Loading two images with same field of view

For a project I’m assisting on, I need to get object data from the nuclei for both Hoechst and BRDU, respectively, and then a count of BRDU positive cells compared to the Hoechst nuclei (since the BRDU positive nuclei are also in that image). The raw images from another grad student in my lab come in sets of 2: one with Hoechst and a second with BRDU with the same field of view. I have two images an an example of the set (00 for Hoechst and 01 for BRDU). To make life a little easier I used imageJ to try to make a composite TIFF file so only one file with 2 channels need be loaded in the pipeline. However, I keep getting a “list index out of range” error when I’m in test mode and with some considerable time testing different ways to upload the images I can’t seem to make the pipeline work. Can anyone lend some assistance in this matter? I would be greatly appreciative.

Thanks!

Dan




Nucleus- 40x objective-- BRDU.cp (13 KB)

Hi Dan,

I don’t seem to get this error with the files you posted. Also, the pipeline posted assumes the composite image is being used, rather than the two individual images you posted.

It would be great if you could post the composite image that goes along with this pipeline, and moreover, one that produces the error that you report. Also, if you could post the actual error message (by clicking the details button and then copy to clipboard), that would also be helpful.

Regards,
-Mark

Hi Mark!

Thanks for the reply. For reference, I’m using a Mac. Here is the error message it gives me when I try to load the composite image:

Traceback (most recent call last):
File “/Applications/CellProfiler2.0.app/Contents/Resources/lib/python2.7/cellprofiler/gui/pipelinecontroller.py”, line 932, in do_step
File “/Applications/CellProfiler2.0.app/Contents/Resources/lib/python2.7/cellprofiler/modules/loadimages.py”, line 2052, in run
File “/Applications/CellProfiler2.0.app/Contents/Resources/lib/python2.7/cellprofiler/cpimage.py”, line 570, in get_image_provider
IndexError: list index out of range

Also attached is the composite file I generated from ImageJ.

I’m not sure if there is something wrong that I have with my files or the way the pipeline is designed, but I surely hope that it isn’t something concerning my computer and anything it may be running that is preventing me from executing this analysis.

Looking forward to hearing from you again!

Best,

Dan

Hi Dan,

I’m attaching a pipeline which shouldn’t give you an error. I had to replace the LoadImages module and then make some changes to the settings on top of it. I also had to use a GrayToColor module to create a color image to overlay the outlines on.

In general, you are better served by using individual grayscale images as input, rather than a composite color image which will need to be split apart in CellProfiler anyway.
-Mark
2013_09_20.cp (13.3 KB)