Autothresholding on Imaris files imported as Virtual Hyperstacks

Hi all,
I am using the BioFormats importer in Fiji (most recently updated version as of Oct 31, 2020). It provides options for import of the various sized 4/5d datasets in the .IMS file s (in my case from Andor’s Fusion software), and I can easily import the highest resolution version (thanks!). However, there seems to be some autothresholding/contrast enhancement applied to the individual images in the dataset, but only when the Virtual Stack option is selected. If I read into RAM it works correctly without this occurring. Since the Virtual Stack feature is important for speed and the only way to go for large dataset that exceed RAM, this is an import current limitation.

I cannot replicate this effect when I select Auto threshold on import using a RAM-based stack. I notice that when selecting the Virtual Stack option the Auto threshold option is disabled.

I have a sample dataset, but all I have is a large one (sorry, but is representative). You can download it here (640 Mb, sorry):
http://worms.zoology.wisc.edu /drop/IJM_archive.zip

Might someone from the BioFormats team take a look?
Thanks!
Jeff

Hi Jeff

Thanks for submitting the sample dataset.
When the Virtual Stack option is used, we cannot determine the min/max for every plane, so the full range of the pixels type is used and the Auto threshold option if turned off.
This is the expected behaviour.

Cheers

Jmarie

Hi Jean-Marie,
Thanks for responding. I may be missing something, but this doesn’t seem right to me. It seems more likely that the Virtual Stack is looking at each slice and applying a contrast stretch or normalization on a per-slice basis that does not happen with the RAM-based import method. If the reverse is true, then the RAM-based import is doing autothresholding even when the autothreshold box is not checked. . I guess I’ll have to check the pixel value histogram on a per slice basis to determine which is happening. Either way, the expected behavior would be that it shouldn’t matter if the import is virtual or RAM-based. perhaps I can look t the code myself to check on this point, if that’s possible.

Thanks for your help!

Best,
Jeff

Hi Jeff

Looking at the image again, it seems that you are correct. I read the dialog wrongly. The full pixels range is used when use Virtual stack is not selected. When it is used, the interval is set to something like 580-296 for the first plane of the first series

Cheers

Jmarie

Hi Jean-Marie,
Thanks for verifying that I’m not crazy (at least about this)! What would you recommend as a next step? I haven’t looked at how the BioFormats files are compiled. Thanks for your help!
Cheers,
Jeff