Supplementary Figure 4: Two-color 4Pi-SMS imaging performance on microtubule samples.

(a-f) 4Pi-SMS images of microtubules labeled with AF647 or CF660C in COS-7 cells and the 3D localization precision. (a,d) z-projections of the data sets. Fourier shell correlation (FSC) estimations of the boxed-regions were provided. (b,e) 20-nm thick x-y slices of the same data. (c,f) Clusters featuring at least 10 localizations from the same dye molecule (emitters that appeared in consecutive frames within a radius of two times the localization precision) were aligned by their center of mass to generate the shown 3D plots. Histograms of the distributions in x, y, z were fit with Gaussian functions, and the standard deviations (𝜎_x, 𝜎_y and 𝜎_z) are reported. (g) The binned 2D intensity histogram of AF647 and CF660C from more than 2 million localizations of each dye based on the same data as shown in (Fig. 1d). The plot shows salvaged fluorescence intensity (y-axis) versus conventional fluorescence intensity (x-axis). The solid lines show the threshold where the intensity value is 2% of each peak intensity. (h) Cross-talk between AF647 and CF660C on a logarithmic scale representing the same data as shown in (Fig. 1e). Localizations outside the two threshold boundaries in (g) are rejected. (i) Two-color images of microtubules (anti-α-tubulin antibody labeled with AF647 and CF660C together) in a COS-7 cell. The lower left and upper right corners show the CF660C and AF647 labeling, respectively. (j) Merged image of the two labels in (i). (k) A 20-nm thick x-y slice of the boxed region in (j). Inset: a 100-nm thick cross-section along the dashed line in (k). Representative images of two (a-b,d-e) or seven (i-k) independent experiments are shown.