(a) Left: Normalized histoplots of the number of detections per frame (for the first 8,000 frames) in 3 different wells. In contrast to the dSTORM acquisition, we can observe a constant number of localizations over the course of the 9-hour acquisition. Right: Normalized histoplots of integrated intensity per detection (I0) grouped by well showing a similar distribution from the first well (A1) until the last well (B7). (b) 18 astigmatism-based 3D DNA-PAINT SMLM images (FOV 20.5 x 20.5 μm, 40 nm/px) of microtubules in Cos-7 cells automatically gathered using our HCS-SMLM approach. It corresponds to an acquisition time of 9 hours (30 min per cell), illustrating (1) the capability to efficiently acquire DNA-PAINT data using our HCS-SMLM pipeline, and (2) the time-consuming process of DNA-PAINT as compared to dSTORM. In 9 hours, the HCS-dSTORM pipeline acquired 5-fold more positions with similar microtubule reconstruction quality compared to HCS-DNA-PAINT (only 18 cells in 9 hours). The DNA-PAINT acquisition generated also a larger database as more molecules were detected (2 to 5 fold) than in a dSTORM acquisition due to a higher duty cycle of DNA probes than organic dyes. (c) Examples of astigmatism-based 3D DNA-PAINT images in well A1, after 4 hours (well A8), and after 9 hours (well B7). Color codes for the Z-position. Scale bar: 5 μm.