An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments.

By Colin Echeverría Aitken, R. Marshall, Joseph D. Puglisi. 2008

857 citations recorded by the source corpus.

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 O(2)-scavenging system should have wide application for single-molecule fluorescence experiments.

Subfields: Bioengineering, Biophysics, Structural Biology

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Citations in the corpus

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence (2013, methodology): “6 μM Neutravidin reagent for 5 min at room temperature and rinsed with Tris-based polymix buffer to remove unbound Neutravidin; 30S preinitiation complexes or DNA oligonucleotides (diluted to 10 or 1 nM, respectively, with Tris-based polymix buffer with oxygen scavenging) (34) were immobilized on su”
Real-time DNA sequencing from single polymerase molecules. (2010, background): “An oxygen scavenging system, consisting of protocatechuate dioxygenase (PCD) and its substrate protocatechuic acid (PCA), is used to remove oxygen and thereby suppress photophysical effects that might be deleterious to the polymerase (Aitken et al., 2008; Eid et al., 2009).”
A practical guide to single-molecule FRET (2008, methodology): “Other options include the use of the protocatechuic acid and protocatechuate-3,4-dioxygenase combinatio”
Photophysics of Fluorescence Probes for Single Molecule Biophysics and Super-Resolution Imaging (2012, neutral): “An alternative system consisting of protocatechuic acid and protocatechuate-3,4-dioxygenase can reduce the oxygen concentration further (34) and does not induce pH drop over time in contrast to the glucose oxidase-based system (35), but in our hands, it tends to act more slowly.”
GTP hydrolysis by IF2 guides progression of the ribosome into elongation (2009, methodology): “5 mM 3,4 dihydroxybenzoic acid, and 250 nM protocatechuate dioxygenase (Aitken et al., 2008).”
A BIRD’S EYE VIEW: TRACKING SLOW NANOMETER-SCALE MOVEMENTS OF SINGLE MOLECULAR NANO-ASSEMBLIES (2010, positive): “The PCD employs a nonheme iron center that catalyzes the conversion of PCA and molecular oxygen into b-carboxy-cis,cis-muconic acid (Aitken et al., 2008), while the antioxidant Trolox suppresses slow blinking and photobleaching of cyanine dyes (Rasnik et al.”
RACK1 on and off the ribosome (2019, methodology): “Complexes were imaged on a prism-based total internal reflection fluorescence (TIRF) microscope as described(Aitken et al. 2008), using 532 and 647 nm excitation lasers with movies collected at 5 frames per second and EM gain set to 650.”
Real-Time DNA Sequencing from Single Polymerase Molecules (2009, background): “An oxygen scavenging system, consisting of protocatechuate dioxygenase (PCD) and its substrate protocatechuic acid (PCA), is used to remove oxygen and thereby suppress photophysical effects that might be deleterious to the polymerase (Aitken et al., 2008; Eid et al., 2009).”