Abstract
The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site.
Highlights
IntroductionSuper-resolution microscopy has greatly contributed to the study of biological specimens with resolutions down to few nanometers while retaining the high specificity of fluorescent labels [1,2,3]
Super-resolution microscopy has greatly contributed to the study of biological specimens with resolutions down to few nanometers while retaining the high specificity of fluorescent labels [1,2,3].The stochastic blinking of individual fluorophores enables the precise localization of molecules in various single molecule localization microscopy (SMLM) methods
We probed whether the distance of fluorescent dye to the docking strand influences the rate of depletion
Summary
Super-resolution microscopy has greatly contributed to the study of biological specimens with resolutions down to few nanometers while retaining the high specificity of fluorescent labels [1,2,3]. The stochastic blinking of individual fluorophores enables the precise localization of molecules in various single molecule localization microscopy (SMLM) methods. In many variants of SMLM, in particular the prominent PALM [4,5] and (d)STORM [6,7], the number of photons available from permanently bound, individual fluorophores determines the achievable localization precision [8,9]. While an individual binding event is still limited by the photon-budget of the fluorescent dye, binding sites can be revisited by fresh probes and contribute to higher resolved images [11]. In DNA-based PAINT (DNA-PAINT) [11,12]
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