I’m looking at fluorescence intensity changes in a group of cells over time. The cells are tightly packed and extremely numerous, hence the value of CellProfiler in this case.
The loaded file is a .cxd - a stack of 400 images over time. Each image contains 5000 cells, which remain entirely static over the duration.
I need to identify each cell as an ROI and then measure the intensity of each individually over each individual stack. Of course, the simplest program would just be to identify cells on each stack and then measure the cells on each stack. However, my pc takes five minutes to analyse the 5000 objects in a single stack in this way, and I’m not even sure it would succeed in getting through all 400 stacks before dying. So, there’s gotta be a better way!
I’ve attached my pipelines. Here’s how I’ve tried to it:
Pipeline 1 loads just a single frame (the first, by default) of the .cxd file. It uses IdentifyPrimaryObjects to find the cells, and then it uses OverlayOutlines to create an overlay of each object. This pipeline works fine – no problems here.
In pipeline 2, I want to ‘load’ the overlayoutlines defined in the previous pipeline onto every sequential stack in my .cxd movie. I will then use MeasureObjectIntensity to extract mean intensity values from each cell object. I haven’t been able to create this pipeline because I can’t work out how to load the OverlayOutlines which hasn’t been created in the current pipeline.
So two key questions:
Can cellprofiler be made to look at every frame in a .cxd movie, or do I need to manually convert that movie to a stack of .tif files before entering pipeline 2?
Can Pipeline 2 be made to load an overlayoutlines file from Pipeline 1?
If not, then perhaps I’ll need to combine these two pipelines, such that stack#1 is used for IdentifyPrimaryObjects and OverlayOutlines, and then these outlines are used for MeasureObjectIntensity upon the rest of the stacks.
I’ve attached a single TIF of my cell view - I can’t give the full .cxd because it’s a couple of gigs sadly…
Thanks very much for your help, in advance!! I’ve been working at this for the last 8 hours and it’s just defeated me