Supplementary Figure 7: Two-color 4Pi-SMS images of mitochondria in HeLa cells.

(a-c) Two-color images of the outer membrane of mitochondria (anti-TOM20 antibody) and mitochondrial DNA (anti-dsDNA antibody) (Supplementary Video 3). Merged image of the two labels in (a) and (b) is shown in (c). The boxed region in (c) is shown in (Fig. 2f). (d-f) Two-color image of the outer membrane of mitochondria (overexpressed GFP-OMP25 labeled with anti-GFP antibody alongside anti-TOM20 antibody). The dataset was reconstructed from 4 optical sections with 500-nm step sizes. (g) A 200-nm thick x-y slice of the boxed region in (f). (h) y-z view of the orange dashed box (200 nm wide) in (g). (i) x-z view of the blue dashed box (200 nm wide) in (g). (j) 3D chromatic shifts at different offset from the focus center (100-nm thick volumes) of each optical section averaged over the 4 sections. (k) 3D chromatic shifts at different z-positions from non-overlapping 200-nm thick volumes after the 4 optical sections were aligned and combined. Chromatic shifts stay within a 3-nm range in both cases. Representative images of four independent experiments are shown (a-c,d-i).