Objects ID'd in Ch1 and use for Ch2 Ch3 intensity quantification from ND2 file with multiple time-points

Hello CellProfiler Fam,

I am imaging 293t cells. The time-lapse images are saved in .nd2 format. The nd2 files have 4 channels and multiple xyPos as well as many time points. The cells have 3 markers(FP1, FP2, and FP3) all in the nucleus. I want to quantify the intensity of FP2 and FP3 under the objects detected from FP1 (best nuclear segmentation and best for tracking cells and highest SNR) per cell over time.

Question 1: I am extracting metadata from the file header of the .nd2 file, but I am getting only 1 xy position with 4 channels. How do I extract all xy pos, all time points, and all 4 channels? I have shared a link (https://drive.google.com/open?id=1Xk4dII1Q4VLLSbhpcuzPlGYrsarqgNTY) for one xy pos of nd2 (whole file was too big).

nd2_object_v1.cpproj (660.6 KB)

Question 2: How to use objects identified from FP1 to quantify intensity in FP2 (channel 2) and FP3 (channel 3)?

I hope I am explaining clearly. This is my first “long” post. I appreciate any help and patience :slight_smile:



I’m not sure how to advise you, but when I use the Metadata extraction from the header on the file you uploaded I DO get all the time points- see below. If you do NOT see that on your machine, can you post what version of CellProfiler are you using, on what OS?

You should then set up your NamesAndTypes module to read the extracted C column to read in your various channels individually, and remove your ColorToGray module.

This has been solved with newer versions and fixing my pipeline. I am running mac.

Thank you very much !