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”.
Creation of Cytoplasm (Tertiary objects) from Nuclei (Primary objects) and Cells (Secondary objects)
Now that you understand the three Identify modules, tell us how you use them to find your favorite objects!