Extended Data Fig. 2 |. tkPAINT enables cell-wide DNA-PAINT imaging under ultrastructure-preserved conditions at sub-3 nm localization precision.

a Ultrastructural disruption in standard paraformaldehyde(PFA)-based immunofluorescence protocols for whole cells18. Transmission electron microscopy (TEM) images of HeLa cells depict how brief fixation times lead to poor structural integrity and permeabilization with Triton X-100 causes reductions in cytoplasmic density as well as apparent organelle loss. Nuclear ultrastructure is relatively well-preserved even for brief fixation and permeabilization (Supplementary Fig. 4). b Physical sectioning enables “on-section” labeling of intracellular antigens without permeabilization. TEM images show Tokuyasu cryosections of HeLa cells prepared following a PFA-based fixation protocol optimized for ultrastructural preservation. Immunogold reveals sites of cytoplasmic LAMP1 and nuclear Pol II S5p; Mitochondria (M), nuclear pores (NP), Endoplasmic Reticulum (ER). Golgi and lysosomes (Ly) are highlighted. c tkPAINT principle: physical sectioning (e.g. using the Tokuyasu method) enables TIRF-based DNA-PAINT imaging of ultrastructurally-preserved specimens, even without permeabilization. The localization precision of 2.8 ± 0.1 nm was measured over four independent repeats (mean and std., respectively). Scale bars, 200 nm in (a), 400 nm in (b), 2 μm in (c) and 100 nm in zoom-in.