Converting fluorescent images to brightfield

There’s a option in InForm (Akoya/Perkin Elmer) that converts fluorescent channels to a pseudo-brightfield image where the stains are displayed as DAB, and DAPI are displayed as hematoxylin. Some pathologists prefer to look at such images than fluorescent images.

Any suggestion how it can be achieved outside InForm?

Hi @manaser

While I don’t have any experience with InForm, it’s surprisingly easy to replicate by taking the single channel fluorescence data then converting it into a colour vector representing first haematoxylin then DAB.

There’s a script linked below that transforms a fluorescent image into a pseudo-brightfield image and it appears to do a reasonable job:

The RGB vectors were found in Fiji by running Image > Color > Color Deconvolution

Haematoxylin: [0.65,0.704,0.286]
DAB: [0.27,0.57,0.78]

If this is the sort of thing you wanted, you can find the ImageJ macro along with the example image here: https://bitbucket.org/davemason/makehdab

As the repo says, I give no warranty or guarantees about the utility or even validity of this approach, only that it seems to look about right.

Hope that helps!

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This is exactly what I needed. Thanks! :star_struck:

However, I don’t have the Color Deconvolution option under the Color in Fiji. I have it listed under Plugin, but that’s throwing error. Any suggestion how to get this option on Fiji/ImageJ?

I also tried to run the macro found in bitbucket anyway, but that’s showing “undefined variable” error on line 15.

Colour deconvolution goes the other way (for breaking HDAB images into their respective channels) so is not actually needed for this example (I’ve edited the original answer to make this clearer). Do you have an updated Fiji installed and not vanilla ImageJ ?

If you post back the exact error message, that would help figure out why it’s not working for you. Bear in mind that it’s more of a teaching aid than an all out solution at this point.

Sorry my bad. It was a silly mistake. Working perfectly now.
Thanks again for the help. Really appreciate it :pray:

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