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Recent developments in fluorescence microscopy have enabled the resolution of images below the diffraction limit of the sub‐200 nm scale for optical analysis. For precise measurement of the distance between the fluorescent dyes, the optical resolution of super‐resolution microscopic techniques needs to be calibrated. Actin filaments, microtubules, and short duplex DNA molecules are used to demonstrate optical resolution, but they are disadvantageous because of their flexibility. DNA origami offers a novel nanostructure with a defined size and can be easily immobilized on a surface in a fixed orientation for single‐molecule analysis. These features turn DNA origami structures into a nanoscopic ruler for the calibration of super‐resolution imaging techniques [82]. Different super‐resolution methods, such as single‐molecule high‐resolution imaging with photobleaching (SHRImP), direct stochastic optical reconstruction microscopy (dSTORM), and blink microscopy, have been used to demonstrate that fluorescent dye‐labeled staple strands immobilized at specific positions on a DNA origami exhibit a defined separation. Two dye‐modified strands were placed in a rectangular origami structure at a distance of 89.5 nm (Figure 1.10a). The immobilized samples were imaged using total internal reflection fluorescence (TIRF) microscopy. The emission patterns of the two fluorophores overlapped in the diffraction‐limited image (Figure 1.10a, left image). However, super‐resolution imaging using blink microscopy allowed for the identification of the positions of individual fluorophores (Figure 1.10a, right image), and the measured distance between the dyes was in good agreement with the original design (Figure 1.10b). This origami nanoscopic ruler was also used for single‐molecule imaging and calibration of other super‐resolution imaging techniques, such as SHRImP and dSTORM. In addition to the 2D origami ruler, a rigid 3D DNA origami ruler was developed for single‐molecule FRET spectroscopic analysis.

Figure 1.10 DNA‐PAINT super‐resolution imaging. (a) DNA origami tile with two fluorescent dye‐labeled staple strands (F in black circle) (left). TIRF image of origami tiles each containing two ATTO655‐labeled staple strands (middle). Super‐resolution image of the same region using blink microscopy (right). Scale bar: 500 nm. (b) Intensity vs time profile (left) and the statistical distribution of the measured distance (right).

Source: Steinhauer et al. [82]/with permission of American Chemical Society.

(c) The design of origami tile with a dye at the corner and a docking strand in the middle. The binding of a red dye modified imager strand to the docking strand. The fluorescence intensity vs time trace of the binding and unbinding event is shown. (d) Diffraction‐limited TIRF and super‐resolved DNA‐PAINT images of triple labeled oligomers with 129.5 nm separation. Distance distribution histogram of triple‐labeled DNA origami (length scale, 120 nm).

Source: Jungmann et al. [83]/with permission of American Chemical Society.

(e) Super‐resolution fluorescent barcodes. Schematic illustration of barcodes for DNA‐PAINT super‐resolution imaging. The DNA nanorod consists of four binding zones (for binding of red, green or blue imager strand) separated by 113 nm. (f) A segment diagram of the nanorod monomers used to create five barcodes. (g) Super‐resolution image of the mixture containing five barcodes in (f). The inset shows the diffraction‐limited image of a barcode.

Source: Lin et al. [84]/with permission of American Chemical Society.