Making new stacks from sequential image files

I have 10 image files with 3 channels each. Each file is at time point 1, 2, 3, etc. I would like to make these into 3 stacks so I end up with a blue stack with 10 frames (i.e. time 1-10), a green stack with 10 frames, and a red stack with 10 frames.

The way I’m doing this is open all images, split the channels, then combine all the blues, the greens, and the reds. This works. But I am wondering if there’s a better way/function to do this.

Thanks for your advices!

I would do:

  • File > Import > Image Sequence, or drag the folder containing the image onto the ImageJ toolbar,
  • Image > Stacks > Stack to Hyperstack… and define the dimensions to be channels=3 and slices=x where x = n/3

These images are actually .nd2–Nikon’s proprietary format so I need to use Bioformats to open. I tried to follow your suggestion by opening each of these image using bioformats (i.e. drag each onto Fiji and click ok when the bioformat window pops up). Then tried the Image > Hyperstacks > Stack to Hyperstack. When I set the number of channels and slices as you recommended, I get this error:

Not sure if this has something to do with the fact that I opened the images using a different method than your suggestion?

Hi @puifais!

You can use Image > Stacks > Tools > Concatenate … to combine multichannel stacks into one hyperstack.

I am not sure how it handles the order of images, so I would verify for yourself that the timepoints are combined in the correct order. Check for problems, e.g., going from timepoint 9 to 10 (because 10 comes before 9 alphabetically).

Once you have the hyperstack containing all timepoints, split channels and you will have your 3 single-channel time series.

The Stack to Hyperstack didn’t work because it rearranges an existing stack into a hyperstack, and as you suspected, you can’t directly open a series of ND2s as a single stack.

Hope this helps.

Never mind. I think I figured out how to do it.

  1. I opened all the images by dragging and dropping onto ImageJ (using bioformats)
  2. I concatenated them all by Image > Stacks > Tools > Concatenate... with All open windows option checked.
  3. I split the channels by doing Image > Color > Split Channels

Boom! Ended up with 3 stacks for each color, with 10 frames each. Thanks!

We must have posted at the same time! Nevertheless, thank you so much!

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