Please help: everything works except identify objects :-(

Dear cell profiler staff,

Congratulations for this nice new release of cell profiler.

I have adapted your example pipe called Neighbours in order to count cells growing in agar. I have 3 cell types, red, blue and green +blue.

Initially I would like to be able to count just blue cells. Everything works except IdentifyObjects, which can not see my cells no matter what treshold method I use. Can you help me to see what’s wrong?

In the future once this is fixed, I would like to measure how many red cells have as a neighbour a green cell and compare with how many has a blue cell, and also orientation if possible (i.e. if there is any self arrangement of cells over time within the microcolony, kind of higher number of individuals (ellipses) whose major axis are parallel). Any suggestion on this will be more than welcome!

Thank you very much in advance for all your help, you are making something really nice in here!

Best regards,

Jose Seoane

Technical university of Denmark

ExampleNeighbors_adapted.cp (8.87 KB)

Hi Jose,

In this case, the ExampleObjectNeighbors pipeline isn’t appropriate for identifying the cells in your image. In the original pipeline, it was preferable to identify the lighter cell borders as compared to the dark cell interior. In your case, you have a dark background and bright cells, so the identification is not optimized.

Attached is a revised pipeline that is better suited to your images. I’ve changed the thresholding method, adjust the size criteria, and adjusted the smoothing filter in IdentifyPrimaryObjects, as well removing the now-unneeded ImageMath module.

Hope this helps!
ExampleNeighbors_adapted_MAB.cp (8.84 KB)

Thanks Mark! Is much better now!


hi Mark,

Just an easy question: how do I produce the files needed for celprofiler analyst?

Thanks again for all your help,


The files needed for CPA are produced in ExportToDatabase. The main items to note are:

  • Select the database type: Use MySQL
    if your database is on a remote server, use MySQL/CSV or SQLite if you want to create a database on your own computer.
  • Select that you want to create a CellProfiler Analyst properties file.

Read the help for the module to see how the other setting work. You can get CPA from here; also on that page is the manual plus example data to get you started.

Remember you can post in the CPA forum if you run into CPA-specific problems. Good luck!