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An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence Experiments

Colin Echeverría Aitken ^*, R. Andrew Marshall  and Joseph D. Puglisi  

^* Biophysics Program, Stanford University School of Medicine, Stanford, California 94305-5126; Department of Chemistry, Stanford University, Stanford, California 94305-5080; and Department of Structural Biology and Stanford Magnetic Resonance Laboratory, Stanford University School of Medicine, Stanford, California 94305-5126

Correspondence: Address reprint requests to Joseph D. Puglisi, E-mail: puglisi{at}stanford.edu.

The application of single-molecule fluorescence techniques to complex biological systems places demands on the performance of single fluorophores. We present an enzymatic oxygen scavenging system for improved dye stability in single-molecule experiments. We compared the previously described protocatechuic acid/protocatechuate-3,4-dioxygenase system to the currently employed glucose oxidase/catalase system. Under standardized conditions, we observed lower dissolved oxygen concentrations with the protocatechuic acid/protocatechuate-3,4-dioxygenase system. Furthermore, we observed increased initial lifetimes of single Cy3, Cy5, and Alexa488 fluorophores. We further tested the effects of chemical additives in this system. We found that biological reducing agents increase both the frequency and duration of blinking events of Cy5, an effect that scales with reducing potential. We observed increased stability of Cy3 and Alexa488 in the presence of the antioxidants ascorbic acid and n-propyl gallate. This new O₂-scavenging system should have wide application for single-molecule fluorescence experiments.

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