Instructions for merging fluorescent images with GIMP
The purpose of this manipulation is to overlay several images of the same cells to make better sense of them. To this end, you will attempt to maximize the appearance of multiple fluorescent signals while still allowing the DIC image to be seen.
Go to File > Open as layers... and select the images to be merged. 2)
If the “layers, channels, paths..” window is not visible, go to Windows > Dockable Dialogs > Layers and the layers window should open. 3)
The layers are listed in this window top to bottom to represent their order of appearance in the image. A layer at the top of the list will be on top of all the other layers, a layer at the bottom of the list will be underneath all the other layers. Rearrange them by left-clicking and holding on the layer and then dragging it in the list so that a black line appears where the layer should go. When you release the mouse button, the layer will go to the new position. 4)
The layer of orders from bottom to top should be: DIC, DAPI, FITC. Once this is done only the FITC image should be visible. 5)
Select the FITC layer on the list of layers and then, using the slider just above the list of layers, reduce the opacity to around 30%. You should see the DAPI stain appear underneath the FITC fluorescence. 6)
Repeat this with the DAPI layer. You should see the DIC image appear underneath the FITC and DAPI images. 7)
Adjust the opacity of the FITC and DAPI layers to optimize the appearance of the fluorescent signal while still including the shape of the visible cell. 8)
Finally, go to Colors > Brightness-Contrast... and increase the contrast by about 30%. You can adjust this to optimize the appearance of the fluorescent signal while still including the shape of the visible cell. 9)
You can save the file using the xcf format which will preserve all the layers so that you can adjust it later if you like. You should save the file using the jpeg format so that the...
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