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Issue 14, 2011
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Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

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Abstract

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.

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

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Publication details

The article was received on 23 Sep 2010, accepted on 14 Jan 2011 and first published on 11 Feb 2011


Article type: Paper
DOI: 10.1039/C0CP01919D
Citation: Phys. Chem. Chem. Phys., 2011,13, 6699-6709
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    Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

    T. Cordes, A. Maiser, C. Steinhauer, L. Schermelleh and P. Tinnefeld, Phys. Chem. Chem. Phys., 2011, 13, 6699
    DOI: 10.1039/C0CP01919D

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