Combinatorial DNA-PAINT

Abstract

State-of-the-art super-resolution microscopy enables nanometer-resolution imaging of proteins, but its multiplexing capacity has been fundamentally constrained. Here, we present Combi-PAINT, a combinatorial DNA barcoding strategy based on DNA-PAINT, that facilitates superlinear scaling of multiplexing with the number of imaging rounds. Instead of assigning one unique docking strand per target, Combi-PAINT encodes targets as combinations of orthogonal sequences, allowing nonlinear scaling of target number with imaging rounds. Using just six imaging rounds, we resolve 41 targets in a field-of-view of 100 x 100 m2 with [~]2.5 nm localization precision and [~]90% decoding accuracy in under 30 minutes, representing the fastest sub-10 nm super-resolution microscopy acquisition to date. We benchmark decoding fidelity and demonstrate robust in situ performance in mammalian cells, achieving 97% decoding accuracy. Combi-PAINT is compatible with existing DNA-PAINT workflows and speed-enhancing techniques, offering a scalable, accessible platform for high-content, single-molecule spatial proteomics.