What causes photobleaching?

Photobleaching occurs when a fluorophore permanently loses its ability to fluoresce. This typically happens as a result of irreversible, photon-induced covalent modification that changes the fluorophore’s structure. In theory, a fluorophore molecule is capable of repeatedly undergoing fluorescence indefinitely, allowing it to generate a signal multiple times. However, in reality, fluorophores produce irreversible covalent modifications on transition from an excited singlet state to the excited triplet state. This renders them structurally unstable and highly susceptible to degradation. The change in structure causes the fluorophore to lose its ability to fluoresce, resulting in photobleaching or fading.