I have been further learning CP and (trying) CPa and I have some more questions about my pipeline. I attached some errors I am getting, to illustrate. Hope my questions are clear and I do appreciate any time invested in answering me. My aim is to use this for our analysis of all of our HCS data, as I found the ScanR program not working for us.
Summary of what I am trying to do.
Fix alignment, rescale and create metadata from the file names in LoadImages.
Find primary (nuclei by DRAQ5) (<-- Mark adjusted this in my original post)
Find secondary (Using nuclei as object, and then distance)
Find tertiary (subtraction to find cyt.)
Calculate the ratio of YFP in the primary / tertiary objects (our positive control treatment induces creation of granules. Can be seen in Screeny#2. Weak but there - I need to identify these cells. In neg controls, there is NOTHING in the cyt.)
Export to database to be able to use CPa to classify etc.
What is “Export to database will overwrite Per-Image and Per_Object tables” (Screeny1). Even If I keep changing the output dir, it keeps giving me this. I am probably doing something wrong.
I added x/y correction (Align module):
a. I don’t see aligned images later in CPa, rather the images still show as the original, unaligned ones, for example when I open an image in CPa. Am I supposed to see them aligned? or is it just behind the scenes?
b. The align module seems to take a lot of processing time for each cycle - is there anyway to simply tell it manually to adjust say y axis by say -7 for every single image set?
c. When I choose “Crop” in the align module, I can see the result of the alignment in the result box but I immediately get error messages as pipeline continues, but when I choose “Keep”, I get no errors, yet the result images shows a yellow image (Screeny2). I realized the yellow is becasue of the Image contrast - when I choose Keep, I need to transfer to Log to be able to view the result of the align. Not sure why. So, which Crop mode is more correct for me, and why the error? You can see how the 7 px shift solves the misaligned channels.
Image processing: In my first post (Exporting to MySQL db issue (solved)) Mark fixed my LoadImages to help better define the nuc. However, this did not seem to take into account the 12bit camera to 16bit save issue. So I added RescaleIntensity, and that module did seem to rescale the images, but then the IdentifyPrimaryObject that Mark adjusted did not work for all he images so I changed it.
What is the difference between the RescaleIntensity module and the check-mark option “Rescale intensities” in LoadImages?
I do need this, right?
I am not managing to define a group. I want to call all column 1 controls, I don’t see where to do this. I thought it’s in “group fields”. In the pull down it has: “Image_Metadata_Well”, but I am not sure what to do with that, how to use that. Specifically, column 1 on my plates are all positive controls and columns 23, 24 are all negative. How to I define those so that I can group them later in CPa.
Calculate statistics give me the following error: Screeny3. This is currently not in the pipline.
6. In CPa: Plate viewer, Scatter plot, Histogram, Density and BoxPlots all give me an error - Screeny4. I can open Classifier, load some cells and view them. I can also open “table viewer” and “open from database”. So it seems I am connecting to the db.