What causes fading in fluorescence microscopy?

Fading, also known as photobleaching, happens when a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification. Fluorophores function by absorbing light at a specific wavelength and emitting light at a different wavelength; the light that is absorbed is the cause of photobleaching. This also leads to the death of the fluorophore. How long a fluorophore keeps fluorescing depends on the type of fluorophore and duration and intensity of light exposure. However, prolonged exposures of fluorescence eventually cause fading and reciprocity failure of sample fluorescence intensity in all fluorophores. It is suggested that when photobleaching occurs, the flourophore’s electrons remain stuck in an excited triplet state, which is a stable and long-lasting state. Fading of specific fluorescent characteristics is typically faster than that of the background, causing a loss of image contrast.