Fig. 5: SIM and MINFLUX nanoscopy with ReTag1 and highly fluorogenic ligands.

a Wash-free three-color SIM image of live U2OS expressing Vimentin-Halo-ReTag1 stained with 200 nM of MaP655-TMP (cyan hot), 500 nM of MaP555-Actin (orange hot), and 2 µg/mL of Hoechst33342 (red) for 1 h. Scale bar: 10 μm. b Normalized fluorescence intensities of bleaching curves fitting from SIM images of Vimentin labeled with 400 nM MaP618m-TMP (red line, shadow indicates the S.D. value) and 400 nM MaP618m-Halo (blue line, shadow indicates the S.D. value). Data are presented as mean ± S.D., n= three individual experiments. c Wash-free wide field and SIM images of live U2OS cells expressing TOM20-Halo-ReTag1 stained with 200 nM MaP655-TMP. Scale bar: 10 μm. The magnified regions (white boxes) and the plot of the fluorescence signal along the line profile in (a) are shown on the right. Scale bar: 1 μm. d Time-lapse SIM imaging of live U2OS cells expressing TOM20-Halo-ReTag1 labeling with 200 nM MaP618m-TMP, scale bar: 10 μm. The biosynthesis (yellow arrow), elongation (red arrow) and fission (green arrow) of the mitochondria in the magnified regions (white box) was indicated with different time scale. Scale bar: 0.5 μm. e 2D-MINFLUX microscopy image of fixed U2OS cells expressing Vimentin-ReTag1 labeled with MaP655-TMP (2.5 nM). Confocal laser-scanning microscopy (CLSM) imaging was used as a reference. Magnification reveals Vimentin-ReTag1 with a localization precision of ∼5.0 nm. Scale bars: 0.1 μm. Source data are provided as a Source Data file.