The consequences of quenching and photobleaching are suffered in practically all forms of fluorescence microscopy, and result in an effective reduction in the levels of emission. These artifacts should be of primary consideration when designing and executing fluorescence investigations. The two phenomena are distinct in that quenching is often reversible whereas photobleaching is not. Quenching arises from a variety of competing processes that induce non-radiative relaxation (without photon emission) of excited state electrons to the ground state, which may be either intramolecular or intermolecular in nature. Because non-radiative transition pathways compete with the fluorescence relaxation, they usually dramatically lower or, in some cases, completely eliminate emission. Most quenching processes act to reduce the excited state lifetime and the quantum yield of the affected fluorophore.

The digital image presented above features a culture of male rat kangaroo kidney epithelial cells that was immunofluorescently labeled with primary anti-beta-catenin rabbit monoclonal antibodies followed by goat anti-rabbit secondary antibodies conjugated to Rhodamine Red-X. The culture was also immunofluorescently labeled with primary anti-tubulin mouse monoclonal antibodies followed by goat anti-mouse Fab fragments conjugated to Cy2 to target the microtubular network, and counterstained with Hoechst 33342 to target nuclear DNA. 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.