I have created a pipeline which:
- opens a ZVI image from the Axiovision SE64 software
- splits said image into four channels (green/BrdU = 1, red/Sox2 = 2, blue/EdU = 3, white/DAPI = 4)
- uses Align function to align the split images
4)allows the user to rotate each channel’s image then crop it with the mouse to determine a region of interest (in my case, the ventricular zone)
- saves the cropped images (SaveImages module)
I am running into a couple of issues with my images:
The ZVI file is not the raw image. In Axiovision, I used their software to subtract the background from the images to be analyzed. I then saved these images as a new ZVI. When I load the image into CP, however, the image loaded is the original (with no background subtracted). Does this have anything to do with how CP is loading these images? If not, do you have any suggestions as to how I could load a 4 channel image which has been processed? I tried to export the image as a 4 channel tif from the Axiovision software, but CP gave an error message when I tried to make a pipeline to open this type of image.
I am using the SaveImages module to save these cropped images for later analysis. The images produced at the end of the pipeline, however, are extremely dark. I can hardly see the cells in the images. I am pretty sure that this is because I have no idea what type of file I should save it as and because I’m not sure what type of color map to use. I tried saving as tif in 8 and 16 bit and it produced the same undesirable result.
Lastly, I noticed as I was running the pipeline that when the Crop and Rotate windows are open, there is no indicator as to which file is being processed/which cycle is currently being run (as opposed to the Align window or the window that pops up after I have cropped an image). Is there are way for this to be displayed in the Crop/Rotate windows as well? The reason is that CP does not seem to process the images in a particular order (Cycle 1 then 4 then 2) and I need to keep track of which image I am on so I can rotate it appropriately.
If not, is there a way that I can order my pipeline so that I do all of the cropping/rotating for one file before CP opens the next one? (Example, rotate all four channels and crop File A then have CP start with File B).
I have attached my pipeline, an example of the output image that is too dark/wrong, and two ZVI images. Also, sorry for the really long post!
Load_ZVI_Rotate_and_Crop_Pipeline.cpproj (79.6 KB)
ZVI2.zip (7.29 MB)
ZVI1.zip (7.21 MB)