I’m not sure how I would upload the images to CellProfiler for analysis.
CellProfiler can easily open multichannel images, simply tell CP in NamesAndTypes that it’s a “Color” image (since it’s >1 channel, then add the ColorToGrey module with the settings “Split” by “Channel” as the first module of your pipeline. That should easily split your image into as many channels as you need.
Also, is this software ok to use for images that are between 90-200 MB?
That will entirely depend on the computer you’re working with. Even if it does work, it’ll be slow, so I’d recommend cropping out a few smaller representative areas of your image to try developing your pipeline on before running it on the whole image.
I also couldn’t help but notice your image has been stitched without illumination correction - this blog post discusses illumination correction of stitched images and why it’s important. Correcting the illumination is important because otherwise a real cell that’s stuck in a dim area of the image may not get counted while background autofluorescence that’s in a bright area of the image will. The post I linked discussed a way to calculate the illumination functions with dyes, but you can also do it computationally if you have enough images. In either case, it’ll be easier to work with the un-stitched images if the microscope will give them to you- it’ll also alleviate the issue that the images may be too big to properly work in CP.
Lastly, would anyone be willing to give some suggestions for modules to use for analysis of the various cell types I have stained for? (CD8, CD4, CD11c, CD19, LYVE-1, and CD31)[…]Any help would be greatly appreciated as I have never done image analysis before. Thank You!
You may want to search around the forum for other people who’ve worked with lymph tissue or just tissue sections in general. We also have example pipelines on our website you can look through.