Multicolor Imaging: The Important Question of Co-Localization

The use of specific fluorescent probes, combined with confocal or multiphoton microscopy and subsequent digital analysis of the images, has greatly improved the accuracy of co-localization of macromolecules in biological samples. This article addresses some of the caveats associated with the preparation and imaging of multilabeled samples and demon-strates the use of digital methods for measuring the extent of co-localization in cells. WHAT IS CO-LOCALIZATION? In a biological sense, co-localization describes the exis-tence of two or more different molecules in very close spatial positions in a specimen. These are often proteins that are vi-sualized using fluorescent antibodies or riboprobes (Figures 1 and 2). The output from most confocal microscopes is a digi-tal image made up of a multidimensional array of many vox-