Z Stacks and Grouping

can you please advise on working with z-stacks. I have .oib z-stack files, and I’m importing in the usual way with Metadata -> Extract from image file headers (updating metadata and table buttons), NamesAndTypes -> Images matching rules, but then I get into the troubles.

I select a rule to match the channel 0 (even though I have only one), and I click + to add the Image Is Stack Frame option, and give it a name (I’ve tried with and without the channel as I have only 0, but the same question arises). Help suggests “In the “Image set matching method” setting, select “Metadata”.”, but for a single channel z-stack you don’t get that option. I continue all the same.

In Groups I use FileLocation and it shows the 3 files I’ve dragged in, grouped into 3 as I’d expect with a count of 5 each (the number of z in each stack), so all looks good.

When I go to perform a MakeProjection and run through the Test Mode it appears to make a projection for all of the individual 5 z’s of each of the 3 files… Is there a way to get it to process the Analysis modules once for each file? Right now I don’t know if it’s working or if it’s just the way it works through.


Hi Neil,

Have a look at the tutorial here called “Loading Image Stacks and Movies”: cellprofiler.org/tutorials.shtml

But getting z-stacks ‘unpacked’ to process can vary a lot depending on how they are arranged. So if you still are having have trouble, you can upload one here and we can see about configuring CellProfiler for you. Dropbox or Google Drive might be necessary for sharing large files.


Hi David, thanks for the speedy reply.

I was following the tutorial you mentioned, and I got stuck when it doesn’t show the “Image set matching method” setting if I only have one channel. I did get to a similar looking end point, where each of the three files I was playing with had a count of 5 (corresponding to the z-sections), but still when stepping through test mode and moving to the next data set it kept bring up all z-sections after I tried to perform a maximum intensity projection.

Thanks for offering to take a look. Here’s a zip of three z-stacks of my data:
My aim was to create a maximum intensity projection, and then for each of the resulting three images calculate the area of the beads (the latter part I’m happy to do, but getting to the point has me stumped).

Thanks in advance for your time.


Hi Neil,

Getting back to this late – have you made more progress? In your metadata, it shows that both the “Frame” and the “Z” metadata indicate the z-stack depth. Also note that CellProfiler’s MakeProjection is best used in a pipeline almost by itself, with a SaveImages module set to write only on the last cycle.

Let us know if you need further help though!