Super-resolution DNA-PAINT imaging with DNA origami

For chamber preparation, a piece of coverslip (no. 1.5, 18 × 18 mm², ~0.17 mm thick) and a glass slide (3 × 1 inch² 1 mm thick) were sandwiched together by two strips of double-sided tape to form a flow chamber with inner volume of ~20 µl. First, 20 µl of biotin-labeled bovine albumin (1 mg/ml, dissolved in Buffer A) was flown into the chamber and incubated for 2 min. Then the chamber was washed using 40 µl of Buffer A. Second, 20 µl of streptavidin (0.5 mg/ml, dissolved in Buffer A) was then flown through the chamber and incubated for 2 min. Next, the chamber was washed with 40 µl of Buffer A and subsequently with 40 µl of Buffer B. Then ~50 pM of the tetrahedron DNA origami structures were flown into the chamber and allowed to bind for 30 min. Alternatively ~300 pM of the modified RRO structures were incubated for 5 min. Afterwards the chamber was washed with 40 µl of Buffer B again. Finally, the imaging buffer with Buffer B and 1× Trolox, 1× PCA, and 1× PCD with the Cy3b-labeled imager strand was flown into the chamber. For the 4-corner motif on the RRO the imager concentration (P1, 9 nt) was 5 nM, for 3 × 4, 20 nm grid on the RRO the imager concentration (P1, 9 nt) was 1 nM and for the DNA origami tetrahedron 3D measurement the imager concentration (P1, 10 nt) was 2 nM. The chamber was sealed with epoxy before subsequent imaging.

For the 4-corner motif measurement (Fig. 1c) the Andor iXon 888 with a readout bandwidth of 10 MHz at 16 bit and 3x pre-amp gain was used. The electron-multiplying (EM) gain was set to 300. Imaging was performed using the Andor Dragonfly spinning disk unit with an excitation intensity of ~180 W/cm² at 561 nm at the sample (laser was set to ~800 mW before fiber coupling).

For the 3 × 4 grid motif experiment (Fig. 1d) the Andor Zyla 4.2 with a readout bandwidth of 540 MHz at 16 bit was used. Imaging was performed using the Andor Dragonfly spinning disk unit with an excitation intensity of ~247 W/cm² at 561 nm at the sample (laser was set to ~1.1 W before fiber coupling).

For the DNA origami tetrahedron structure experiment (Fig. 1e–i; Supplementary Fig. ⁵) the Andor iXon 897 with a readout bandwidth of 5 MHz at 16 bit and 5× pre-amp gain was used. The EM gain was set to 100. Imaging was performed using the Yokogawa W1 spinning disk unit with an excitation intensity of ~226 W/cm² at 561 nm at the sample (laser was set to ~38 mW). No additional magnification lens was used resulting in an effective pixel size of 160 nm.