The role and interplay of triplet states and radical ion states in single-molecule fluorescence spectroscopy has recently been elaborated providing us with new insights into the photophysics and photobleaching pathways of fluorescent dyes. Adjustment of fluorophore redox properties in combination with specific redox properties of the environment, i.e. addition of reducing and oxidizing agents, allows control of the emission properties: it has become possible to suppress blinking and to also induce blinking in single-molecule fluorescence transient by selectively opening and closing specific excited state pathways. Induced blinking is, for example, of interest for super-resolution fluorescence microscopy based on the subsequent localization of single fluorophores. For oxazines this control even allowed the separation of the influence of reducing and oxidizing agents, enabling switching the fluorescence of single fluorophores. Here, we study the factors that contribute to the kinetics of the photophysical pathways more closely with a focus on the photophysics of the oxazine ATTO655 labeled to DNA. Our data show that the oxazine ATTO655 interacts with DNA, shielding it efficiently from reagents in solution. Besides redox reactions, the pH also influences the blinking kinetics and especially the off-times. Moreover, we present the extension of ATTO655 as a single-molecule redox sensor to a ratiometric fluorescence-resonance-energy-transfer based sensor. Therefore, we designed FRET probes that showed the highest possible contrast of FRET changes and demonstrate reversible FRET-switching of Cy3B-ATTO655 DNA constructs.
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