0.1 μm beads

To estimate our uncertainty in detecting distances across channels, we used a cell line with multiple integration of TetO arrays that can be tagged with TetR-eGFP and TetR-tdTomato. Spot detection is done as for our dual-color lines. We corrected for chromatic aberration using TetraSpeck Microspheres, 0.1-μm beads (Thermo Fisher Scientific, T7279), and then matched spots across channels by solving the LAP using scipy.optimize.linear_sum_assignment function with the Euclidean distance between spots as a measure of distance. Spots across channels with distances higher than a threshold are filtered out to avoid mismatches. We used a threshold of 300 nm for matching the spots registration. We applied a second round of chromatic aberration correction using the set of registered points themselves. The resolution limit (uncertainty) is then estimated as the average distance between registered spots which corresponds to 130 ± 70 nm.

Images were collected on Nikon Ti2 with Perfect Focus System with a sCMOS camera (Hamamatsu Orca Flash 4 v3 (Hamamatsu Photonics, Hamamatsu, Japan)) and Adaptive optics unit with MicAO module from Imagine optic for 3D imaging. SR Apochromat TIRF 100x NA = 1.49 oil immersion was used to provide the highest quality point spread function. Resolution limits of the instrument were define as: lateral (x,y) = 20 to 30 nm and axial (z) = 50 to 60 nm. Excitation was performed using 647 nm (125 mW at the fiber tip) and 561 nm (70 mW at the fiber tip) lasers acquiring 20,000 frames per image with an acquisition time of 10 ms per frame. Imaging was performed in dSTORM super-resolution buffer (Abbelight, Cachan, France) and TetraSpeck Microspheres, and 0.1 μm beads (Thermo Fisher Scientific, T7279) were used for calibration.