Fig. 4. Dual-color live-cell labeling of endogenous targets and live-cell super-resolution imaging by fluorescent nanobodies. (A) Dual-color labeling of endogenous lamin and vimentin in living cells. α-LaminATTO655 and α-VimentinATTO488 were simultaneously transferred into HeLa Kyoto cells. 3 h after cell squeezing, spatially distinct labeling of endogenous lamin (red) and vimentin (green) was visualized by multiplexed imaging. Magnification (box, top) of the α-VimentinATTO488 stained vimentin network showed the three-dimensional organization around the nucleus. The respective confocal z-planes are indicated in the scheme. (B) Reconstructed dSTORM image of endogenous lamin specifically labeled with α-LaminATTO655 fNb (5 h after squeezing). The sensitive and high degree of fNb labeling allowed for visualization of the native nuclear envelope with nanometer precision. (C) Comparison of dSTORM analysis with the averaged fluorescence intensity. Cross section of the intensity profiles (along the dashed line in B) displayed the enhanced resolution by dSTORM. Images were taken by CLSM with the Airy Scan detector or dSTORM. Dashed lines indicate the cell border. Scale bars: 5 μm.
