How to run PixFRET plugin in macro in ImageJ?

Hi there,

I’m trying to calculate the FRET using the PixFRET plugin for ImageJ. As I have a number of image sets, I’d like to use a macro for automation. However the command


which was recorded during the manual execution, only opens the plugin dialog window. There are no other commands recorded to execute actual FRET calculations.
I thought maybe I should use some arguments. But where can I find a list of available arguments for the plugin? Or should I do something differently?

Thanks in advance


I’m pretty sure @imagejan is the person to best help here…


I am not able to access Google Drive at work, but maybe the script here could give you some hints (if you can get to it).



Thank you for the suggestion. I’ve already checked Benjamin’s script, and while I’m trying to adapt in for my needs, it is not exactly what I was expecting. In this script, Benjamin used IJ robot plugin for the automation of button clicking. It is much better than manual clicking, but still not an optimal solution as it can’t be run in BatchMode.

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Hi, @Dobrokhotov1989

If you need just calculated FRET image, please try using our plugin.
It can calculate 2ch stack images. *2ch(Donor, FRET), slices, frames


Thank you for the suggestion. I’ll check it.
This plugin calculates FRET index without any correction for bleed-though and cross talk, isn’t it?

Hi, @Dobrokhotov1989

As you say, bleed-through and crosstalk are not taken into account as the calculation method is simply a ratio based on two pixel values.
To explain IMDwithHSB briefly, the ratio for each pixel is calculated and converted into a Hue value. The values ​​of Saturation and Brightness are calculated according to the brightness of the acceptor.
I believe it is a good way to display.


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