Not preserving fourth channel when opening CZI to TIF files in Imaging J

1_MIP_TIFF_today.tiff (1.7 MB)

I have tiled .czi images that I want to stitch together. To do so, I save as .TIF files, and upload to Fiji.

However, once I open the file I realize it automatically opens them as RGB images. When I convert them to composite images, it only shows three channels (green, red, blue). I have four channels in my .CZI images, so it seems my far red and my red are being combined in Image J.

Is there a way to preserve all four channels? I read that you could have more than 3 channels on section 9 of the image J guide (ImageJ User Guide - IJ 1.46r | Color Images) but it’s too vague for me to understand how to make that happen.

Any help or tips would be greatly appreciated!

You exported an RGB image - the image you uploaded is not 4 channel, it is 3. So FIJI is opening it correctly.

How are you creating the RGB images?


Thanks for getting back to me.
On zen blue 3.3, I create a maximum intensity projection and save it as TIF file. I then use bio-format importer on Image J to open the file.

Here are the settings in case this is where I went wrong.

This is what I get.

If I open up the CZI file of the z-stack, the 4 channels are shown correctly (however then I can’t stitch different images together correctly).

I could stitch the maximum intensity projections correctly, but then I lose all four channels (i want to keep the composite image, and not turn them into RGBs so this is where I’m getting stuck).

Also, opening the CZI file of the maximum intensity projections made on ZEN have their own issues which I opened up another topic about.

Sorry I know this is a lot! I’m a beginner of fluorescent image analyzes, so I hope I covered my bases enough.


Those settings are not important in this case - you converted the image to RGB prior to opening it in FIJI. If you were using Zen, exporting a merged channel image creates a non-scientific TIFF picture.

If you export the Original Data you will create single channel images that you can then merge in FIJI.

Some versions have the option to export as OME.TIF * and I just double checked, the ome.tif is not an RGB, it is a multichannel image.


I would generally start with the CZI file, though.