DNA and chromatin can be stained with dyes that bind externally to the double helix. The most popular fluorochromes in this category are 4',6-diamidino-2-phenylindole (DAPI) and the bisbenzimide Hoechst dyes that are designated by the numbers 33258, 33342, and 34580. These probes are quite water-soluble and bind externally to AT-rich base pair clusters in the minor groove of double-stranded DNA with a dramatic increase in fluorescence intensity. Both dye classes can be stimulated by the 351-nanometer spectral line of high-power argon-ion lasers or the 354-nanometer line from a helium-cadmium laser. Similar to the acridines and phenanthridines, these fluorescent probes are popular choices as a nuclear counterstain for use in multicolor fluorescent labeling protocols. The vivid blue fluorescence emission produces dramatic contrast when coupled to green, yellow, and red probes in adjacent cellular structures.

The culture of rat kangaroo kidney epithelial (PtK2) cells featured in the digital image presented above was immunofluorescently labeled with primary anti-vimentin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Rhodamine Red-X. Vimentin is a protein present in the intermediate filaments of cells derived from the mesoderm. The cell culture was also counterstained for DNA with DAPI. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.