I have been using Icy to try and segment 3D gastruloids stained with florescent markers and have come up with a protocol that splits the channels, applies the ‘Edge preserving Denoising and Smoothing’ filter before HK-Means thresholding to get a binary image. I am then using the binary image to generate a distance map. Subsequently, because there are a lot of overlapping and clustered cells, I wanted to use watershed to separate the cells with the distance map and binary image as inputs, however, I have tried playing around with the watershed block for some time now and it always results in an error and ‘bug report’.
Apologies if anything doesn’t make sense, I am fairly new to Icy to some of my problems may simply be because I have missed something basic.
I would be grateful for any suggestions on how to resolve this issue or suggestions of alternative methods of segmenting highly clustered cells.