Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore perf...

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Bibliographic Details
Published in:Physical chemistry chemical physics : PCCP 2011-04, Vol.13 (14), p.6699-6709
Main Authors: CORDES, Thorben, MAISER, Andreas, STEINHAUER, Christian, SCHERMELLEH, Lothar, TINNEFELD, Philip
Format: Article
Language:English
Subjects:
Brightness
Chemistry
Dyes
Exact sciences and technology
Fluorescence
General and physical chemistry
Microscopy
Microscopy, Fluorescence - methods
Optimization
Oxidants - chemistry
Oxidation-Reduction
Physical chemistry
Reducing Agents - chemistry
Spectroscopy
Spectrum Analysis - methods
Storage stability
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Summary:Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs.
ISSN:1463-9076
1463-9084
DOI:10.1039/c0cp01919d