How to figure out pixel size for TIFF images

Hello all,

I am working with a TIFF image (pixel type: uint8 (rgb)), and the pixel size was set at 169.333 um. After reading some posts and answers, I know this is wrong. However, I cannot seem to figure out what it should be set to. I cannot get the cell detection to work and my resolution is atrocious, I believe due to this. If anyone knows how to figure out what the true pixel size is, I would really appreciate the help!

There is no such thing as a true pixel size. You would need to get that from the microscope/objective/measure with slide that has a built in ruler, etc.

Many microscopes take images in an original format that contains this metadata. If you have the original image, you can use that to determine the pixel size.

Worst comes to worst, you can estimate it based on the objective/NA, but even with that you can get variation and your measurements would be only estimates.

If the image itself looks bad, that has nothing to do with the 169.333, which I think is the default when no pixel size was found.

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The original image has a 50 um marker on there, so I measured that with the line annotation to figure it out. Thank you for your help! Just for future reference, how would I find the metadata from the original format? Also, is there any way I can fix this resolution with Qupath, or is this an irreversible issue that’s due to the scanner?

If you know the pixel size, you can double click on the pixel size in the Image tab to set it (assuming you have no annotations selected). Or there are scripts on the forum you can use to apply it en mass.

Also you can adjust the metadata in FIJI using the Image->Properties…
Record that macro, and apply it to batches of images.