Segmentation of candidate PIEZO1 particles from iPALM localizations. A representative x100 differential interference contrast image (from n = 5 cells) of a HEK293 cell expressing TCO*K 103 PIEZO1 labelled with tetrazine-Alexa Fluor 647 (AF647) (top left; scale bar, 10 m). A 3 nm per pixel rendering of plasma membrane localizations from the magenta inset on the top left (scale bar, 3 m) (top middle). Binary AF647 localizations (magenta points) with candidate PIEZO1 molecules meeting nearest neighbour requirements highlighted with cyan boxes (scale bar, 3 m) (top right). Representative 3 nm per pixel renderings of candidate triple-labelled PIEZO1 molecules that meet minimum interlocalization separation requirements are also shown (scale bars, 30 nm) (bottom).
