Raw image data - DNA-PAINT on 1-micrometer PS particles

Fluorescence microscopy data of single molecule localization microscopy (SMLM) characterization of 1 micrometer polystyrene particles using DNA-paint.

Neutravidin-coated 1 μm polystyrene particles (ThermoFisher Scientific F8777) are immobilized on a clean gridded glass coverslip (Ibidi 10816). To this end, the coverslip is first cleaned using 15-minute sonication steps in Triton-X100 and KOH interleaved with sonication in Milli-Q water for rinsing. The coverslip is then dried with nitrogen. A silicone gasket (Grace Bio-Labs SKU 103250) cut to the size of the coverslip is deposited to create a chamber and the assembly is cleaned with oxygen plasma. The cleaned coverslip is incubated with 100 μL of biotinylated BSA (Sigma Aldrich A8549) at 0.1 mg/mL for 30 minutes then rinsed with 1xPBS. Subsequently, the coverslip was incubated for 1 h with 100 μL of 100:1 diluted stock concentration of the particles, then rinsed three times with 200 μL 1xPBS. Thereafter, the sample was incubated with 100 μL of 20 μM biotinylated DNA docking strands for 1 h, washed twice in 1xPBS, and once with the imaging buffer. The docking strand sequence is 5'-biotin-TTATACATCTA-3' (IDT). The imager strand sequence is 3'-atto655-TATGTAGAT-5' (IDT). The imager solution is prepared by diluting the imager strand DNA to 50 pM in imaging buffer consisting of Buffer B with 10 mM MgC₂. Both concentrations are adjusted to obtain single events on beads and minimize the background in epifluorescence imaging.