1.Load the Stack file ie., .tif file into ImageJ -- File -> Open -> Browse the Stack   file(.tif)

2.Convert the Stack file of 16-bit image into 8-bit image-- Image -> Type -> select 8-bit

3.Apply Threshold on the 8-bit image--  Image -> Adjust -> Threshold

4.Now apply Segmentation with the plug-in “Segment blob in 3D Viewer” -- Plugins ->         Segmentation -> Segment blob in 3D Viewer


5.Selection of a Seed Point -- plugins -> segmentation -> levelsets
6.Overlapping/Touching Cell Structures -- Process -> Binary -> Watershed
  Analyze -> Analyze Particles -> Show -> Outlines -> Display Result

7.Applying the Skeletonize  3D -- Plugins -> Skeleton -> Skeletonize 3D