Utilities: matching quick clarification

[Will760]

Hello team & community,

I just wanted to clarify something. I have some images that I would like to split into epithelial niche (basically the epithelial layer + ~100um underneath) and then everything else excluding any non-tissue areas (non-epithelial niche/stroma for e.g.). I have more-or-less done this now for all images in imageJ and can a create binary image for the epithelial niche and one for the stromal niche. For cell segmentation, I am using cellprofiler/ilastik.

I assume the correct tool to use here is the matching command in the utlities section of the steinbock.

Is my understanding correct that I would do this twice, once for epithelial niche and once for the other, this would leave me with two .csv files that are essentially a list of cells within either region. I guess I can then take this into R (I am following the multiplex imaging data analysis guide) and assign a tag to col_data that indicates which niche each cell is in. From this point I can create subsets of spe as spe_epithelial and spe_stroma for downstream analysis.

I have a few queries about this:

for the tumour_masks directory (I assume I can call this whatever I like) does this just need to contain a binary mask for each image (with exactly matching names) in .tiff format.

for cell_masks: this would just be the directory containing the 'masks' output from CellProfiler right?

Sorry if this is extremely simple of me, I just wanted to be sure I understood before embarking upon this journey :)

Thanks all!