How to create channels in a composite.tiff image in QuPath, when the image was obtained in inForm

I just started using QuPath, I opened a composite_image.tif obtained from inForm software. It should contain 4 Opal channels plus DAPI, but QuPath recognises it as RGB.

How can I open it so that Qupath recognises all the channels? To be able to do multiplex analysis.

Hi @Laia, how does Fiji recognize the image?
QuPath should not recognize it as RGB unless it really is RGB… rather, it should see the correct 4 channels.

(However note that some images can include an RGB thumbnail in addition to the full image – importing the image to a project should give you both. I am also assuming you are using v0.2.0-m12 here; v0.1.2 may be different.)

Hi @petebankhead

I am using v0.2.0-m12 QuPath version. And indeed Fiji recognises the image as RGB… is there a way to overcome this, or one should start all over from inForm software from the original slide scan .msi files or the .im3 files?

Hmmm, it sounds like it really is an RGB image then… unless there is an issue with how Bio-Formats reads it. I don’t expect this to be the case, although it might be if you read the same image back into InForm and find that it is not RGB there.

Sorry I haven’t used InForm myself and don’t know what export options it provides. But if the image really is RGB then it does sound like you’ll need to export again somehow.

If you can share the image I could also have a look at the metadata.

I’ve gone through this a bit on the file receiving side, and there are several ways to export from InForm. The first files I got were RGBs, as it sounds yours are. You can also export individual component files and rebuild a stack in FIJI.
I think they did figure out the settings to export the unmixed components as a multichannel tiff, but I am not sure on the steps and am asking about that now. It is certainly doable though, and does require re-export from InForm.

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thanks to both!

At the end, I re-exported, and got a file called component_data.tiff, when opening this one with QuPath it does recognise it as composite, and with all the opals!!!

Now, my problem is when I do cell detection nothing happens, but going step by step!

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Have you created a whole image annotation to do the cell detection inside of? Also, I think the default export kept accurate metadata, but you may want to check whether the default pixel size “makes sense,” as shown in the Image tab.

Also, if this is your first foray into Vectra analysis, there is a script to rebuild Vectra field of view tifs into whole slide images you might want to consider. I’m not sure which versions of QuPath it might work for as I haven’t used it in a little while, so I’ll refrain from linking it for now. Just wanted to make you aware of the possibility.