Problem with merging channels on imageJ


I’m using fiji/ImageJ and I am trying to combine a FLIM image and a 4-channel confocal image of the same cell. First FLIM was taken while the cells was alive and after fixation I relocated the same cell with some staining. But since the images were taken with different microscopes and magnitudes, some arranging needs to be done before merging. I have done the scaling, made both the images approximately same to be fit perfectly when merged(arranged the angle, width,height etc.) but I cannot merge these images together. For instance FLIM image needs to be moved 100 micron to the left on the x axis and then added as a 5th channel to the 4-channel stack image. It would be great if you could help me on that!

One other thing is that (I can live without it but would be perfect if we can solve), when I select “merge channels” and merge 4 images into a stack and select “make a composite”, there are 4 channels in a stack but in each channel I see all those images merged together. But I would like to see each image separately and only see the merged image as another channel instead. Is that possible?

Hope I am clear with what I mean…

Thank you very much in advance!

Hi @melody,

You split the stack to images, select and copy the FLIM image, create a slightly larger image, make a selection on the new image and place that 100 µm to the left, paste the clipboard (ie. flim contents), clip the new image to the desired size and then do a merge on all 5 channels of the now equal-sided window. Is that what you mean, and if not, what do you miss?

Thank you @eljonco !

I guess that would have worked too, I solved the issue as adding a new slice(empty) to the stack, simply copied a selection of that flim image and pasted it to the new slice. When I first paste it, it allows me to move the image as I want, so I used other channels as a reference to make it fit. Now they are all merged, with flim image on the 5th channel.