Figure 4.

Comparison of Halo and SNAP Tagging in Live-Cell Super-Resolution Imaging

(A) STED images of HeLa cells that are transiently expressing ManII-Halo or ManII-SNAP and that have been labeled with SiR-CA or SiR-BG, respectively (scale bar: 2 μm). The insets show the confocal image of the region highlighted with the green box. The vertical dark and light green lines indicate where the kymographs shown in the middle were taken (scale bar: 60 s). The plots show the average fluorescent signal as a function of position between the arrows shown in the confocal and STED images (dots, measured values; lines, fit).

(B) Average initial intensity of STED movies of HeLa cells treated as described in (A) (n = 4 cells).

(C) Average intensity over time of STED images of HeLa cells treated as described in (A) (n = 4 cells).

(D) STED images of HeLa cells that are expressing Halo-CLC or SNAP-CLC and were labeled with SiR-CA or SiR-BG, respectively. The green boxes highlight clathrin-coated pits with a hollow center. Magnifications of the clathrin-coated pits highlighted with the dashed green boxes are shown in the upper right corner (scale bar: 1 μm). The plots show the average fluorescent signal as a function of position between the arrows shown in the STED images (dots, measured values; lines, fit).

(E) STED images of Drosophila egg chambers expressing Halo-SNAP-aPKC that have been labeled with JF646-CA (top) or JF-646-BG (bottom). The insets show a confocal image of the area in the green dashed box. The values indicate the full-width half-maximum of line profiles taken between the arrowheads. Scale bar: 2 μm.

(F) First frame of an STED video of Drosophila egg chambers that have been treated as described in (E). Scale bar: 1 μm. The green lines indicate where the kymographs shown next to it have been taken. Scale bar: 100 s.

(G) Average initial intensity of STED movies of Drosophila egg chambers that have been treated as described in (E) (Halo, n = 4; SNAP, n = 6).