Raw image data - DNA-PAINT on 100 nm gold nanospheres

For immobilizing, gold NPs (CTAB-coated 100 nm nanospheres obtained from Nanoseedz, product NS-100-50), 500L of stock solution was centrifuged at 1500 rcf for 3 mins and the pellet was re-dispersed in 250 L of 1mM CTAB in milliQ water to obtain 2x concentrated solution. Right before spin-coating the particles on the thiolated coverslips, the nanospheres are heated to 30 degrees Celsius for 1 minute and sonicated for 30 seconds to minimize agglomeration. The nanospheres are then immobilized by spin-coating (at 2000 rpm for 1 min) on the thiolated coverslip. The coverslips with GNS are generously rinsed with methanol, phosphate-buffered saline (PBS), and water, to get rid of the residual CTAB and weakly-bound particles.The GNS were then incubated with a mixture of 5 M of single-stranded DNA docking strands (5' SH-CAT CAT CAT ACG CTT CCA ATA ATA CAT CTA-3', from Integrated DNA Technologies) and 1mM tris(2-carboxyethyl)phosphine hydrochloride in citrate buffer (10 mM, pH 3, 1 M NaCl) for surface functionalization. After two hours, slides were rinsed with PBS and buffer B (5 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 8.0) to wash away free DNA strands. The slides were stored in buffer B in a humidity chamber, until the optical measurement.An inverted total internal reflection fluorescence (TIRF) microscope with a 100x/NA 1.49 objective (Nikon) was used for the DNA-PAINT experiment. Once the glass slide with GNS was mounted on the sample stage, buffer B was carefully removed, and then 50L of 200 pM of complementary imager strands (3' TAT GTA GAT C-5', from Integrated DNA Technologies) with ATTO655 diluted in Buffer B was drop-casted with a pipette onto the slide. The sample was illuminated with a 637 nm laser (Coherent® OBIS™ FP 1193837 | 637nm LX 100mW) with a power density of 3.98 kW/cm², the intensity time traces were recorded with a CMOS camera (Prime BSI Scientific CMOS, physical pixel size 6.5 m). The dichroic mirror (ZT640rdc-UF2, Chroma Technology) and several filters such as a notch filter (ZET635), a long-pass filter of 650 nm (Thorlabs) and a bandpass filter (center wavelength 700 nm, bandwidth of 75 nm, Thorlabs) are used in the detection path to get high signal-to-noise ratio. Hundreds of PSFs were obtained from single gold nanospheres from 18000 frames recorded at 10 fps.