Fig. 4: OLS is amenable to a variety of SMLM techniques and acquisition schemes.

a, JF₅₄₉ and JF₆₄₆ co-labeled Halo-KEAP1 U2OS cells imaged within the same FOV. Scale bar, 5 μm. b, Ten-point dose response of KI-696-treated Halo-KEAP1 U2OS cells co-labeled with JF₅₄₉ and JF₆₄₆. c, Diffraction-limited image of a full OLS FOV of immunofluorescently labeled tubulin with AF647-conjugated secondary antibody (Methods). Scale bar, 20 μm. d, Zoomed-in region of interest from c. e, STORM reconstruction of full OLS FOV, as in c. f, Zoomed-in region of interest from e as in d. Scale bar, 5 μm. g, Line profile from yellow lines in diffraction-limited (d) and STORM (f) gray value (a.u., arbitrary units) to compare spatial resolution of microtubules. h, Localization precision histogram for AF647-labeled and CF568-labeled secondary antibodies used to stain microtubules with OLS illumination with a 0.4-ms integration time. i, Representative image of correlative FRAP/SMT where a central region is bleached using the OLS line scan before spot recovery after photobleaching. Areas outside and inside of the FRAP region are used to measure SMT. Scale bar, 20 μm. j, T_1/2 FRAP for Halo-KEAP1 U2OS cells treated with DMSO or 1 μM KI-696 labeled with 400 pM JF₅₄₉-Halo ligand. The black line denotes the median, and each spot represents an individual FOV from a single 384-well plate. k,l, Spot density after recovery over time for each of DMSO (10 FOVs; k) and 1 μM KI-696 (8 FOVs; l). Standard deviation shown in confidence bands for each condition. m, Diffusion coefficient from SMT for 400 pM JF₅₄₉-Halo ligand concentration in bleached (inside) and unbleached (outside) region with 8 FOVs sampled for each condition. Whisker plots are rendered using the Tukey method with the median represented as a horizontal solid line.