Problem Generating Analysis Stack Tiffs

[cassandraman]

Hello,

I've recently been re-running the pipeline and have come across an error with the step "Generate the analysis stacks" to generate the initial tiff folder images. They appear to be only cropped sections of the full (square) image. I've included a screenshot of the tiff folder preview and an opened image file:

I have run the pipeline from the beginning several times to confirm, but this is reoccurring, either affecting all the tiffs or only a portion of them. I have tried to continue with the pipeline, but when I get to Ilastik it is clear that I cannot properly capture the cells due to the small section.

I'd really appreciate any help in troubleshooting this error! Thanks, Cassandra

[votti]

Hi Cassandra,

This does seem strange :/

Unfortunately I am still on holidays traveling till next Wednesday. So till thebI can only give limited support.

Can you try to open the _full.tiff image in ImageJ/Fiji to confirm their unexpected shape?

Otherwise this does seem like a potential issue with the .mcd file parsing. What software version is your machine running? I suppose the images opened in the mcdviewer Fluidim software look ok?

Cheers!

On Wed, Jul 15, 2020, 18:01 cassandraman notifications@github.com wrote:

Hello,

I've recently been re-running the pipeline and have come across an error with the step "Generate the analysis stacks" to generate the initial tiff folder images. They appear to be only cropped sections of the full (square) image. I've included a screenshot of the tiff folder preview and an opened image file: [image: Screen Shot 2020-07-15 at 10 53 55 AM] https://user-images.githubusercontent.com/51934558/87578614-84db3880-c689-11ea-982d-35d3836f60c5.png [image: Screen Shot 2020-07-15 at 10 55 25 AM] https://user-images.githubusercontent.com/51934558/87578734-b522d700-c689-11ea-9dda-57bdda785794.png I have run the pipeline from the beginning several times to confirm, but this is reoccurring, either affecting all the tiffs or only a portion of them. I have tried to continue with the pipeline, but when I get to Ilastik it is clear that I cannot properly capture the cells due to the small section. [image: Screen Shot 2020-07-15 at 10 48 37 AM] https://user-images.githubusercontent.com/51934558/87579176-5f9afa00-c68a-11ea-9101-8f7352249614.png I'd really appreciate any help in troubleshooting this error! Thanks, Cassandra

— You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub https://github.com/BodenmillerGroup/ImcSegmentationPipeline/issues/43, or unsubscribe https://github.com/notifications/unsubscribe-auth/AA4R2KFJTRH7QHC356TEFDTR3XVHNANCNFSM4O2YQY7A .

[cassandraman]

Thanks for your quick response!

I was able to confirm the cropped shape of the _full.tiff image in Fiji. Im running macOS Catalina 10.15.5. I have to ask around and confirm on the mcdviewer images, as I am not familiar with the software.

This is the first time it has happened with this dataset in particular, but has been occurring very consistently for another dataset I'm working with. Really appreciate your help on this!

[votti]

"This is the first time it has happened with this dataset in particular, but has been occurring very consistently for another dataset I'm working with"

So it's an error that occurs a) sometimes and sometimes not for the same files b) only for a subset of files?

Would you be able to share an example .mcd file where this happens? To me it does sound like an issue with the data or metadata contained within the .mcd file, so seing a file would greatly facilitate resolving the issue.

Best

On Thu, Jul 16, 2020, 00:35 cassandraman notifications@github.com wrote:

Thanks for your quick response!

I was able to confirm the cropped shape of the _full.tiff image in Fiji. Im running macOS Catalina 10.15.5. I have to ask around and confirm on the mcdviewer images, as I am not familiar with the software.

This is the first time it has happened with this dataset in particular, but has been occurring very consistently for another dataset I'm working with. Really appreciate your help on this!

— You are receiving this because you commented. Reply to this email directly, view it on GitHub https://github.com/BodenmillerGroup/ImcSegmentationPipeline/issues/43#issuecomment-659086313, or unsubscribe https://github.com/notifications/unsubscribe-auth/AA4R2KDLZ6A73OTATWRGIM3R3ZDONANCNFSM4O2YQY7A .

[cassandraman]

Apologies for the delayed response.

This error has been occurring consistently with a subset of files, and has only recently occurred for a different set of files (which ran normally previously).

Really appreciate your offer to take a look at an example .mcd file, but it looks like its too big to link here (its over 100 MB). Is there another way I can share it with you?

Thank you again for your help!

[votti]

Hi @cassandraman

Please send me the file with a service like https://wetransfer.com/

You find my email address here: http://www.bodenmillerlab.com/people/vitozanotelli/

Best! Vito

[cassandraman]

Hi Vito,

I have just sent over two example files which have presented this problem. Please let me know if I can provide any other information that may help. Thank you--I appreciate your help on this!

[votti]

Hi @cassandraman ,

I had a quickly look and it didnt look really suspicious: e.g. mcd folder SP19_14141C1 contained 5 acquisitions:

From the metadata (cpout/acquisition_metadata.csv):

Description |  AcquisitionROIID | MaxX | MaxY
-- | -- | -- | --
ROI_002 | 2 | 1000 | 1000
LaserTest | 3 | 49 | 13
LaserTest - split | 4 | 49 | 22
LaserTest - split - split | 5 | 49 | 20
LaserTest - split - split - split | 6 | 49 | 95

Only acquisition with acquisition ID 2 seemed to be a 'real' acquisition with dimensions 1000x1000. The others seemed to be some test acquisitions which have the tiny size you observe. This included the acquisition with the name: SP19_14141C1_s0_p3_r5_a5_ac_full.tiff (AcquisitionROIID = a5 ) that you were concerned about.

The 'real' acquisition (AcquisitionROIID 2, SP19_14141C1_s0_p3_r2_a2_ac_full.tiff) has the expected dimensions.

Is there any other specific acquisition that you think to be concerning?

[votti]

I think this was solved?

[cassandraman]

Hi Votti,

Thanks for reviewing the file--we went back and were able to resolve the problem.

Thanks for your help!