RNAISH covers nuclei and disrupts cell detection

My RNAISH stain has two patterns. It either presents as punctate dots or completely covers the nuclei so that I can no longer detect or count the cell.

Any tips or tricks on how to approach these images? I’m open to FIJI or Qupath workflows. I’m guessing I’ll need to ask the wetlab to adjust the staining parameters to “lighten” things up or adjust the illumination during image capture. I’m definietly going to ask that they darken the hematoxylin.

I was hoping to get area of stain per cell but it looks like I’ll have to settle with area of stain per tissue, as I cannot accurately detect cells.

Sorry I don’t have access to a higher quality image that I can distribute.

Any advice is appreciated!

Depending on how important it is, and whether you can pick up the red dye in a fluorescent channel, you might be able to also counterstain with DAPI and image that way. I think Eosin and FastRed both show up, at least.

From that image, though, I don’t think you can pull out the cell underneath. The only other option might be to assume each big blob is one cell. Try using Optical Density sum to find a set of values that works for both the red and Hematoxylin “cells,” while making sure to only count Nuclear stain for the red cells (classification). Or create a color vector that weights blue much more heavily than red such that you can use the Hematoxylin option in cell detection.
Either way, you lost the nuclear information for the blob cells, so you won’t be able to judge cytoplasmic/nuclear distribution. A combination of darkening the hematoxylin and lightening the red might help in the future.

You might be better off with the area of stain anyway, as judging by that picture it would be difficult to figure out which cell to assign some of the spots to without any kind of cell membrane marker.