I have created a pipeline (see attached image for outline of modules used) where I segment primary objects (my spheroids) and then I track these objects with the trackobject module and then I segment other primary objects (my T cells) and convert the objects into a binary image and at the end I use the measure object intensity module to measure the T cell image in my individual spheroid objects. The ultimate idea is to count the number of T cells inside my spheroids over time (these are movies that I have uploaded as individual planes and then organised it into movies with the groups module). My spheroids have heterogeneous GFP expression and are quite hard to segment which is why I thought that tracking them which supposedly gives them a unique ID would solve the problem of when my one spheroid is segmented as 2 objects but I need it to be counted as one. When the tracking image popped up it would label the 2 segmented objects as 1 because of the overlap criteria I set up, however I have now figured out that for all the downstream measurements (including counting of T cells inside spheroids) CP still uses the 1 spheroid segmented into 2 and treats the 2 halves as different objects despite them being tracked as a single object.
For now assume that although the root of the problem is the segmentation of the spheroids that we cant change that.
I have attached a sample image in which it shows the segmentation of spheroids (there should only be 2 spheroids but CP segments one of them into 2 giving a total of 3 spheroids), their tracking (the 2 spheroids are labelled as 1 and 2 which is why it surprised me that all of the analysis is still done on the original segmented image not the tracked one), and the T cell image (which isn’t that relevant for this but just thought I would include it to give you an idea of what it looks like).
My idea is to merge objects that have been tracked as one but segmented as 2 but I haven’t figure out how to do this yet. Let me know of any other ideas. I don’t need the tracking module for actual tracking, I just needed it to give my individual spheroids (there are several per image) unique IDs so I can then look at their individual T cell infiltration over time.
Thank you so much for your help in advance!