Cell profiler help identifying larval tubes?

Hello all, I am trying to use cell profiler to distinguish individual “tubes” in which microinvertibrates/nymphs reside. Having a hard time applying the appropriate thresholds to clear out some of the noise and reduce the “primary identified objects” identified to only include the individual tubes. I’ve attached images of a couple of the tanks, if anyone can help with suggestions on pipelines and thresholds that may be helpful to use I would greatly appreciate it.

Hi @ridetiludie,

Would you be able to upload a copy of the pipeline you’re currently using? If you’re running this on the raw photos you might need to add some steps to remove the tank walls and other objects which aren’t of interest. Is there a particular region of each image that you’re interested in?

I have attached a copy of the pipeline I was using. I am new to this program and am sure there is some issues with the pipeline. I was converting the original photos to grey scale and trying to enhance contrast of the darker tubes and the clean/lighter areas near them. Thanks for the help.
Tanytarsus pipeline.cppipe (12.2 KB)

Okay, if you switch your ColorToGrey module to ‘Merge’ mode rather than ‘Split’ it actually doesn’t do a bad job. However, you’re discarding a lot of objects with your diameter filters. From there you might want to try changing the declumping parameters, particularly only filling holes after declumping. You may also like to try out the EnhanceOrSuppressFeatures module, which has a mode dedicated to finding tube-like objects. It might also be worth adding a crop module to exclude the edges of each tank.

Unfortunately it’s difficult to provide specific settings without knowing exactly what you want to detect. I assume it’s the dark structures that are of interest, but do they need to be a particular shape or size?

The dark structures are what I am interested in however the sizes can vary a bit. The dark structures are tubes that are formed by larvae chironomids which vary in shape and size somewhat drastically from first instar (first larvae after hatching from egg) to later instars prior to forming a pupae and metamorphosing/emerging into an adult fly. The first couple of instars may be to small to detect via photograph.