I have fluorescence images from a microscope -- they are in TIF format. I simply want to merge these together, I.e. overlay them. I have images in red, green and blue channels. My typical method of doing this is simply to make a new RGB image in photoshop and paste each of the channel images into their respective channels. I do Ctrl+A to select the image of interest, then Ctrl+C and Ctrl+V it into the channel. My question is: how can I do this with brightfield image as well? The brightfield is sort of like another channel, since it does not fit in red, green or blue channels.

How can I merge/overlay these images, such that all fluorescent channels plus the brightfield (which is a greyscale image) are visible?

My images are 16bit and I'd like to merge them like that. My understanding was that ImageJ forces one to go to 8-bit but I'd prefer not to.

  • What is the purpose of combining them like that? Are you just viewing the composite onscreen to get better insight about the subject? – Matt M. Sep 26 '11 at 8:17

You could try using Lab color space for this.

After you have created your RGB channels as you have described, convert your image to Lab Color mode (Image -> Mode -> Lab color) and paste your brightfield channel into L. You can then optionally convert back to RGB mode.

If you don't want to completely overide the brightness as defined by the RGB values, you could experiment with copying the L channel to a new grayscale image and overlaying your brightfield on it before pasting a flattened copy back to your L channel.

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