Unmix Colors Custom Setting


Hi there,

Is there a way to figure out what the absorbance of your stain is, so you can set a custom filter in the “unmix colors”?

I have some liver slides stained with ORO, and on my test run (of a batch of about 5 samples they showed me) unmixing with eosin worked pretty well. The problem is, with this larger batch of samples I have now, There are some where I have a little off-target staining, but the off target is a little more blue. I was hoping I could create a mask to filter out this off-target stain, but to do that, I’d need to figure out the absorbance.

I just thought if any of you had any tips and techniques for figuring out what you want to use for your custom stain numbers, it would be helpful to share with everyone.


I ended up solving the problem a different way. I selected a different stain for the ORO unmix, and then also added a stain to unmix, since they were counterstained in blue. I just masked the blue parts, and got much more accurate readings.

I still would like to learn how to figure out the absorbances for custom stains though. I can get the RGB values from paint, but I don’t know how to convert those into the absorbance values.


You can crop down a small area of your blue staining, save it as a separate image, then use the ‘Estimate’ function in UnmixColors to load up this cropped region- the module will then calculate the stain absorbance for you automatically. That’s probably the easiest way to do it.