: Adaptive (Should Global be used??)
- Thresholding method: Otsu (Is this the right choice…?)
- Two-class or three-class thresholding: Two-classes
- Minimize the weighted variance or the entropy: Weighted variance
- Select the smoothing method for thresholding: Automatic (Is it better to use a manual method? If so, how to play around with it…?)
- Threshold correction factor: 1.0
- Lower & Upper bounds on threshold: 0 - 1.0
- Method to distinguish clumped objects: Intensity
Thank you for your help!
Updated_Counter_export.cppipe (8.0 KB)