It can be confusing when you’re trying to set up your first pipeline to figure out which modules to use to generate your objects! A helpful way to understand the difference between Identifying Primary, Secondary, and Tertiary objects:
- Primary objects are segmented independently from any other object you’ve designated (like segmenting nuclei from your DAPI channel).
- Secondary objects are created around a primary object, and so they usually need two pieces of information – a primary object (like the nuclei to build the cells around) and an image (your whole-cell image channel). If you don’t have a good whole-cell image, however, you CAN use IdentifySecondary objects to create a cell by expanding out from your nucleus (or other primary object) in every direction by a certain number of pixels.
- Tertiary objects are created by looking at the difference between two other objects (such as saying the cytoplasm is the difference between the whole cell and the nucleus) – hence they’re the “third”.
Now that you understand the three Identify modules, tell us how you use them to find your favorite objects!