COLORIZING & OVERLAYING IMAGES WITH ADOBE PHOTOSHOP


Your image should be saved as an 8-bit grayscale images, without color information. The following are instructions for one way to use Photoshop to colorize and overlay two or more images taken from the same sample so that you can see how the different fluorescent objects relate spatially to each other. You should start with 2 or more images taken with different filters from the same position in the specimen.

1) Open each of the images as a separate window in Photoshop. Immediately save a copy of each image USING A DIFFERENT FILE NAME so that you don’t modify the original images. For example, if your first image is called something like "tubulin.tif," resave it as "tubulin copy.tif."

2) Optimize the intensity levels in the images. From the pulldown menu, select Image>Adjust>Levels. You will see a HISTOGRAM, which is a graph that shows the distribution of intensities in the image. The triangular sliders below the histogram affect how the intensities are displayed on the screen.

a. Move the left slider to a point where the background is black but none of the useful information has become invisible.
b. Move the right slider up and down, and select a position where the image is bright but not saturated (containing areas of solid white).
c. When you are happy with the image, click Okay. Repeat for each image.

3). Create an RGB merged image

a. Type Cmnd-A and then type Cmnd-C to copy one of the images.
b. Type Cmnd-N to create a new file. It will automatically be the same size and resolution as the one you copied. Change the mode to "RGB" and click "OK".
c. In the Layers/Channels/Paths window click on "Channels", and select (click on) the channel of the same color you would like your image to be. For example, paste a rhodamine image into the red channel. Type Cmnd-V to paste your image into that channel.
d. Repeat with your other images. If there are few than 3 channels used, fill the remaining channels with black by selecting them, choosing Edit > Fill. Select Black and click "OK".
e. To see your merged image, click on "RGB".

4). Save the resulting image.

a. Save the window with the overlaid images as a Photoshop file, USING A NEW FILENAME (this will preserve each of the images as a separate layer).
b. To import the image into other image programs, such as PowerPoint, you should save it in a more versatile image format, such as JPEG or TIFF.
c. Now select File>Save As and select an image format. High-resolution JPEGs are particularly useful for importing into other programs such as PowerPoint.

This web site is updated by Tim Melton Site modified Aug 2003.