Setting a static ROI over a time series of images

Hi there,

I am trying to measure the intensities of molecules tagged with fluorophores over a series of time-lapsed images (.tif movie). I would like to identify a set of objects in the first frame using a set of criteria and then use those regions to track the integrated intensities for the rest of the frames. Effectively, I want to construct bleach curves for each object over the entire time series.

So far I have a project that identifies and filters the objects, then it uses the TrackObjects module to assign unique labels for each object between frames. Unfortunately this does not seem well-suited for my purposes. The objects do not move from frame to frame but some of them blink and get assigned new labels. Also, as soon as an object bleaches it is no longer tracked. I would like to be able to measure the background intensities after they have bleached as well.

Is there a way to use the regions of interest from the first frame to measure the intensities over the entire series? Any advice would be greatly appreciated.

Here is the pipeline and the first ten frames:
Cy5_08s_fluors.cppipe (15.4 KB) (1.0 MB)


Hi, I have Just recently done this with cellprofiler to asses calcium data. Although, i use my last image with to obtain my cell measurements.

See these two links, 1 2, to observe my struggles.

Basically you will need two pipelines. The first will be to select, and save objects. (Read through my posts, and you will find you need to save the objects, as 16bit grayscale if you have over 256 objects).

Then the next pipeline, only consists of two modules, MeasureObjectIntensity, and ExportToSpreadsheet.

You will need to master the import modules; Metadata import, NamesAndTypes, and if you are processing more than one experiment Groups.

This is where i create and save my objects, deep in there somewhere. (1.6 MB)

And this is how i create time lapse data.
f2.example.cpproj (109.8 KB)


Thank you very much for your help! I had a bit of trouble assigning names to the input images for the second project file (only one frame displaying or throwing errors) but it seems to be working now.

Thanks again,