Fig. 4. Confocal images of HeLa cells labeled with VL nanobody by repeated photoporation.

a HeLa cells were photoporated once (1×) with 40 µg/mL VL nanobody Atto 488 (green) and b co-stained with anti-vimentin primary Ab and goat anti-mouse secondary Ab (Alexa Fluor® 568) (magenta) after fixation and permeabilization. c The merged image of (a) and b. The same procedure was repeated for cells labeled by d–f twice repeated (2×) and g–i third repeated (3×) photoporation steps. Live-cell labeling becomes uniform upon repeated photoporation. The scale bar is 10 µm. k Image analysis shows that upon repeated photoporation, the area of green fluorescence increases while the red fluorescence decreases. A total of 39, 49, and 40 cells were analyzed in the 1×, 2×, and 3× repeated photoporation steps, respectively