Segmentation of phalloidin stained actin

Hey there! I’m running into problems trying to classify C2C12 differentiated myotubes via identifying the outline of myotube. First, the myotubes were stained with Alexa Fluor phalloidin at a given concentration. When I go to pull up the images, the staining worked but worked too well. It appears that the there are multiple objects on different Z-planes/intensities. What I want to do is just to classify the outline of the myotube from stain and so far nothing is giving me what I want.

The best thing that has worked so far is to do the following:

1.) RescaleIntensity
2.) EnhanceOrSuppressFeatures
Enhance > Neutrites
3.) IdentifyPrimaryObjects

And that gets me to a point where its able to identify a whole myotube but then it gets a little too good at working because it clusters very small, fine phalloidin stained actin into an entire mass.

I’ve attached an example image of what I’m working with just so whoever views it can get an idea.

plate1_ A - 01(fld 1 wv FITC - FITC).tif (8.0 MB)