title: Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy.
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.
authors.affiliation: Applied Physics-Biophysics & Center for NanoScience (CeNS), Ludwig Maximilian University of Munich, Amalienstr. 54, 80799 Munich, Germany. thorben.cordes@physik.lmu.de
authors.forename: Thorben
authors.initials: T
authors.lastname: Cordes
authors.forename: Andreas
authors.initials: A
authors.lastname: Maiser
authors.forename: Christian
authors.initials: C
authors.lastname: Steinhauer
authors.forename: Lothar
authors.initials: L
authors.lastname: Schermelleh
authors.forename: Philip
authors.initials: P
authors.lastname: Tinnefeld
mesh_terms: D008856:Microscopy, Fluorescence / Q000379:methods*; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D019163:Reducing Agents / Q000737:chemistry; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D016877:Oxidants; D019163:Reducing Agents