Photobleaching

Photobleaching describes a decay of Fluorescence intensity caused by excitation light. It is typically caused by reactive oxygen species, which react with the excited states of Fluorophores. Excessive photobleaching in TRIC experiments usually only occurs at high Excitation Power. A consequence of strong photobleaching is an increased noise of the binding signal and therefore a lower Signal-to-noise ratio or Signal Quality. As long as the signal- to-noise ratio is tolerable, photobleaching does not pose a problem. In some cases, ligand binding can change the photobleaching properties of the fluorescent Target. This means that the Photobleaching behavior changes with the ligand concentration. Please see the article on Ligand-Induced Photobleaching for further information. One way to eliminate photobleaching is to remove molecular oxygen from the reaction mixture, e.g. by using enzymatic systems. Some Fluorophores or Fluorescent fusion proteins have oxygen- independent photobleaching mechanisms, therefore removal of oxygen does not completely abolish photobleaching. In these cases, it is recommended to increase the concentration of fluorescent target and decrease the excitation power to prevent excessive photobleaching.