Photobleaching in TRIC/MST experiments
Photobleaching describes a decay of fluorescence intensity caused by excitation light. It is typically caused by reactive oxygen species that react with the excited states of fluorophores.
Excessive photobleaching in MST experiments typically only occurs at high excitation powers, predominantly with the NT.115Pico and NT.LabelFree optics. A result of strong photobleaching is an increased noise of the binding signal, and therefore a lower signal-to-noise ratio. If the signal-to-noise ratio is tolerable, photobleaching does not pose a problem.
One way to eliminate photobleaching is to remove molecular oxygen from the reaction mixture, e.g. by using enzymatic systems. Some biological fluorophores (e.g. tryptophan or fluorescent proteins) have oxygen-independent photobleaching mechanisms, so that removal of oxygen does not completely abolish photobleaching. In these cases, it is recommended to increase the concentration of fluorescent target and decrease the LED excitation power to prevent excessive photobleaching.
Ligand-induced photobleaching
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. In those cases, the photobleaching signal has to be used for calculation of the affinity! Some compounds and other substances however, can act as reducing or oxidizing agents, and thereby alter photobleaching in a concentration-dependent manner without actually binding the target.
Recommendation:
To discriminate between binding specific and unspecific effects on photobleaching, it is recommended to repeat the binding experiment with a negative control target (a non-binding target, such as binding impaired mutants or unrelated proteins, or fluorophore only). In this control, the ligand does not bind the target. If it still changes photobleaching behavior, it is acting as reducing or oxidizing agents, and the effect on photobleaching rate is unspecific. If there is no more effect on photobleaching behavior, the effect is binding-specific and affinity information can be extracted from the photobleaching rate.