Merge channels: more than 7!

Dear all,
I’m doing spectral confocal Imaging with more than 8-10 colors.
In ImageJ, I didn’t find the possibility to merge more than 7 channels.
Could you help me please?
Best regards.

Welcome to the ImageJ forum, @Seb!

You can always use

  • Image > Stacks > Images to Stack to fuse single images into a stack (or Image > Stacks > Tools > Concatenate… to fuse several stacks), and then:
  • Image > Hyperstacks > Stack to Hyperstack to define the number of channels and slices

Note that the Composite display mode (e.g. from Image > Color > Channels Tool…) won’t work when working with more than seven channels, but Color mode works, and you can define custom LUTs per channel.

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I should add that once you have a multi-channel hyperstack with custom LUT, then “Image > Color > Stack to RGB” will flatten the HS to make a single merged image.

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Dear all,

thanks for your help.

I foolwed our advices. Now, I have a stack with 10 channels and each of them has its own LUT.
Now, how can I make a composite image please?

Best regards.
Sébastien

I think Image > Color > Stack to RGB only works when you can use Composite display mode (≤ 7 channels, as @imagejan mentioned). I can’t get it to merge 10 channels.

You’re right I forgot a detail. I use the “Stack to RGB” command to convert a multi-channel (>30 channels) hyperstack into a regular RGB stack, then Stack > Z project with the “Sum Slices” option to get a flattened image.

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Hi I just joined, I am new to imageJ I have about 10 Channels, I followed these steps but how do I give 10 different colors to each channels? I got up to 7 and rest of my images are still grayscale.

As I mentioned in my post above, you can use look-up tables (LUTs) for each channel:

Thank you, it works.

Hi,

I´m trying to merge 8 channels; I did as christlet and imagejan have suggested, however the final image present low resolution. I think the image loses quality.

Hi

I tried this (stacks > tools > Concatenate) but it gives me this error: Images do not all have the same dimensions or type. I scaled all the images in the different stacks to be the same and also tried changing the image type but still no joy. I was wondering if you can help?

I have seen something like that qhen i really did think that all of my images were the same type, but due to a quirk of the export, one of my single channel imaes was actually RGB despite only showing blue. Not sure your problem is the same, but ai would recommend first making sure an RGB image hasn’t snuck in.

Thank you for your reply - I just checked - everything is 16 bits :frowning:

Also I can merge different combination of images to create two or even three different stacks. But when trying to merge the stacks it gives me this error…

Concatenate is only for objects that are the exact same, as I understand it. So you do have the same number of images per stack for all stacks, in all dimensions?

Good point - so when I created three different stacks and merged them it works but the three stacks create three different channels (if that makes sense) and not one composite image with 9 channels. I am trying to use concatenate to merge 9 channels together since colour > merge can only merge 7 channels. Am I being stupid here?

I am not so great with FIJI sadly, but I don’t think that will work. You can create a 9 image stack and set the LUT for each channel, which is a process that can be macroed, I think.

Not really sure what the end goal is. If you just want to see the 9 channels, the stack should work. If you want to make a picture, you should be able to snip the view of the stack?

I don’t understand the end goal though, since composite images haven’t seemed to have much use from an analysis standpoint (vs a stack as a single TIFF).

It is not really for analysis purposes but rather a figure for the paper as our working hypothesis. It is just to show the overall change between three different scenarios. What I want is a 9 image composite figure exactly the same that can be done using image> colour> merge channels but for 9 channels instead of 7.

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You could try opening the 9 stack in qupath for visualization and channel coloration. Not sure what the FIJI options are. I usually go that way, plus it is easy to flip individual channels on and off for subset images.

Ah no worries - figured it out. The problem appeared to be in saving the individual stacks as tiff and somehow it was changing the scales when I opened them and even though I re-scaled them it still didn’t work. If I create two stacks fresh from the images then I can merge the stacks and get one composite with 9 channels :slight_smile: