Protocol for Adding Color to Light Microscope Images Using PhotoShop

This protocol works well if you have images of a double or triple fluorescently stained structure that you wish to represent with 2 or 3 different colors, respectively. It is important that all the images were taken with the same objective and positioning to allow for proper overlay and alignment of images during the colorizing process.

1. Open up Adobe PhotoShop. Go to File and Open all of the desired black and white images you obtained from the confocal or the fluorescence microscope that you would like to represent in the colorized image.
2. Go to Image and then Mode and then change to RGB color. The image comes in as indexed color but changing to RGB color will allow you to colorize the image. (Images obtained from the Web Atlas are already in RGB format.)
3. Usually the image you obtain from the microscopes is not at the same image quality in terms of brightness and contrast in PhotoShop. Thus the Levels of the image must be adjusted. Go to Image and then Adjust and then Levels. You will see a small window with a histogram of light and dark levels present within your image. You want to adjust the output levels with the provided arrows to reveal the best contrast and structure. You also want to make the image background as black as possible without losing imaging quality. Any grey present will be reflected in a poor image reconstruction. This is done by placing the black arrow at the far-left tail of the histogram and the white arrow at the far right tail of the histogram. Now use the gray arrow to move within the boundary set by the other arrows. Once satisfied click OK.
4. Go to Window and then Show Channels. This shows the image you are working with in 4 channels: RGB, Red, Green, and Blue.
5. Now go to the second image (image 2) and copy it. Make sure that image 2 is also in RGB Color mode (follow the same instructions as above). To copy this image go to Select in the main menu bar and then All. Now go to Edit and then Copy.
6. Now go back to image 1 by simply clicking on image 1. If you want image 2 to be green, paste it into that channel by going to Edit and then Paste. If you now go to the RGB channel in image 1 you see a green image 2, but a purple image 1. This is because image 1 is in both the red and the blue channel and when both of them are over-laid, it becomes a purple image. Let's say you want a red image 1. Go to the Blue channel by clicking on that channel, then Edit in the main menu bar, then Fill, then select Black under the Use box with 100% opacity and the Mode set to Normal. Then, click OK. You should see that the blue channel is now completely black and will not contribute to your image. Now go back to the RGB channel. You see that image 1 is red and image 2 is green.
7. If you have 3 images representing a triple staining, just paste each of the images into the channels that represents the color you want without having to worry about filling the unused channel in black.

Page 4 of 4 -