Fluorochromes were introduced to fluorescence microscopy in the early twentieth century as vital stains for bacteria, protozoa, and trypanosomes, but did not see widespread use until the 1920s when fluorescence microscopy was first used to study dye binding in fixed tissues and living cells. However, it wasn't until the early 1940s that Albert Coons developed a technique for labeling antibodies with fluorescent dyes, thus giving birth to the field of immunofluorescence. Over the past 60 years, advances in immunology and molecular biology have produced a wide spectrum of secondary antibodies and provided insight into the molecular design of fluorescent probes targeted at specific regions within macromolecular complexes.

The culture of African green monkey kidney cells featured in the digital image above was immunofluorescently labeled with primary anti-vinculin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Cy3 (red emission). Vinculin is a protein associated with focal adhesion and adherens junctions, which are membrane-associated complexes that serve as nucleation sites for actin filaments and as crosslinkers between the external medium, plasma membrane, and actin cytoskeleton. The specimen was counterstained for F-actin with Alexa Fluor 488 conjugated to phalloidin, and 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.