1. Open the images in ImageJ
2. Adjust the contrast if neceesary: Image/Adjust/BrightnessConstrast
3. Change or check all Images are in 8-bit format. Image/Type/8-bit
4. Image/Color/Merge Channels and the Merge Channels box will appear
5. Select the fluorescent images in the appropriate R, G and B channels and the DIC or similar image in gray channel. Select “Create Composite” and click “OK”
6. Click on the new ‘composite’ image to activate it
7. Image/Type/RGB Color
8. Save it as “Tiff” file

Overlap