the nuclei displayed are usually from an atlas but you can also visualize in native space (see the popup in the main GUI) and then atlas-based segmentation is used (read e.g. Ewert et al. 2019 NeuroImage for more info on that).
Regarding the format, things are stored in custom files that are either Matlab files or Nifti images.
Maybe it makes sense for you to watch the walkthrough tutorial on our website which explains a few of these basic questions.