"3D" pseudocolor for ratiometric images (FRET)



Hi Jan. I can’t deal with run(“Fill”, “slice”) following the macro, because I didn’t find the command “slice”. Could you please give me more information about that? Thank you!


I can’t still solve the problem.


In this instance, the second argument, here “slice”, is what you want to actually be filled. As you have a stack, you have the option to fill all the images in the stack, or just the current slice.

If we take the above macro, we want to only fill the saturation channel (slice 2) with the white foreground color, not all 3 channels of the HSB stack.

It might be easier to try and follow these steps manually, as you can then visualise the corresponding dialog boxes etc…

After selecting the entire saturation channel, Edit > Fill would then produce a dialog box like this:

Clicking no would produce the following: run("Fill", "slice");
Clicking yes would produce the following: run("Fill", "stack");

Hope this helps!


Thank you so much. This is exactly what I needed.:slight_smile:



Hi, I am generating an ratiometric image now, kind of like Fret image in this topic. I’m wondering how to do it. I tried the image calculator, it would not give me the right results as you showed here. Did you successfully generate the image? Would you please let me know how to do it. Many thanks!


Hi @olivia, welcome to the forum!

I’m afraid I dropped the ball on this and didn’t get any farther than what you see in this thread. We had the opportunity to do FLIMFRET which served us much better than ratiometric - so I stopped getting new ratiometric FRET data.

I was never able to get to the quality depicted below, but I’m pretty sure my images were just not as good to begin with (old equipment).


I would suggest you post your data here (or in a new thread) so we can better know what is possible in your case, and what you were not able to do with the image calculator.


@Sverre, thank you for your advice, I will post a new thread, and forward it to you.


This is the topic I just posted

Thanks a lot!