LIF Losing Channel with FIGI?

Hi All,

I have a 4 channel (DAPI, eGFP, Cy3, Cy5) z-stack image I took on a Leica Confocal, resulting in an LIF project file that is fairly large. I can put it into the LAS X application and see/export it as a TIFF and then put that file into ImageJ.

My usual process (done on OIF files) is doing the max-projection z-stack followed by separating the channels. However, for some reason when I do this the end output is only 3 channels. They’re called “red”, “blue”, and “green”, but they don’t perfectly overlay with the 4 channels, so I am confused.

Also, when I do this with OIFs, it splits into channels it labels C1-, C2-, C3-, and C4-, which is different from the labels I get from doing the same steps with the LIF.

Does anyone know what is going on or a way to work around this? I just want to split the channels into four greyscale pictures for later image analysis and don’t want to lose any image quality.

Thank you for any and all help!

Hi Alex,

It’s sounds like you are exporting your images as RGB files! There probably is a way to export with full bit depth and all your channels but if you are using FIJI, then the Bio-Formats plugin that comes packaged with it should be able to open your .lif libraries without any need for exporting in another program.


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Thank you so much for your help! I suspected something like that happening as well, but had no idea what to look for. I will try looking into the plugin to see if it’s up and running as expected.


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Is the issue that they are too large, that you do not want to open them in ImageJ on your computer?
If not, LIF files can be opened in ImageJ/FIJI, using Bioformats importer.