DAPI (4',6-diamidino-2-phenylindole) is a popular nucleic acid and chromosome counterstain. Similar to the Hoechst line of blue-fluorescent dyes, DAPI is associated with the minor groove of dsDNA, studies indicating that the probe binds preferentially to adenosine and thymidine (A-T) base pair regions. When bound to DNA, the quantum yield of DAPI is substantially enhanced, a benefit believed to be linked with displacement of water molecules from the stain and the minor groove. The dye is semipermeant to live cells and little or no background staining of the cytoplasm typically occurs with its use. Though soluble in water, the solubility of DAPI is limited in phosphate-buffered saline.

The log phase culture of pig kidney epithelial (LLC-PK1) cells appearing in the digital image above was immunofluorescently labeled with primary anti-tubulin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Cy3, targeting the intracellular microtubular network. In addition, the cells were labeled for cytoskeletal F-actin with Alexa Fluor 488 conjugated to phalloidin, and for nuclear 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.