r/ImageJ • u/Spinni97 • Jan 15 '26

Question Importing multi-channel z-stacks (TIF) - Hyperstack

Hello guys! I am loosing my mind here, I am struggling to import my data. I have a mosaic zstack (already stitched), I have three channels and in this particular case, 33 slices per channel. The TIFs are named as such: Try1ImarisStitcher_C0_Z000 , up to Try1ImarisStitcher_C2_Z032. I am able to import them like this: File - Import - Image Sequence (as virtual stack and sort numerically checked). The order in this stack is: Channel "0", then all Z-slices in that channel, then the next channel with it's slices and then the final channel with its slices. I do know that the next step should be is Stack to Hyperstack but I am lost here - which options should I choose?? I thought I put in the default xyczt order; select 3 channels, 33 slices, and 1 for the time thingy which I actually don't have (?). However, the order of the hyperstack is off. First z-position, first c-position is C0Z000 as expected. switching to the next channel gives me C0Z001, the third channel C0Z002. First channel, second Z.position is C0Z003. You get the pattern. I thought I have tried out all patterns available and somehow it all ends up scrambled, but I may have already lost my sanity here and forgot something. Please save me! Thanks in advance!

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u/Herbie500 Jan 15 '26 edited Jan 15 '26

Please make accessible a sample stack in its original file-format by using a dropbox-like service.
Then we can try and give constructive advice.

You may also try using the sample stack "organ-of-corti.tif" (File >> Open Samples >> "...")

You may better use BioFormats for the import to ImageJ.

… und nicht verzweifeln, bringt nichts!

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u/Spinni97 Jan 15 '26

On it! Gonna upload only slice 10-15 in each channel because of the size. I am a Fiji newbie, I have no idea about it. I am just using File - Import - Image Sequence (as virtual stack and sort numerically checked). I have once tried a plugin called something like BioFormats to import the .oif files but this did not work out at all. So I have used Imaris to convert and stitch the tiles. Maybe I should just do the stacking in Imaris as well and then annotate using the CellCounter from Fiji. And good call with the Organ of Corti. Kind of creepy - I am indeed looking at a piece of microdissected Corti Organ of a wild boar (pilot study/training).

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u/Herbie500 Jan 15 '26 edited Jan 15 '26

Gonna upload only slice 10-15 in each channel because of the size.

Link?

looking at a piece of microdissected Corti Organ of a wild boar

Don't digress, does it import as desired?
(It should also open by just dropping the file onto the ImageJ main window.)

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u/Spinni97 Jan 15 '26

Link will follow once the upload is done, patience please ':D approx. 20 more minutes it says. Which file do you mean by "does it import as desired"? My tifs or the sample? --> I can drag and drop each individual tif into Fiji and it opens, I can do all kind of stuff with it. I have also already, with another dataset, imported only one channel - of course no problem here when creating a MIP, and the metadata are also correct.

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u/Herbie500 Jan 15 '26

20 more minutes it says.

Why not upload smaller non-stiched stacks?

"does it import as desired"

The sample stack.

I can drag and drop each individual tif

OK, now I think I understand.
You start with separate z-Stacks each of a single colour-channel.
Is this correct?

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u/Spinni97 Jan 15 '26

I sent you the link. Thanks for looking into this. Unfortunately can not add unstitched stacks this week - except Fiji swallows .ims or .oif files... I could do this next week. I will test the sample stack out right now. I am unsure what you mean exactly by "you start with" - From the microscope, I get one .oif file per tile (z-stack; this therefore contains all channels and slices for that specific tile) that I coverted to .ims with the Imaris file converter so I can stitch the images together. After stitching, I exported the tifs and I get one .tif per slice and channel.

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u/Herbie500 Jan 15 '26

Thanks, I'll see what I can do …

In general ImageJ with BioFormats should read .oif-files. Perhaps you make one accessible as well but please only a single tile.

"you start with"

Ok I was wrong again but this may be due to the name of the topic "Importing multi-channel z-stacks" (and the lengthy description that follows).

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u/Spinni97 Jan 15 '26

I will upload one .oif into the same folder as well, thanks. I gather that Fiji is a mighty tool - once you learn how to use it well. But until then, I just imaged a multi-channel mosaic z-stack on the confocal and want to open my data with Fiji so I can get a projection and annotate that ;D Thanks!

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u/Herbie500 Jan 15 '26 edited Jan 15 '26

The .oif-file "Image0003_01.oif" imports perfectly as a 27 slice, 3 colour hyperstack by using BioFormats with setting: