Fig. 2: mScarlet3-S2 enables 2D and 3D STED imaging.
Actin structures in U-2 OS cells were labeled with Lifeact (an actin-binding peptide) fused to FPs. Cells were fixed 24 h after transfection. a, Confocal (left and right) and STED (middle) images of actin structure imaged with mScarlet3-S2, mScarlet3-M163H or mScarlet-H, respectively. The STED images (middle column) are magnified views of the boxed regions in the confocal images. The right column shows confocal images after STED imaging. Scale bar, 5 μm. b, Ratio of averaged fluorescence intensity in boxed areas after STED imaging (right column of a) versus before STED (left column of a). For each FP, five images were analyzed. Individual dots represent independent measurements derived from two independent experiments. Data are presented as mean ± s.e.m. Significance was calculated by two-tailed Student’s t-test; **P < 0.01 (P = 0.00185), ****P < 0.0001 (P = 4.19 × 10−9). c, FWHM measurements of actin filaments of confocal (Conf.) and STED images (arrows, a, top row). d, Color-coded 3D distributions (XY and YZ views) of ER labeled by mScarlet3-S2, mScarlet3-M163H or mScarlet-H in U-2 OS cells. Scale bar, 5 μm. e, Stacked bar chart of the accumulated fluorescence intensity of whole-stack scanning. f, A 3D reconstruction of the entire ER in U-2 OS cells labeled with sec61β-mScarlet3-S2. g, Structures of sec61β-mScarlet3-S2-labeled invagination reconstructed by Imaris software. The arrowhead and arrows indicate structures resembling NR and NR-like channels. h, XZ view (11.4 µm y-slice) showing NR-like intranuclear channels in confocal (top) and STED (bottom) modes. The hollow arrowhead indicates the NF, while the solid arrowhead and solid arrow denote the NR and NR channels, respectively.
