Files format for multiplexe QuPath analysis

Hi everyone

I’m trying to use QuPath to analyse imaging mass cytometry data. I have tried to import images in different formats (16bit and 32bit multi and single tiff from MCDViewer, .mcd and txt) but I can’t manage to import one single file with all the channels included as required for the multiplexed analysis. I have seen that the sample data used in the QuPath tutorial is either tif or qptiff (not sure about it).

I’d be very grateful if you could answer these two questions:
Which formats can I use in QuPath for imc multiplexed analysis?
How can I transform my Hyperion files which are .mcd in any acceptable format for QuPath?

Regards
Juan

Hi Juan,

OME-TIFF is probably the best format to use for highly multiplex images. MCDViewer can export image data into that format (see: https://www.fluidigm.com/binaries/content/documents/fluidigm/resources/mcd-viewer-v1.0.560.6-user-guide/mcd-viewer-v1.0.560.6-user-guide/fluidigm%3Afile - chapter 2, use the Multi checkbox). Are those OME-TIFF exports not readable by QuPath? If not, I would complain heartily to Fluidigm because that means they have a bug.

Cheers,
Damir

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The MCDViewer exports should be readable in QuPath, though I used to have to script merging the channels into a single image. Not sure if they handle export to a multichannel image yet?

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Hi Damir and RA

Thank you for your comments, they are very helpful to me. QuPath can read my ome.tiff files but there’s a problem somewhere because when I click on [Brightness & contrast] I can only see one channel (with all the markers together, I think) instead of one for every marker. So I can’t give different colors to different channels.

I don’t know whether I’m doing something wrong or there’s some issue with these ome.tiff. I noticed that the sample data is not ome.tiff but just tif. I don’t know how relevant that is. Damir, should I contact Fluidigm?

RA, how did you? Di you export as single tiff and then merged the channels? How could I produce a similar script?

Regards
Juan

I used FIJI. The script would grab all of the tiffs in a folder, create a Stack with them, and then adjust the Image->Properties to make sure they were all Channels (and the pixel size was correct). If you leave the default Properties when creating a stack, you end up with a Zstack, as seen here:

This only helps if you have each of the channels as a separate file. If you do have one file with all of the markers piled together in a single channel, I am not sure how you would split that up.

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Hi Juan,

The MCDViewer manual appears to indicate that it can export into ome.tif format and it even specifies which fields in the OME-XML header it will populate so that sounds very promising. Do you have a way to post an example exported ome.tif file somewhere for us to look at? I’ll be happy to do a quick check.

Cheers,
Damir

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