We want to identify a certain type of lymphoid cells in cancer tissue however for that we need to identify and exclude three different types of cells (T, B and Natural Killers) using immmuno-histochemistry separately applying antibodies (CD56, CD2 and CD 20) on separate )sections/slides by assuming there will not significant difference among four sections at 4µm thickness.
Brief procedure is to make 4µm thickness slides from the same tissue sample obtained from FFPE blocks, first slide will be exposed to CD2 to identify T cells, take photograph using light microscope and export the image to Fiji and identified T cells will be given a color,
Similarly the same procedure will be done on rest of three separate slides to identify B cells, NK and ILC 3 using rorgamma t antibody giving each cell type different colors. At the end we'll have 4 different Figi colored images.
Now the tricky part is how can we merge these four images together to get one single image with all four types of cells exhibiting different colors, so that in a single final merged image we can show all four types of cells WITH DIFFERENT COLORS.
Or are there other sofwares which can help us achieve this objective?
Hi Muhammad,
I suppose your your single channel images (e.g. CD56) is a grayscale image and not a color image?
In that case you can adjust each single channel image (CTRL+Shift+C) and then use the command MainMenue\image\color\mergeChannels\
you will get a menue which you can use to assign each open image with the contrast settings you choose to be displayed in a certain color scale in the merged color image. You should end up with a composite image which has still the original bit depth per channel. You can then also flatten the image if you choose Image\type\RGB but your original dynamic range is then lost in this color image.
If your images are already color images (I would not think so, but...) you can use the command image calculator in MainMenue\Process..
If this does not help it might be better if you share a set of your 4 images.
off note: It should be possible to choose 4 fluorescent markers with different spectral properties so you could generate your 4channel image from one section...
Best
Heiko
Heiko Dussmann, Thank you very much, really much help. I'll take the liberty to ask you more if I need more help I hope you are okay with that :)
Have a nice Christmas and season greetings
Tahir
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