Investigation on photobleaching of fluorophores: Effect of excitation power and buffer system

Abstract Photobleaching is an irreversible photochemical process that makes fluorophores not to emit permanently, limiting the observation time of the molecules. Thus, preliminary photobleaching can interfere with most fluorescence spectroscopy and microscopy, especially single‐molecule fluorescence applications. To minimize photobleaching of fluorophores, low excitation power as well as specialized imaging buffers that improve the photostability of fluorophores is highly recommended in single‐molecule fluorescence measurements. Here, we investigated how photobleaching can get affected by the excitation power and buffer components in the single‐molecule buffer system such as Trolox and glucose oxidase/catalase (GOC). We found that the photobleaching rate showed higher‐order photon interaction, indicating that photobleaching rate is decoupled to the amount of fluorescence. We also observed that the photobleaching rate was significantly reduced by fourfold when the combination of Trolox and oxygen scavenging system using either GOC or N 2 bubbling was employed in single‐molecule buffer.