Fig. 1: The idea and principle of the π-conjugated polymer-based signal amplification (POSA) method.

a Schematic of the DNA amplification and optical amplification using oligonucleotide-barcoded π-conjugated polymer nanoparticles (OCPNs). OCPNs are self-assembled from oligonucleotide grafted fluorescent π-conjugated polymers, showing much higher brightness than molecular dyes. b Workflow for spatial proteomics imaging analysis by POSA with five steps: (1) Various targets (T₁ to T_n) are immunolabeled by orthogonal barcode sequences (BS) encoded primary antibodies (PA_n-BS_n); (2) Three distinctly colored OCPNs (PF OCPNs, PFBT OCPNs, and PFTBT OCPNs) with orthogonal imaging sequences (IS₁-IS₃) are hybridized to the corresponding BS sequences on the antibodies to image T₁ to T₃ simultaneously; (3) The fluorescent signals are erased by the introduction of erasing sequences (ES) to displace OCPNs from antibodies using toehold-mediated strand displacement reactions; (4) More targets are sequentially imaged in the next cycle; (5) The images are reconstructed and recolored to overlay all targets after imaging cycles.