Document Detail


An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments.
MedLine Citation:
PMID:  17921203     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Colin Echeverría Aitken; R Andrew Marshall; Joseph D Puglisi
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, N.I.H., Extramural     Date:  2007-10-05
Journal Detail:
Title:  Biophysical journal     Volume:  94     ISSN:  1542-0086     ISO Abbreviation:  Biophys. J.     Publication Date:  2008 Mar 
Date Detail:
Created Date:  2008-02-14     Completed Date:  2008-04-02     Revised Date:  2013-06-06    
Medline Journal Info:
Nlm Unique ID:  0370626     Medline TA:  Biophys J     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1826-35     Citation Subset:  IM    
Affiliation:
Biophysics Program, Stanford University School of Medicine, Stanford, California 94305-5126, USA.
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MeSH Terms
Descriptor/Qualifier:
Ascorbic Acid / pharmacology
Carbocyanines / chemistry
Catalase / metabolism*
Enzyme Stability
Fluorescent Dyes / chemistry*
Free Radical Scavengers / metabolism*
Glucose Oxidase / metabolism*
Hydroxybenzoates / metabolism
Microscopy, Fluorescence
Nanotechnology / methods*
Oxygen / metabolism*
Photobleaching
Propyl Gallate / pharmacology
Protocatechuate-3,4-Dioxygenase / metabolism
Reactive Oxygen Species / metabolism
Grant Support
ID/Acronym/Agency:
GM51266/GM/NIGMS NIH HHS; T32 GM008294/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Carbocyanines; 0/Fluorescent Dyes; 0/Free Radical Scavengers; 0/Hydroxybenzoates; 0/Reactive Oxygen Species; 0/cyanine dye 3; 0/cyanine dye 5; 121-79-9/Propyl Gallate; 50-81-7/Ascorbic Acid; 7782-44-7/Oxygen; 99-50-3/protocatechuic acid; EC 1.1.3.4/Glucose Oxidase; EC 1.11.1.6/Catalase; EC 1.13.11.3/Protocatechuate-3,4-Dioxygenase
Comments/Corrections

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