Channel Surfing: Creating Different Color Combinations from Multiple-Label Images

Mapping the distribution of more than one macromolecule in a tissue is now a relatively routine technique that uses multiple-label fluorescence (2,7) and one of several different digital imaging methods (9,10), including wide-field, confocal, deconvolution, or multiple-photon microscopy. Methods of specimen preparation continue to be refined, especially as new fluorescent probes are developed and become more widely available (6). A convenient method for displaying three grayscale images collected from the same region of a triple-labeled specimen uses the channels feature of the popular image manipulation program, Adobe Photoshop. Here, the three grayscale images are pasted into the red, green, and blue channels of a red/green/blue (RGB) image where they appear as a three-color merged image (7,8). For the method to be successful, the grayscale images should be collected from precisely the same region of the specimen so that they are in register with one another. Some shifting of the images relative to each other can be achieved in Photoshop to bring them back into register. It is preferable, however, to use both a microscope that collects three images at three different excitation wavelengths simultaneously and high-quality objective lenses (2). To glean the most information from combining the three grayscale images, one may resort to “channel surfing” using Photoshop. Here, grayscale images are simply rearranged within the channels of Photoshop to produce images with different color combinations. Images are subsequently viewed and printed using a digital color printer. They can then be compared, and the best one chosen that satisfies the criteria of scientific information along with the all-important aesthetic appeal. For our example of channel surfing, I have chosen a confocal image of a triple-labeled third instar Drosophila haltere imaginal disc. The imaginal discs are made up of pouches of cells at the time of larval hatching and are destined to form appendages such as the legs, the wings, the halteres, the genitalia, and the eyes. The halteres are the balance organs of the fly, located behind the wings, and are related to the wings by their development and their evolution (12,13). At the third instar stage of development, the imaginal discs are relatively flat epithelial sheets of cells. They are currently the subjects of much genetic and molecular dissection, which has greatly benefited from the improved resolution and image registration afforded by confocal imaging of multiply labeled specimens. The spatial distribution of up to three macromolecules has been mapped during development using this approach (3,8,12). Our specimen has been labeled with antibodies to three proteins using secondary antibodies labeled with rhodamine, fluorescein, and cyanine 5 (3) and imaged using a confocal laser-scanning microscope (Bio-Rad MRC1024; Bio-Rad