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- Title
Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy.
- Authors
Blumhardt, Philipp; Stein, Johannes; Mücksch, Jonas; Stehr, Florian; Bauer, Julian; Jungmann, Ralf; Schwille, Petra; Fitter, Jörg
- 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.
- Subjects
BINDING sites; FLUORESCENT dyes; PHOTONS; HIGH resolution imaging; REACTIVE oxygen species; NANOSTRUCTURES; DNA damage
- Publication
Molecules, 2018, Vol 23, Issue 12, p3165
- ISSN
1420-3049
- Publication type
Article
- DOI
10.3390/molecules23123165