Merge multiple Z stacks



Hi all,
I have a multistack TIFF file with 3 channels (RGB) for each individual stack (13) - and all these are in the same exact plane. What I’m trying to do is create a singleplane TIFF file that is just the resulting composite of the Red Green and Blue channels of all the stacks together. Is there a way to do that? I’ve tried concatenate, split merge channels but nothing seems to have worked.
Any advice would be more than appreciated.
Thanks again


“Merge channels” has worked for me in the past. What is the type for each image?


Merge channels doesn’t really help me in this case.
I’ve attached to this thread the original file. Think that could be of help in showing you what I’m dealing with!
Thanks again!


Hi Roberto,
First of all, the file you shared has a total of 33 slices, which means you want to extract 11 individual RGB stacks, is that correct?

Since this is a stack, you would need to use the Stack Tools.
What I would do is to first divide the stack in 11 parts using the Stack Splitter (Image > Stacks > Tools > Stack Splitter).
Then I would split again the individual small stacks in 3 separate images (Image > Stacks > Stack to Image) and reassemble them in a single composite image using Image > Color > Merge Channel.

You could also do “Stack to Image” from the beginning and then Merge 3 channels at a time to make the composite image.

If you are good with Macros you can create one to do all the steps at once.

I hope this helps!



I have tried to follow Anna Fasoli’s solution but some of the features may not be available depending on which version of ImageJ you have. Here is what I was able to do using ImageJ 1.50b.

  1. Open stack
    • I use drag-and-drop
  2. Image > Hyperstacks > Stack to Hyperstack…
    1. Set Order to xyczt
    2. Set Channels (c ) to 3
    3. Set Slices (z) to 11
    4. Set Frames (t) to 1
    5. Set Display Mode to Composite
    6. Click OK
  3. Image > Color > Stack to RGB
    1. Check Slices (#)
    2. Check/Uncheck Keep source as desired
    3. Click OK
  4. Save


Hi there! I was running into a similar problem, but needed a different solution. So I figured I would share the solution here in case someone runs into a similar issue as I did.

I had two images that I wanted to merge together of two different desired colors. The images were taken in stacks of the same exact plane, without any movement or alteration. I stacked the images using the microscope program I had using a macro. When I used imageJ to then use the image calculator, no problem, the images came together nicely with the two different light ranges being seen in the single photo. When I tried to merge the colors to assign their respective colors, the error I got was that the images were not the same size.

So what I needed to do to correct my problem, was to scale the images first. Turns out that the automatic scaling for each photo is dependent on factors that are totally unknown to me. However, just assigning the scaling to match first and then merging the channels worked for me! Since the scaling stays true to the real dimensions of the photo, there is no issue of altering the photo is some artificial way. The scale command is under image.

Hope this can help someone!


If the issue was the stored scale values, you could have also just removed the scales from both images.


go to image, stack, images to stack
then go to image, stack, z-projection
use average or max