Open Microscopy Environment

Hi!
I am new to using LOCI plugin in Fiji.
I wanted to open a series of .tif files using the 'Bio-format importer' > 'group files with similar names' in order to get a z-stack with 3 channels merged (RGB). I do not have the merged pictures but only the separate channels for each focal plane.
For this I was choosing a hyperstack output and composite colour mode. However, I always get the error 'the data has too many channels', regardless of the colour mode I choose. Why is this?...

I have been trying to get these stacks using different plugins but so far no luck... I always seem to get strange stacks with info from only one channel and have not figured out how to get the z-stack with the 3 channels merged. Is this even possible or do I have to make the merges first and only then get the stack? Geting it all at once would really save me some time...

Thanks for the help!

I would guess based on the error message that the Z slices are being detected as channels, but without seeing the files that you are trying to open it's difficult to say for certain.

Would you be willing to send one of your datasets to the OME team for investigation? If so, please upload a Zip file containing the entire dataset to:

http://qa.openmicroscopy.org.uk/qa/upload/

Once we have the dataset, it will be easier to find the best way to open it.

Hi mlinkert!

I have uploaded some samples so that you can see what I am talking about. Three z-focal planes with three channels each.
I really appreciate your help!

Thank you for uploading an example dataset.

Bio-Formats nearly does the correct thing with this data; 3 Z sections are detected, as are the 3 channels specified by the file names. The problem is that each of the 9 files contains a single RGB (i.e. 3 channel) image - so there are in total 9 channels. As far as I can tell, the images for *ch00.tif have valid data in the green and blue channels (though both appear to be identical); the images for *ch01.tif have valid data in the red channel, and the images for *ch02.tif have valid data in the green channel.

There is no way to have all 9 channels composited together in ImageJ; to get a composite image, you would need to select the channels with valid data. Probably the easiest approach is to open each of the "real" channels individually - use the "Group files with similar names" and "Split channels" options, then replace "Series001_t0_z<0-2>_ch0<0-2>.tif" in the "File pattern" box with "Series001_t0_z<0-2>_ch00.tif" (repeating with ch01 and ch02). Each of the RGB channels will then be opened separately; close whichever ones do not contain valid data, leaving the others open. Once everything is open, use "Image > Stacks > Images to Stack" to put everything back together into a single stack. You may find it useful to write/record a macro to accomplish some or all of those steps.

Additionally, if there is a file named "Series001.lei" or "Series001.txt" in the same folder as the .tif files, you might try opening that instead of using the "Group files with similar names" option.

If those suggestions don't work, please let us know.

-Melissa

Thanks a lot Melissa.

I will try your suggestion and let you know if it works!

Catarina

Hey Melissa.

Spliting the channels for each series just like you suggested is giving me three stacks, of which I choose the one that has information for each channel.
This means that in the end I get three stacks and not single images. Is it possible to merge stacks?? I have looked for a solution but so far no luck...

If I understood your suggestions correctly, even if I had single images, which I had to convert into stacks in the end, this issue would still stand given that I want to merge the stacks.

Cheers,
Catarina