Unable to properly open multi-color TIFF images

We have a new microscope (Olympus) with one camera (Andor iXon 888 1k x 1k EMCCD) and an OptoSplit III device to split the chip into 2 or 3 channels. We acquire images via metamorph. When we try to open the images via Bioformats, they are not recognized as multi-color images, but rather just as if they are 1-color full chip images.

We have an equivalent set up on an older microscope (with some differences, described next), which we also control via metamorph, and Bioformats is able to open those images correctly. One difference between the old and new microscope images is that from our old microscope the images are 512x1024 and the split is vertical (so each color is 512x512), while from our new microscope the images are 1024x1024 and the split is horizontal (so each color is 512x1024). In the case of 3-way splitting, each color would be ~341x1024 on our new microscope.

For context, the differences between our old and new microscope are as follows:
(1) Old microscope has two photometrics EMCCDs while new microscope has 1 Andor EMCCD camera and a splitter.
(2) Old microscope is a hybrid from Nikon and Spectral Applied Research, while new microscope is from Olympus.

But again we use metamorph in both cases, with equivalent stream acquisition settings, and the files are stored in TIFF format in both cases.

Could you help us get those images read properly using Bioformats (we really like Bioformats)? I can supply example images if you let me know where to upload them. Thank you.

Hi,
You can send files to us at https://www.openmicroscopy.org/qa2/qa/upload/
If you could upload an example from both microscopes, that would allow us to see what’s changed.
It may also be helpful to have a screenshot of how you expect the images to appear. Are you opening images via BioFormats in ImageJ/Fiji or some other way?
Regards,
Will.

Hi Will,

Thank you for getting back to me. I am in the process of uploading example images via the link that you sent me. I am uploading 4 files:

(1) m01_newMicroscope_DoesNotOpenProperly.tif: This is an example movie from our new microscope, which BioFormats does not open properly.

(2) m02_oldMicroscope_OpensProperly.tif: This is an example movie from our old microscope, which BioFormats can open properly.

(3) exampleOldMicroscope.jpg: This is just a screenshot of opening movie “m02” (from old microscope) using BioFormats in Fiji. In the screenshot you see the metadata at the top, and you can see that it correctly identifies that it is a 2-channel movie. In the bottom left of the screenshot, you see the movie as opened correctly via BioFormats. So you see that it has 2 channels and the right 512x512 size. In the bottom right of the screenshot, you see the movie as it appears if I simply drag and drop it in Fiji, i.e. not using Bioformats. In this case, it is not read properly, and instead of 2 512x512 channels, it appears as if it is 1 512x1024 channel.

(4) exampleNewMicroscope.jpg: This is a screenshot of opening movie “m01” (from new microscope) using Bioformats in Fiji. As you can see, it thinks it is a 1 channel 1024x1024 image series, instead of 2 channels, each 512x1024 in size (horizontal cut). In the OME metadata you see that sizeC = 1, but it should be 2.

Thank you for your help, and please let me know if I can provide you with any more information.

Best,

Khuloud

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Thank you very much for those files and your accompanying explanations Khuloud. You’re absolutely right that they cause Bio-Formats difficulties: we’ll investigate accordingly.

I was wondering if you had any further insight or update regarding opening our multi-color TIFF files with BioFormats.

Also, in relation to Will Moore’s original question about how we open the files, I should say that we open them using BioFormats both in Fiji and in Matlab.

Thank you for looking into the matter,

Khuloud

Hi @kjaqaman1, there is currently a Trello card which we will be using for tracking this issue (https://trello.com/c/kDDb9pEu/365-multicolor-tiff-issues-with-new-olympus).

I was retesting it this morning and the uploaded tiff files currently are not identified as MetaMorph files, is there an associated stk or nd file to go with these files?