![]() ![]() ![]() DNA-PAINT 5 exploits the specificity of DNA by using single-stranded oligonucleotides as labels (“docking strands”) to which fluorescently-labeled complementary “imager” strands bind. In contrast, PAINT imaging is based on reversible binding of a fluorescent species to the target structure. In STORM and PALM, the blinking required for super-resolution reconstruction is obtained by complex photo-physical switching and activation of target-bound fluorophores. One of the major branches in the field is referred to as single molecule localization microscopy (SMLM) and includes methods such as photo-activated localization microscopy 1 (PALM), Stochastic optical reconstruction microscopy 2 (STORM), point accumulation in nanoscale topology 3 (PAINT), and their descendants 4. The advent of super-resolution microscopy has revolutionized life science research by providing access to molecular structures with light microscopy, which were previously hidden below the diffraction limit. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. ![]() However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary ‘blinking’ without the use of rather complex optical or chemical activation schemes. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. ![]() Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. ![]()
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