Help with cell counting and pipeline building

Dear All,
I’ve been trying to modify the pipeline of the mammalian cell tutorial for my purposes. Essentially I’d like to titrate viruses: the cells are infected with different dilutions of the virus, fixed, stained with virus antibody.
Now I’d just need to count the positive cells (green), and the negative ones (no signal in green, only in blue). The good news is that I don’t care about the cell shape - I only want to know the presence of absence of green in the vicinity of the blue signal.
This is one hurdle; I’m not sure how to tell the program I have two types of cells.

My input files are in zvi format, and two channels: blue and green. So far the best I could come up with is to manually export them into tiff’s with ImageJ… this results in a file system of the dilution_ch01.tif, dilution_ch02.tif for the two channels. Is there a better way to do it?

The other problem is that I get the following message when I try to run my modified pipeline:
Encountered error while processing:
Pipeline error: error while processing MeasureObjectIntensity: Feature cells.intensity_integratedintensity_origblue has already been set for image cycle
I guess it comes to halt when it starts to process the next set of images in the directory; I’m not sure what to adjust so that it simply jumps to the next pair of images.
Your help is greatly appreciated.

We have recently added the ability for CellProfiler to recognize .zvi files in LoadImages, although this has not been officially released. However, if you want to obtain the latest build of CP from here, you can give it a try. However, we make no guarantees that this build is fully stable prior to the full release.

[quote=“spongya77”]The other problem is that I get the following message when I try to run my modified pipeline:
Encountered error while processing:
Pipeline error: error while processing MeasureObjectIntensity: Feature cells.intensity_integratedintensity_origblue has already been set for image cycle
I guess it comes to halt when it starts to process the next set of images in the directory; I’m not sure what to adjust so that it simply jumps to the next pair of images.[/quote]

It seems that you may have either (a) a second MeasureObjectIntensity module measuring cells from OrigBlue, or (b) you’re measuring the intensity from cells twice in the same MeasureObjectIntesnsity module. In either case, you should only make a unique measurement once per cycle. Is this the case in your pipeline?

Regards,
-Mark

Thank you for your answer, and sorry for the late reaction; suddenly the transfer report on my PhD got absolute priority…

Thank you for the suggestion as well - indeed there were two cell measurements.

The one thing that confuses me still is that I don’t know how to make it process the next set of images - it seems like finishing with the first two (prim + sec), and then stops. It seems like it get stuck on the first cycle… it might be just my computer’s too slow, though.

Thank you again,
A

The latest release of CP (10997) now reads .zvi files. Please let us know if this helps with your assay.
-Mark