Importing a .zvi multiplex image as mosaic image

Hello all,

I am trying to import mosaic images into qupath. Normally, I export .tifs from the imaging software, and Qupath does fine importing the concatenated image. However, I am now working with some fluorescent mosaic images, and I can’t export them as .tifs without the pixel type being set to RGB. So, I thought I would just import the .zvi mosaic into QuPath, which I know for a fact preserves the proper pixel type. However, Qupath automatically splits the mosaic into its composite tiles. I want to preserve the concatenated aspect of the image, as I am trying to create spatial data of various immune markers.

I imagine there is probably an easy solution to this, but I haven’t been able to find anything with my lacking knowledge in computer stuff. Any advice is appreciated!

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I don’t know enough about .zvi files, but my guess is that Bio-Formats provides them as different scenes.

QuPath isn’t really splitting anything… but it doesn’t yet support merging multiple scenes into a single one. This is actually quite difficult to do for any arbitrary Bio-Formats-supported image, since it potentially raises various coordinate-interpretation / overlapping / what-is-the-most-sensible-background-value complications.

Hi Pete, I think you’re right. Apparently the imaging software doesn’t actually merge the tiles of a mosaic until export, so it makes sense that opening the .zvi through QuPath just opens the tiles…

So then, my problem is finding a way to export the image so that it is saved in its concatenated form while preserving the 16bit pixel type. A recent reply on a post by @Research_Associate suggested .tifs as a file fromat that should be able to do this, but for whatever reason our lab’s imaging software exports .tifs in the rgb format… or at least, it’s being interpreted as rgb when I load it in QuPath. So, not sure what to do…

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If you are using ZVI, is that Zen you are exporting from? And if so what settings are you using?

What do you want to do with the mosaic? in QuPath - what type of analysis can you not do with separate scenes?

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Yes - I’m exporting it as a .tif with 0% compression, merged-image only, and i specifically have “convert to 8-bit” turned off. This leads me to believe that the .tifs are being exported as 16-bit but aren’t making it to qupath on my computer that way.

So, my lab exports images from our microscopy computer using DVDs. Is it possible that writing the exported tifs onto the DVD format coverts them into 8bit?

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It wouldn’t be the writing itself, but the any export process might have a conversion step. It should be easy enough to check by opening the DVD images in Zen again, even Zen Lite, or FIJI maybe.

Another option might be using BigTIFF or OME-TIFF as export options (OME showed up in some more recent versions).


Thank you - I’m going to play around with some stuff and see if i can figure it out. I’ll post if theres a final answer, but I won’t clog the forum feed considering this has turned out to be a personal problem :slight_smile:


Hi there, I am having the same issue. Were you able to separate the red channel from the far red channel as a tiff or open the .zvi as a single merged mosaic image? Would really appreciate any suggestions!

Hi Kelsey,

So this was a problem for me because our imaging computer runs an old version of Axiovision. If you have Zen, or maybe even a newer version of axiovision, you should be able to export the images as OME-Tiffs or another more sophisticated file format. In fact, newer softwares probably can export regular TIFs as 16bit - it just seems to be a problem with the old Axiovision software we use.

In my case, the only workaround is to bring my .zvi’s over to a neighboring lab’s computer and export them to 16bit tifs there. I hope you find a workaround too :slight_smile:

Thanks so much, David. I’ll try that! Ours is probably quite old too! :slight_smile:

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