Term used to describe chemical degradation of a fluorophore due to its interaction with light, resulting in a non-fluorescent product.
The excited state chemistry of a molecule is often very different from that of the ground state. By exciting a molecule with a photon, once often induces a redistribution of the electrons in the molecular orbitals, resulting in changes in the conformation and introduction of electrophiles and nucleophiles at new positions on the molecule. If this molecule is a fluorophore of some sort, then it may occupy the excited state for nanoseconds or even microseconds. This time, albiet short, is long enough for other molecules to interact with the excited fluorophore and possibly cause a chemical reaction. If the interaction is reversible ... say protonation/deprotonation, then returning to the ground state will cause the reverse reaction to occur, restoring the fluorophore to its original condition. In some cases, the interaction is not reversible, such as a ring in the molecule breaking open. As a result, the fluorophore returns to the ground state as a new molecule. If this molecule can no longer fluoresce, then it is considered a photo-degradation product; it has been photobleached. Photobleaching may change the absorbance of the molecule such that it can no longer use light of that wavelength to reach the excited state. It can also change the rate of nonradiative decay, meaning that the excited state no longer emits a new photon, but instead returns to the ground state vibrationally or through other pathways. In a more empirical sense, photobleaching may just mean that the chemistry of your fluorophore has changed in such a way that it emits light in a new range in which you are not interested in.
Photobleaching can be a good thing
or a bad thing
. If you are interested in studying the intact fluorophore, photobleaching can be a way of losing material throughout the course of your experiment. I've personally watched tryptophan
fluorescence in one of my proteins
become dimmer and dimmer with time as the spectrofluorometer
bleaches more and more of my sample. People who study single molecule fluorescence
are limited by the number of times they can excited a particular molecule before it undergoes photobleaching. This is why single molecule work has focused mostly on the use of rhodamine
dyes. They have a high quantum yield
and do not photobleach readily.
Photobleaching is not always a problem, however. It has been applied in fluorescence microscopy to study motion of molecules in living cells. One technique - Fluorescence Recovery After Photobleaching (FRAP), can be used to measure the diffusion constant of molecules attached to cell membranes. Fluorescent probes are chemically attached to a membrane protein or to a lipid, which are then reconstituted into a live cell. This cell is then placed under a microscope which is attached to a laser. The laser shines an intense beam of light on a small spot of the cell surface, photobleaching all the fluorophores in that spot. Then over time, the photobleached molecules wander out of the spot and new ones wander in. The rate at which this occurs can be used to calculate the diffusion constant in the membrane. This technique has also been used to monitor how actin filaments assemble at the surface of a cell. By monitoring fluorescently labelled actin monomers, a laser was used to bleach actin filaments that were pushing against the cell surface of a moving cell. It was found that new actin monomers were constantly being recruited to the cell surface, as the photobleached dark spot moved towards the inside of the cell.
Green Fluorescent Protein undergoes a phenomenon called 'blinking'. Exciting GFP with an intense beam of light will cause it to turn off for an extended period of time. However, the same molecule will later become fluorescent again. This phenomenon has been studied extensively, but it is still not entirely clear whether this is a result of chemical photobleaching that reverses, or whether it is just conformational fluctuation of GFP between 'on' and 'off' states.