Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular-scale (sub-5-nm) spatial resolution, reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1–2 d.
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We thank B. Rieger, S.S. Agasti, S. Strauss, D. Haas, J.B. Woehrstein and E. Woehrstein for helpful discussions. This work was supported by the German Research Foundation (DFG) through an Emmy Noether Fellowship (DFG JU 2957/1-1), the European Research Council (ERC) through an ERC Starting Grant (MolMap, grant agreement no. 680241), the Max Planck Society, the Max Planck Foundation and the Center for Nanoscience (CeNS). M.T.S. acknowledges support from the International Max Planck Research School for Molecular and Cellular Life Sciences (IMPRS-LS). T.S. acknowledges support from the DFG through the Graduate School of Quantitative Biosciences Munich (QBM). F.S. acknowledges support from the DFG through the SFB 1032 (Nanoagents for the spatiotemporal control of molecular and cellular reactions).
R.J. is a cofounder of Ultivue, a startup company with an interest in commercializing DNA-PAINT technology.
Integrated supplementary information
(a) The main window showing the origami canvas with the hexagonal tiles. (b) Extensions dialog to set extensions corresponding to each selected color. (c) Plate export dialog to specify the export format of the plates. (d) Pipetting dialog to select a folder with *.csv files to generate a list of sequences that need to be pipetted and to create a visual pipetting aid. (e) Folding table to calculate volumes that are needed for pipetting.
The main window has two preview windows, the left one to display the positions of structures in the full frame, the right one to display an individual structure. Structural parameters such as number and structure definition can be set in the group box “Structure”. All PAINT-related parameters, i.e. mean dark and bright times are set with the “PAINT parameters” group box. The group box “Imager parameters” is used to define properties of the simulated imaging probe.
(a) The main window after the analysis of a movie file. Yellow boxes indicate the identification of a spot, green crosses show the fitted subpixel coordinate. (b) The contrast setting dialog. (c) The parameters setting dialog.
(a) The main window with two picked regions of interest (yellow circles). (b) The display settings dialog for the render scene in (a). (c) The info dialog for the picked regions in (a). (d) The tools settings dialog.
(a) The main window showing properties (columns) of localizations (rows). (b) Filtering in a histogram of a property column. (c) Filtering in a two-dimensional histogram of two property columns. The green areas in (b) and (c) have been selected with a pressed left mouse button. After releasing the mouse button, any localization with property values outside the green range will be removed.
(a) Two stripes of double-sided sticky tape are placed on a 76x26 mm microscopy slide with a distance of ~ 8mm. A coverglass is placed on top of the sticky tape stripes. After pressing the coverglass thoroughly against the sticky tape, overlapping tape can be removed. (b) To immobilize DNA nanostructures, fluids are pipetted from one side while simultaneously being sucked out with a lab wiper from the other side. (c) The coverglass is sealed with epoxy glue and can be used with the coverglass facing towards the objective in a microscope stage once the glue is hardened.
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Schnitzbauer, J., Strauss, M., Schlichthaerle, T. et al. Super-resolution microscopy with DNA-PAINT. Nat Protoc 12, 1198–1228 (2017). https://doi.org/10.1038/nprot.2017.024
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