Fluorescence-lifetime encoding for scalable single-protein-resolution imaging

Resolving individual subunits within protein complexes requires precision beyond DNA-PAINT's ~15 nm limit. RESI reaches this by averaging localisations across orthogonal DNA barcodes, but the imaging rounds needed scale quadratically with subunit number. RE-FLIM reduces this cost threefold by resolving three lifetime-distinct barcodes per round. Validated on DNA origami at 3 nm separations, RE-FLIM provides, to our knowledge, the first protomer-resolved view of HIV-1 Env trimers in intact cell membranes.