Biomedical Imaging Research Unit School of Medical Sciences Faculty of Medical and Health Sciences The University of Auckland Private Bag 92019 Auckland, NZ Ph: 373 7599 ext. 87438 http://www.auckland.ac.nz/biru/ [email protected] Jacqui Ross 08 October 2009

Combining Images with Cyan/Magenta/Yellow LUTs

These instructions demonstrate how to apply a Look Up Table (LUT) to a fluorescence or confocal image and then how to combine images with Cyan, Magenta and Yellow LUTs as well as Red, Green or Blue. As mentioned in the introductory notes, LUTs assign a color to be used for each of 256 possible displayed pixel values (for 8bit images). There are special LUTs for applications such as calcium imaging, which help to highlight intensity changes.

1. If your image is not 8bit, then you need to first change it to 8bit as below. If you have an RGB image, you could split the channels instead. This results in three 8bit images.

2. Applying a LUT is very easy in ImageJ. All you have to do is go to Image – Lookup Tables and choose the table that you want to use, e.g. Cyan as shown below.

3. This image is now in 8bit colour. If you just want to use the Cyan image and you don’t want to combine it with other images, you can then change it to RGB (Image – Type – RGB).

4. If you want to combine this image with another image, e.g. Yellow as below, you need to download the Colour Merge plugin. This is different from the one which is built in to ImageJ. It is available within the MBF bundle (http://rsbweb.nih.gov/ij/plugins/mbf‐collection.html). You don’t have to use the entire bundle, just download the Plugins .zip file and then copy the plugin into the IJ Plugins folder. You could also copy it into a separate folder (e.g. Colour or Colour Functions as per MBF) within the Plugins folder. Then relaunch ImageJ. You will now have a Colour Merge plugin available for use. 5. Make sure that both of your images are open as below:

6. Then go to the plugin (e.g. Plugins – Colour – Colour Merge).

7. The window below comes up. You just need to select the two images and which LUT you want to use for each. In this case, I am selecting Current to keep the LUT that I just applied.

8. However, you could select a different LUT by clicking on the drop‐down box as below:

9. The image below shows the resulting image.

11. If you use the “Difference” operator, then you get the result as below. Instead of adding the images, this option produces a difference image. Sometimes this shows the overlap more clearly.

12. You can merge additional images by keeping the Colour Merge image open (selecting Current to keep the colours) and then selecting the next image.

13. The image below shows the Cyan, Magenta and Yellow images combined. 14. Please note that the image looks a bit odd because I have just rotated one of the images to create the third image for combining!