How to add a composite image stack as the additional channel to the a multi-channel Z stack?

I have 3-channel z stack images from confocal microscopy, and I want to check their co-localizations. So I need to have their composite images as the fourth channel.
It is quite easy to get the composite z stack by “split chennels/merge channels”, but then how do you attach it to the original z stack?

Hi @Ye_Jiahao,

you could split the channels of the image, generate new image stacks e.g. by multiplying channels using the Image Calculator ( ) and finally merge the images as channels together again.

However, for colocalisation there are actually useful tools like

So why not using these tools?

This link may also be interesting for you:


Hi, Robert, Thanks a lot, especially for the info. on plugins for coloclization analysis. I will try them out!

If I am not mistaken, you are suggesting me an alternative way to merge multiple single-channel image into a composite image. But I actually stuck when I try to combine this composite image stack(i.e.256x256; c:1; z:30) with the original stacks (i.e. 256x256; c:3; z:30) into a new stack (then 256x256, c:4, z:30). It is very easy to do this with Leica LAS Lite, but I have not find a convenient with FIJI.


Just split your stack with three channels (using Image > Color > Split Channels… ) and combine these three individual images with another 4th image. I mean, in the menu Image > Color > Merge Channels, you can enter up to 7 images. They all need to be of size 256x256; c:1; z:30 in your case :wink: