Hi everyone,
I am working with cells labelled with Mcherry (membrane) and EGFP (cytoplasm). Following my experiment, I want to quantify the green signal which relocated to the membrane. How can I extract and quantify in Fiji the amount of green signal on the membrane (e.g. where the red signal is)?
Thanks in advance for all your answers and suggestions!
Sissi
You can split the channels (image>color>splite channels) and then threshold the red channel (image>adjust>threshold). Then press "apply". This will now become a binary image that you can use as a mask. Copy it and paste it on the green channel image, after having first adjusted the paste control to "AND" or "transparent white". The result will be an image where the green channel will be visible in all the areas that are also red. You can quantify this normally: thershold it, but do NOT click "apply" (otherwise you will end up with another binary image), and then go to analyze>measure, after having selected your parameters through "set measurements". Hope this helps! Keep in mind that colocalization is best done on 3-D stacks, personally I do it using Imaris. But even if you do it on 2D images, I think you can have good results in an experiment like yours, because the error will exist in all cases, so any difference you see will reflect a real, biological, difference between the conditions.
Alexandros A Lavdas thanks for your precious answer, that was very helpful!
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