MakeProjection/SaveImages Modules Help

I am currently running CellProfiler version 7522 on a OSX 10.6 (I also have access to a XP 32 bit and a Vista 64 bit systems as well)

My pipeline is being used to calculate FRET using time-lapse images, which I have working fine. I am also curious about the FRET values at each pixel not just in ROI’s, which I have done successfully with the ImageMath module, which I then make a projection out of the ImageMath images (not necessary for analysis, but the investigator would like to have a visual representation of it), which is where I run into a problem.

When I run this over one field, I get a perfect projection image, but when I go to do multiple fields, the projection image keeps compiling a projection from all previous fields, even when I have each field separated by individual folders. Is there some module I am missing that would assist with this?

I have attached a set of of test images with two time points from the same field along with the pipeline. (I apologize for the length of the pipeline, we believe in calculating all items under the sun, just in case.)

FRETPIPE10E.mat (3.34 KB)
Test (4.11 MB)


It sounds like you may need LoadImageDirectory. It has the same functionality as MakeProjection, but operates on each directory individually. So if you separate each field of view into a different directory, then you can make a projection of each directory individually. Due to the Matlab function we use to do this, unfortunately you can’t pre-process the images before this module- you have to load them directly. So you’ll have to save off the ImageMath-ed images to the correct directories, then use this module to make the maximum projection. You also have to experiment a bit with the text-matching in the settings and how you name your files.

As an example: your files are named like this: 091007-PC3-E10-P002_T00_A-HSP90b.tiff and I’m guessing P0002 is the field of view and T00 is the timepoint (or something like that). so you’d put all T0(1-n) under a directory named P001 and T0(1-n) under P002 and so on. In LoadImageDirectory, the settings would be as follows (assuming your default image folder is the level where you can see all the P00x subfolders):

Enter the pathname to the folders containing images: .
Enter the text that the folders have in common: P0
Analyze all subfolders within each folder? Yes
How do you want to load these files? Exact match
Enter the text that one type of file has in common: Dapi
What kind of projection would you like to make? Maximum
(you can leave the rest of the settings about quality control as default, unless you want to get into removing out-focus-planes from the projection)

You would then have one module for each channel (DAPI, WGA, etc). You could make a projection, of all the timepoints, for each field of view separately.