Static or complex quenching arises from non-fluorescent complexes formed between the quencher and fluorophore that serve to limit absorption by reducing the population of active, excitable molecules. This effect occurs when the fluorescent species forms a reversible complex with the quencher molecule in the ground state, and does not rely on diffusion or molecular collisions. In static quenching, fluorescence emission is reduced without altering the excited state lifetime. A fluorophore in the excited state can also be quenched by a dipolar resonance energy transfer mechanism when in close proximity with an acceptor molecule to which the excited state energy can be transferred non-radiatively. In some cases, quenching can occur through non-molecular mechanisms, such as attenuation of incident light by an absorbing species (including the chromophore itself).

The cytoskeletal F-actin network and nuclear DNA present in the culture of COS-7 cells featured in the digital image above were targeted with Alexa Fluor 488 conjugated to phalloidin and the intercalating dye propidium iodide, respectively. The specimen was also labeled with Alexa Fluor 350 conjugated to wheat germ agglutinin, a fluorescent lectin that selectively binds to sialic acid residues, which are found in both mucoproteins and glycoproteins. 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.