Variable height stacks in OME TIFF?

If a microscopy image consists of channels with different Z height (In particular e.g fluorescent channels with stacks, and phase contrast image with a single plane), is there a (standard?) way to represent this in ome tiff, such that the channels would not loose their relation in XY coordinates.

Hi @KaupoPalo and welcome to the forum,

there are a few ways to handle sparse planes in OME-TIFF. To point you at the most relevant examples, I have two questions:

  • following your example, I assume the phase contrast plane is acquired at the same Z height as one of the fluorescent planes. Typically, that would be the top, middle or bottom plane. Is that a correct assumption?
  • from a client perspective, assuming both channels were overlaid on each other and the user was going over the Z-dimensions, would you expect the phase contrast single plane to always be displayed on top of the relevant fluorescence plane? Or would you expect it only to show up on the Z height where it matches the fluorescent channel and have blank planes otherwise?

Best,
Sebastien

Thanks for the answer. The thing is that (Digital) Phase Image characterizes the stack as whole (in some ways it is like a 2D projection). In some vague sense one can attribute a Z value to it too.

I am not really thinking in terms of what a software should do with it to display. If someone wants to use it, then this should be handled very specifically.

I rather would like the tiff file to contain the information in unambiguous way.

If to fantasize what a possible application could do then e.g. one could determine a 2D ROI mask from the phase image, and then “lift” it like a tube into the volume.

The question is not solely bound to Z stacks. One can also do a time series, while taking the full set of channels much less frequently than the actual frame rate. This seems to collide with the 5D paradigm.

Hi Kaupo,

yes there are several modalities where imaging is sparsely acquired which challenge the traditional 5D volume expectations. As long as the sparseness is at the level of entire planes, there are a few strategies available to represent such data.

The first option is to create a 5D image with two channels and the single phase contrast plane associated duplicated in the metadata across the entire Z-stack for phase channel. This can be achieved by having each TiffData element for the phase channel pointing to the same IFDs of the same TIFF file. This strategy had been discussed in our legacy forum.

The second option is a variant where the phase contrast plane would only be associated with a single Z index. An example in the case of sparse time-series acquisition is demonstrated by the time-lapse images published under the IDR study idr0065 where the fluorescence channel is acquired every two time frames. In the companion OME file, available here, all the planes of the fluorescent channel where no acquisition was recorded are defined as a TiffData element with no associated UUID/Filename element. The main caveat is that this strategy is only valid if the OME-XML is stored as a separate metadata-only companion file. If the metadata is stored in a TIFF file, the assumption is that a TiffData element with no associated UUID/Filename element is referencing the TIFF file itself for the binary data.

Finally, if the digital phase image is best seen as a projection, a third option is to express it as a separate image. This is the strategy that was used for the IDR study idr0047 - see an example of 3D stack, the associated projection and the companion OME-XML that defines these images. Here, the main issue is that the XY relationship between the two images becomes effectively implicit.

Hopefully this gives you pointers for a few options. Let us know if you need more specifics.
Sebastien

Thanks so much Sébastien for taking the time to give a comprehensive answer! Very helpful. Useful hints all of them and make sense to me. As a newcomer to OME tiff, it is a bit difficult to grasp if I am missing something. Interesting enough the version #1 of repeating a single plane over is smth that we have considered in our workflows too.

Glad the answer was helpful. As a side note, I just realized we don’t have any public example for the option 1 with plane repetition. Just let us know if such an example would be valuable as it should be easy enough to derive one from the public samples.

Thanks for bringing these questions on the open, we fully appreciate some aspects of the OME model/specifications require some experience. Discussing the representation of non-trivial acquisition modalities helps us clarifying the features and limitations of the file format and its documentation but it also provides valuable input for the ongoing community discussion about the next-generation file formats - see Next Generation File Formats for BioImaging.