Fig. 3: Erasing the fluorescent signal with a reducing agent (ChemiPlex).

a Schematic of ChemiPlex cleaving process. The fluorescent groups in pre-formed complexes of 1.Ab and Chemi-2.Nb, can be cleaved and removed after treatment with TCEP. b Laser scanning confocal images from U2OS-Nup96-GFP cells labeled against vimentin (magenta) using preformed complexes with Chemi-2.Nb carrying the Atto643. The sample was treated with TCEP for 15 min, and the vimentin’s fluorescent signal was recorded at 0 (before adding TCEP), 2.5, 5, 10, and 15 min upon TCEP application. c The fluorescent intensities recorded at each time point were normalized to the intensity values before applying TCEP. d Representative 6-Plex confocal from U2OS-Nup96-GFP cells after application of 6 iterative cycles of ChemiPlex. The plot profiles depict the fluorescence intensity across the denoted line for each target before and after signal removal, while the bar graph shows the signal removal after treatment with TCEP (mean ± SD. n = 3 independent regions). Images from mitochondria, focal adhesions, vimentin, clathrin vesicles, peroxisomes, and nuclear speckles were recorded using the same Atto643, fluorophore; for all staining details, see Methods and Supplementary Table 6.