Zen will only create a pyramidal structure if the file size is over a certain limit, though I am not sure what that is. If over that limit, Zen (Blue) automatically creates it when you split scenes, write files, and look at the image (I think you need to open it). The greater issue for me tends to be that the initial multiscene files might be JPEGXR compressed, and after performing operations on the image (like stitching the tiles together), the resulting image is full size. So a 200MB scene might become 6GB, while still maintaining all of the resolution of a 200MB compressed image. Compressing it a second time makes things worse. If I know I wanted separate files and was starting with a multiscene CZI, I would probably save the initial image at full quality, split the scenes, and then save with JPEGXR compression as the final step. That may or may not be helpful to you!
For the ImageJ stuff, you can run quite a few plugins from ImageJ through QuPath, though I have not specifically tried that one (Weka segmentation). You do need to add the plugins to QuPath’s version of ImageJ though. In Preferences, there should be a field for “ImageJ plugins directory.” I have created a few different scripts that use the Analyze Particles… feature to get objects returned to QuPath through ImageJ, so that is definitely possible. Maybe these scripts will help:
A quick intro that also includes a link to Pete’s intro:
A more general script to handle finding brighter regions rather than darker regions
This one calls one of two different IJ scripts, and inserts variables for thresholds given by the user. It was mostly intended to help with newer 7-8+ channel fluorescent images:
Finally, for percentage area, I also use a script that sums the areas of objects of a particular set of classes, and then divides them by the total area of their parent annotation, and adds that percentage to the parent annotation measurement list. It may take some editing for your use. For example you may want to add an extra for loop so that it cycles through only objects of a particular class, and then creates multiple percentages.
I’ll leave the pixel classifier question to Pete, and I am not sure QuPath really performs any image modifications… though many things can be scripted! Here is one discussion:
Ugh, edited for formatting, did noooot like the way the links worked! Will need to figure that out when I have more time!