A tutorial that uses a CellPainting assay to find segmentation parameters for larger “parent” objects (nucleus, cell, and cytoplasm) and show you ways to pull out smaller features in your image by segmenting organelles within the nuclei. You will also be shown how to use RelateObjects so that you can relate the average counts, distances, and measurements of the smaller “child” organelles to their larger “parent” objects.

The images used come from the Broad Bioimage Benchmark Collection (https://bbbc.broadinstitute.org/bbbc/BBBC022). They are fields of U2OS cells (10 sites imaged in 5 channels), and represent 5 mock treated wells from a single 384 well plate experiment.