Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy
AbstractThe 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. View Full-Text
Externally hosted supplementary file 1
Description: Raw data for DNA-PAINT, SI-FCS and confocal FCS measurements.
Share & Cite This Article
Blumhardt, P.; Stein, J.; Mücksch, J.; Stehr, F.; Bauer, J.; Jungmann, R.; Schwille, P. Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy. Molecules 2018, 23, 3165.
Blumhardt P, Stein J, Mücksch J, Stehr F, Bauer J, Jungmann R, Schwille P. Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy. Molecules. 2018; 23(12):3165.Chicago/Turabian Style
Blumhardt, Philipp; Stein, Johannes; Mücksch, Jonas; Stehr, Florian; Bauer, Julian; Jungmann, Ralf; Schwille, Petra. 2018. "Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy." Molecules 23, no. 12: 3165.
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.