Segmentation of touching objects (S. aureus)

Hi,

I would like to automatically detect fluorescent objects (bacteria) in a z-stack using ICY.

I’ve been using HK-mean to detect my objects but I’m struggling to separate touching objects using distance map and watershed. My touching objects are either not separated or the separation doesn’t make sense and I can’t fix it by playing around with the parameters. Does anybody know what the problem is and how to fix it?

This is an example image: Staph.tif (395.9 KB)

And this is the protocol editor I’ve tried: staph detection.protocol (9.9 KB)

Thank you for any help!
Sina